WO2007048022A2 - Antibody-polypeptide fusion proteins and methods for producing and using same - Google Patents

Antibody-polypeptide fusion proteins and methods for producing and using same Download PDF

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Publication number
WO2007048022A2
WO2007048022A2 PCT/US2006/041215 US2006041215W WO2007048022A2 WO 2007048022 A2 WO2007048022 A2 WO 2007048022A2 US 2006041215 W US2006041215 W US 2006041215W WO 2007048022 A2 WO2007048022 A2 WO 2007048022A2
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WIPO (PCT)
Prior art keywords
antibody
amino acid
acid sequence
antibody fusion
peptide
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PCT/US2006/041215
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French (fr)
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WO2007048022A3 (en
Inventor
Anke Kretz-Rommel
Naveen Dakappagari
Martha Wild
Cecilia Orencia
Katherine S. Bowdish
David R. Gies
Jeremy P. Springhorn
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Alexion Pharmaceuticals, Inc.
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Publication of WO2007048022A2 publication Critical patent/WO2007048022A2/en
Publication of WO2007048022A3 publication Critical patent/WO2007048022A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • BACKGROUND When a healthy host (human or animal) encounters an antigen, normally the host initiates an immune response.
  • This immune response can be a humoral response and/or a cellular response.
  • humoral response antibodies are produced by B- cells and are secreted into the blood and/or lymph in response to an antigenic stimulus. The antibody then neutralizes the antigen by binding specifically to epitopes on its surface, marking it for destruction by phagocytic cells and/or complement- mediated mechanisms.
  • the cellular response is characterized by the selection and expansion of specific helper and cytotoxic T-lymphocytes capable of directly eliminating the cells which contain the antigen.
  • Antigen Presenting Cells process the encountered antigens differently. Exogenous antigens are typically processed within the endosomes of the APC and the generated peptide fragments are presented on the surface of the cell complexed with Major Histocompatibility Complex (MHC) Class II. The presentation of this complex to CD4 + T cells stimulates the CD4 + T helper cells. As a result, cytokines secreted by the helper cells stimulate B cells to produce antibodies against the exogenous antigen (humoral response). Immunizations using antigens typically generate antibody response through this endosomal antigen processing pathway.
  • MHC Major Histocompatibility Complex
  • intracellular antigens as well as some exogenous antigens, are processed in the proteasome and the resulting peptide fragments are presented as complexes with MHC Class I on the surface of APCs.
  • antigen presentation to CD8 + T cells occurs which results in cytotoxic T cell (CTL) immune response to remove the host cells that carry the antigen.
  • CTL cytotoxic T cell
  • Dendritic cells derived from blood monocytes by virtue of their capability as professional antigen presenting cells have been shown to have great potential as immune modulators which stimulate primary T cell response (Steinman et al. (1999) Hum Immunol 60(7): 562-7; Banchereau and Steinman (1998) Nature 392(6673): 245-52).
  • This unique property of the DCs to capture, process, present the antigen and stimulate naive T cells has made them very important tools for therapeutic vaccine development (Laupeze et al. (1999) Hum Immunol 60(7): 591-7).
  • novel antibody fusion constructs for selective delivery of an amino acid sequence (e.g., a peptide or polypeptide) to cells.
  • the novel constructs are useful as vaccines, e.g., for targeted delivery of antigen to antigen presenting cells for the treatment of cancer, autoimmune diseases, inflammation or infectious diseases.
  • methods for using the antibody fusion constructs for a variety of therapeutic applications such as elimination of cancer or other unwanted cells using antibody fusions comprising cytotoxic peptides incorporated into internalizing antibodies specific for receptors on cancer cells or other unwanted cells in a disease state.
  • Other therapeutic applications include targeted delivery of chemotactic peptides or other therapeutic peptides, such as growth factors or fragments thereof.
  • the antibody fusion constructs described herein may also be used as a screening tool for identifying internalizing antibodies or cytotoxic peptides.
  • receptor specific antibodies may be used.
  • the peptide epitope may be grafted into a location in the antibody that does not disrupt protein folding and allows for robust production of the antibody fusion proteins.
  • the antibody fusion will be taken up by the target cell and processed correctly to release the appropriate peptide epitope.
  • selection of an antibody constant region in which Fc Receptor binding is minimized e.g., immunoglobulin (Ig) G2G4 may be desirable to avoid or reduce non-specific cellular uptake by Fc receptor bearing cells.
  • Controlled intracellular release of a peptide from the antibody fusion construct may be achieved by incorporating the peptide into the antibody between proteasomal cleavage sites.
  • the proteasomal cleavage sites may be naturally occurring in the antibody or may be introduced into the antibody.
  • Exemplary proteasomal cleavage sites comprise two or more lysine and/or arginine residues. Incorporation of a polypeptide into an antibody between flanking proteasomal cleavage sites permits insertion into any region of the antibody and allows maximal flexibility in selecting a site of incorporation that favors efficient folding and expression of the protein.
  • peptides may be incorporated into the constant domain of an antibody at a region exhibiting sequence similarity with the peptide and/or at a hydrophobic region within the constant domain. As many peptide epitopes are hydrophobic, a region of the antibody constant domain that is both hydrophobic and has sequence similarity with the peptide may be selected for incorporation. Introduction of a polypeptide into a region of hydrophobicity and/or sequence similarity may facilitate efficient expression and proper folding of the antibody fusion construct.
  • an antibody fusion wherein the antibody comprises a polypeptide incorporated into the constant region within the hinge region or close to the hinge region.
  • the polypeptide may be flanked at one or both ends by proteasomal cleavage sites, such as two or more lysine and/or arginine residues.
  • the antibody fusions provided herein may comprise antibody fragments such as, for example, a Fab or a single chain antibody (ScFv) fused to at least a portion of a heavy and/or light chain constant region.
  • the antibody fusions provided herein may comprise an amino acid sequence attached to the C-terminus of the heavy and/or light chain of the antibody.
  • Antibody fusions that have at least one amino acid sequence incorporated into the constant region and at least one polypeptide attached to the C- terminus of the heavy and/or light chain are also provided.
  • the antibody fusions may have a peptide incorporated into the constant domain and/or fused to the C-terminus of the constant domain in association with a cleavable peptide linker.
  • cleavable linkers are also flexible.
  • the antibody fusions may comprise one or more polypeptides incorporated into the same or different locations in the antibody molecule.
  • an antibody fusion comprising a disease specific carrier protein having multiple disease specific epitopes incorporated into the carrier protein.
  • the disease specific carrier protein may have one or more disease specific peptide epitopes incorporated at regions of sequence similarity and/or hydrophobicity and/or between proteasomal cleavage sites.
  • the carrier protein may be linked to an antibody having a desired specificity by chemical conjugation or
  • the specificity of the antibody may be selected for targeted delivery of the disease specific carrier protein to a desired cell type.
  • the method involves contacting the cells with an antibody fusion comprising an antibody and a cytotoxic peptide.
  • the antibody is specific for a protein expressed on the surface of a cell or cell population that is to be targeted for destruction.
  • the cytotoxic peptide is released inside the cell resulting in cell death.
  • the antibody fusions comprising a cytotoxic peptide may be used for treating cancer.
  • the antibody fusions useful for treating cancer comprise an antibody specific for one or more tumor associated antigens and/or an antibody that binds to a cell surface protein involved in undesirable immune suppression, such as, for example, regulatory T-cells.
  • the antibody fusions described herein may be used for targeted delivery of a variety of polypeptides including, for example, growth factors or fragments thereof, and chemotactic agents. Targeted delivery of such polypeptides is useful for controlled stimulation of cell growth, cell differentiation, or other cellular functions.
  • screening methods for identifying internalizing antibodies or cytotoxic peptides are provided.
  • libraries of antibodies comprising a peptide toxin linked to a plurality of antibodies may be constructed. Such libraries can be screened against cancer cells, or any other cell target of interest and internalizing antibodies may be identified using cell death as a read out.
  • a library of potential cytotoxic peptides may be attached or incorporated into an internalizing antibody. The antibody fusion library is mixed with cells and cytotoxic peptides may be identified using cell death as a read out.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S.
  • FIGURE 1 shows the amino acid sequence of immunoglobulin G2G4 fused constant region (SEQ ID NO: 1) showing 2OS proteasome cleavage sites and hydrophobic areas.
  • SEQ ID NO: 1 shows the amino acid sequence of immunoglobulin G2G4 fused constant region (SEQ ID NO: 1) showing 2OS proteasome cleavage sites and hydrophobic areas.
  • the letter “S” underneath the sequence indicates 2OS proteasome cleavage sites predicted by the algorithm “NetChop.” Highlighted areas indicate hydrophobic patches predicted by the Kyte-Doolittle algorithm.
  • FIGURE 2 shows the amino acid sequence of immunoglobulin Gl constant region (SEQ ID NO: 2) showing 2OS proteasome cleavage sites and hydrophobic areas.
  • FIGURE 3 shows an alignment of the amino acid sequences for the immunoglobulin Gl constant region (SEQ ID NO: 2) and the immunoglobulin G2G4 fused constant region (SEQ ID NO: 1). Sequence differences are indicated asterisks. Hydrophobic patches are highlighted.
  • FIGURES 4A-B illustrate immunological responses to tetanus toxin (TT).
  • Figure 4A shows the antibody responses to TT protein. Serum from all donors except donor 14 was collected two weeks after vaccination. Donor 14 was vaccinated in the year 1997, but had equivalent levels of antibody responses (see donor 14 - prevaccine 2) as the vaccinated donors.
  • Figure 4B shows the T-cell responses to TT peptides two weeks after vaccination. 100,000 PBL were incubated with lug/ml peptide and 25ng/ml TT protein for five days. Proliferation was enumerated by 3 H-Thymidine incorporation during the last 18 hours.
  • FIGURES 5A-B illustrate antibody fusions that were constructed using sequence similarity and insertion strategies.
  • - Polypeptide TT (632DR) was introduced into the constant region of the DC-SIGN/L-SIGN antibody (clone ElO) by insertion and sequence similarity replacement.
  • Figure 5 A shows a schematic of the sites of introduction of the TT polypeptide (632DR) into the constant region of the antibody and the local sequences involved in each construct.
  • the local sequence for insertion construct is SEQ ID NO: 3
  • the local sequence on the Ig constant region (ElO) for the sequence similarity replacement construct is SEQ ID NO: 4
  • the TT polypeptide sequence incorporated into the constant region (632DR) is SEQ ID NO: 5.
  • Figure 5B shows the amino acid sequence of the IgGl constant region (SEQ ID NO: 2) of the antibody. The sites for incorporation of the TT polypeptide (632DR) by insertion and replacement are indicated by arrows.
  • FIGURES 6A-B show expression and binding characteristics of antibody fusions illustrated in Figures 5A-B.
  • Panel A is a Western blot showing the expression of antibody fusions generated by insertion and replacement (as described in Figure 5) as compared to native antibody. 20 ul of supernatant from each clone was run on a native SDS-PAGE gradient gel (4%-15%), transferred to nitrocellulose and detected with anti-light chain-HRP conjugate. The peptide fusion generated by insertion shows a size difference of about ⁇ 2kDa as compared to the native antibody.
  • Figure 6B illustrates the binding of the insertion and replacement antibody fusions to DC- SIGN receptors on cells (0.5 million/sample) as compared to native antibody. The experiments were conducted using K562 cells (K562) or K562 cells overexpressing the DC-SIGN receptor (K562/DC-SIGN) or the L-SIGN receptor (K562/L-SIGN).
  • FIGURES 7A-B show affinity purified antibody fusions (insertion clone) and the binding affinity of the antibody fusion insertion clone as compared to the native
  • FIG. 7A shows a native SDS-PAGE gradient gel (4%-15%) of affinity purified peptide inserted antibodies. As shown on the gel, the affinity purification eliminated partially formed antibodies (e.g., as compared to supernatant fraction shown in Figure 6A).
  • Figure 7B shows the relative binding affinity of the peptide inserted antibody as compared to the native antibody. Binding affinities were determined using K562 cells overexpressing DC-SIGN or L-SIGN receptors.
  • FIGURE 8 provides the results of T-cell activation after targeted delivery of TT peptide to immature dendritic cells (iDCs).
  • T-cells from vaccinated donors were used. 10,000 iDCs were incubated with 10 ug/ml (66 nM) of native antibody (ElO), 10 ug/ml (66 nM) of peptide inserted antibody (E10-632DR), lug/ml (500 nM) of the 632DR fragment of TT protein, or medium, for lhr at 37 0 C. The iDCs were then washed and added to 100,000 T-cells and incubated for five days.
  • ElO native antibody
  • E10-632DR peptide inserted antibody
  • lug/ml 500 nM
  • FIGURES 9A-B illustrate the dose dependent response of native and peptide inserted antibodies for T-cell activation and iDC binding.
  • Figure 9A illustrates the dose dependent response of T-cell activation to the targeting antibody (E10-632DR) and blocking of T-cell activation by competition with native antibody (ElO). 10,000 iDC were incubated with antibodies and free peptide for 1 hour at 37 0 C, washed and added to 100,000 T-cells and incubated for five days.
  • FIGURE 10 shows a graph illustrating that antibody targeting produces a sustained immune response.
  • the cells were treated as described in Figure 9 A, except that T cells were added 2 and 4 days after antibody targeting. Highly significant
  • FIGURE 11 shows the competitive binding of native antibody (ElO) and peptide inserted antibody (E10-632DR) to the Fc receptors.
  • U937 cells were incubated with 15 ng/ml biotin-hlgGl together with various concentrations of competing unbiotinylated antibodies. The percentage of cells bound to biotin-hlgGl was determined using streptavidin-phycoerythrin by flow cytometry analysis.
  • FIGURE 12 is a schematic of a peptide inserted antibody designed by sequence similarity replacement in the CH3 domain of the heavy chain constant region. The local amino acid sequence at the site of peptide introduction is shown (SEQ ID NO: 6).
  • FIGURE 13 is a schematic of a peptide inserted antibody having a peptide sequence inserted into the CHl domain of the heavy chain constant region and flanked by arginine (top) or lysine (bottom) residues.
  • the local amino acid sequence at the site of peptide introduction is shown for both the top (SEQ ID NO: 7) and bottom constructs (SEQ ID NO: 8).
  • FIGURES 14A-G show several embodiments of antibody peptide fusions having a tetanus toxin (TT) peptide introduced into the G2G4 constant region.
  • Figure 14A shows the sequence similarity between a region of the CH3 domain of the G2G4 constant region (SEQ ID NO: 9) and the TT 230 peptide (SEQ ID NO: 10).
  • Figure 14B shows the local sequence and proteasomal cleavage sites (S) for the TT 230 peptide embedded into the CH3 domain of the G2G4 constant region (SEQ ID NO: 11).
  • Figure 14C illustrates two variations of the embedded TT 230 peptide having proteasomal cleavage sites introduced at the N-terminus (top; SEQ ID NO: 12) or at the N-terminus and C-terminus (bottom; SEQ ID NO: 13).
  • Figure 14D shows the sequence similarity between a region of the CH3 domain of the G2G4 constant region (SEQ ID NO: 14) and the TT 702 peptide (SEQ ID NO: 15).
  • Figure 14E shows the local sequence and proteasomal cleavage sites (S) for the TT 702 peptide embedded into the CH3 domain of the G2G4 constant region (SEQ ID NO: 16).
  • Figure 14F shows the sequence similarity between a region of the CH2 domain of the G2G4 constant region (SEQ ID NO: 17) and the TT 632-651 peptide (SEQ ID NO: 18).
  • Figure 14G shows the local sequence and proteasomal cleavage sites (S) for the TT 632-651 peptide embedded into the CH2 domain of the G2G4 constant region (SEQ ID NO: 19).
  • FIGURE 15 shows the local sequence alignment for two antibody peptide fusions having the TT 830-844 peptide attached to the C-terminus of the antibody heavy chain via a cleavable linker having two arginine (top; SEQ ID NO: 20) or two lysine (bottom; SEQ ID NO: 21) residues.
  • FIGURE 16 shows the amino acid sequence for human Glutamic-Acid
  • Decarboxylase 65kDa protein (GAD65) (SEQ ID NO: 22). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
  • FIGURE 17 shows the amino acid sequence for human Heat Shock Protein 6OkDa (HSP60) (SEQ ID NO: 23). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
  • FIGURE 18 shows the amino acid sequence for Human Insulinoma Associated protein (IA-2) (SEQ ID NO: 24). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
  • IA-2 Human Insulinoma Associated protein
  • FIGURE 19 shows the amino acid sequence for Human Proinsulin (PI) (SEQ ID NO: 25). Hydrophobic patches are highlighted, 20S proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
  • FIGURE 20 shows the amino acid sequence for Melanocyte Lineage-Specific PI
  • Antigen gplOO (SEQ ID NO: 26). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitopes are boxed and lower case.
  • FIGURE 21 shows the amino acid sequence for Melanocyte Associated Tumor Antigen, Tyrosinase (SEQ ID NO: 27). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
  • FIGURE 22 shows the amino acid sequence for LMP-2 Membrane Protein (SEQ ID NO: 28). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
  • FIGURE 23 shows the amino acid sequence for Carcinoembryonic Antigen (CEA) (SEQ ID NO: 29). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
  • CEA Carcinoembryonic Antigen
  • FIGURE 24 shows a flow diagram illustrating a method for designing disease specific carrier proteins with multiple embedded disease specific epitopes.
  • FIGURE 25A shows the local sequence and proteasomal cleavage sites (S) for a peptide toxin attached to the Fab heavy chain of clone ElO (SEQ ID NO: 30).
  • Figure 25B is a table showing the proteasomal cleavage scores for the sequence shown in Figure 25A. The site with the highest cleavage score is highlighted.
  • FIGURE 26A shows the local sequence and proteasomal cleavage sites (S) for a peptide toxin attached to the C-terminus of the Fab light chain of clone ElO (SEQ ID NO: 31).
  • Figure 26B is a table showing the proteasomal cleavage scores for the sequence shown in Figure 26A. The site with the highest cleavage score is highlighted.
  • FIGURE 27A shows the local sequence and proteasomal cleavage sites (S) for a peptide toxin embedded into the hinge region of chimeric IgGl clone ElO (SEQ ID NO: 32).
  • Figure 27B is a table showing the proteasomal cleavage scores for the sequence shown in Figure 27A. The site with the highest cleavage score is highlighted.
  • FIGURE 28 shows a table providing the sequences of the hinge region from various human, mouse, rat, guinea pig and rabbit antibodies (SEQ ID NOs: 33-50).
  • FIGURE 29 shows a variety of goat anti-Fab purified chimeric antibodies that were run on an SDS-PAGE gel and stained with Coomassie blue. Lane a:
  • FIGURE 30 shows a half antibody (Hab) that was run on an SDS-PAGE gel and stained with Coomassie blue or western blotted.
  • Lane a Molecular weight marker
  • lane b E10chGl-HC632tt/LC947, Coomassie blue stain
  • lane c ElOchGl- HC632tt/LC947, Western with goat anti-human kappa chain antibody
  • lane d E10chGl-HC632tt/LC947, Western with goat anti-human gamma chain antibody.
  • FIGURE 31 shows immune responses elicited by DC-SIGN antibody (clone
  • ElOchlgGl grafted with TT epitopes, 632DR and 947DR.
  • Panel A 10 ⁇ g/mL antibody
  • Panel B 0.1 ⁇ g/mL antibody.
  • FIGURE 32 shows the immune responses elicited by DC-SIGN antibody (clone E10chIgG2G4) grafted with TT epitopes, 632DR and 947DR.
  • Panel A 10 ⁇ g/mL antibody
  • Panel B 0.1 ⁇ g/mL antibody.
  • FIGURE 33 shows internalization of CD19 single chain antibody, clone 2G12 on RAJI cells. Percentage of internalization indicated on top of the bars was determined as (Geo. mean fluorescence at 4°C - Geo. mean fluorescence at 37°C)/(Geo. mean fluorescence at 4°C) X 100.
  • FIGURE 34 shows binding of peptide toxin embedded full length IgGl antibodies (clone 2G12) to RAJI cells.
  • FIGURE 35 shows binding of peptide toxin embedded single chain antibodies
  • FIGURE 36 shows inhibition of RAJI cell growth by peptide toxin embedded CDl 9 antibody, clone 2G12.
  • Panel A High Dose Full length Toxin Embedded Antibodies
  • Panel B Low Dose Full length Toxin Embedded Antibodies
  • Panel C High Dose Single Chain Toxin Embedded Antibodies
  • Panel D Low Dose Single Chain Toxin Embedded Antibodies
  • Panel E Synthetic Free Peptide Toxins.
  • an “immune cell” refers to those cells critical for immune response in an individual and which are commonly found in the lymphatic system, and in particular, in lymph nodes. Such cells include T cells (or T lymphocytes), B cells (or B lymphocytes), natural killer (NK) cells, macrophages and dendritic cells.
  • each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50- 70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • IgG, IgA and IgD isotypes have a "hinge region" which is an amino acid sequence of from about 10-60 amino acids that confers flexibility on the immunoglobulin molecule.
  • the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.
  • Immunoglobulins may be organized into higher order structures.
  • IgA is generally a dimer of two tetramers.
  • IgM is generally a pentamer of five tetramers.
  • Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. MoI. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
  • an “antibody” refers to an intact immunoglobulin, or to an antigen-binding portion thereof that competes with the intact antibody for specific binding.
  • Antigen- binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding portions include, inter
  • 1Q235000J 12 alia, Fab, Fab', F(ab') 2 , Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • CDR complementarity determining region
  • An Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CHl domains; a F(ab') 2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of the VH and CHl domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341 : 544-546, 1989) consists of a VH domain.
  • a single-chain antibody is an antibody in which VL and VH regions are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).
  • Diabodies are bivalent or bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak, R. J., et al., Structure 2:1121-1123, 1994).
  • One or more CDRs may be incorporated into a molecule either covalently or noncovalently.
  • a minibody is a bivalent or bispecific antibody in which two scFv monomers are joined by two constant domains (see e.g., Hudson, PJ. and Sourisu, C, Nature Medicine 9: 129-134 (2003)).
  • An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites (e.g., bivalent), a single-chain antibody or Fab fragment may have one or two binding sites, while a "bispecific" or "bifunctional” antibody has two different binding sites.
  • the term "human antibody” includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In a preferred embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). These antibodies may be prepared in a variety of ways, as described below.
  • hinge refers to a region of the heavy chain that comprises amino acid residues 224 to 251 (Kabat numbering scheme). This region encompasses the genetic hinge (e.g., amino acid residues 224- 243 using the Kabat numbering scheme) as well as amino acid residues C-terminal to the genetic hinge that are structurally flexible.
  • Exemplary hinge region sequences include, for example, the hinge regions for human IgG, IgA and IgD isotypes and mouse, rat, guinea pig and rabbit IgG isotypes that are provided in Figure 28 (see also, Burton DR, Molecular Genetics of Immunoglobulin, Chapter 1, Calabi, F. and Neuberger, M.S., eds; Elsevier Science Publishers B.V. (1987)).
  • the hinge region may be divided into three subregions referred to as the upper hinge, the middle hinge, and the lower hinge ( Figure 28).
  • chimeric antibody refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
  • tumor-associated antigen refers to a polypeptide which is preferably presented by tumor cells and thus allows a distinction between tumor cells and non-tumor cells.
  • Tumor associated antigens are proteins expressed inside or on the surface of tumor cells which are putative targets for immune responses. They often differ from normal cellular counterparts by mutations, deletions, different levels of expression, changes in secondary modifications or expression in other stages of development. The proteins are preferably expressed on the cellular surface and, in addition, presented as processed peptides on the tumor cell surface by MHC class I molecules.
  • tumor-associated antigens include, for example, CAl 25, CA19-9, CA15-3, D97, gplOO, CD20, CD21, TAG-72, EGF receptor, Epithelial cell adhesion molecule (Ep-CAM), Carcino-embryonic antigen (CEA), Prostate specific antigen (PSA), PMSA, CDCPl, CD26, Hepsin, HGF (hepatocyte growth factor), Met, CAIX(G250), EphhB4 (Ephrin type-B receptor 4), EGFRl, EGFR2, PDGF, VEGFR, DPP6, syndecan 1, IGFBP2 (Human insulin-like growth factor binding protein 2), CD3, CD28, CTL4, VEGF, Her2/Neu receptor, tyrosinase, MAGE 1, MAGE 3, MART, BAGE, TRP-I, CA 50, CA 72-4, MUC 1, NSE (neuron specific enolase), ⁇ - fetoprotein (AFP), SSC (
  • sequence similarity refers to the proportion of amino acid matches plus conservative amino acid substitutions between two amino acid sequences over a window of comparison.
  • percentage of sequence similarity is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of matched positions by adding (i) the number of positions at which the identical amino acid occurs in both sequences and (ii) the number of positions at which the sequences contain conservative amino acid substitutions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence similarity.
  • sequence identity means that sequences are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino acid basis for polypeptides) over a window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
  • conserved residue refers to an amino acid that is a member of a group of amino acids having certain common properties.
  • amino acid substitution refers to the substitution (conceptually or otherwise) of an amino acid from one such group with a different amino acid from the same group.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure, Springer- Verlag 1990). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of
  • One example of a set of amino acid groups defined in this manner include: (i) a charged group, consisting of GIu and Asp, Lys, Arg and His, (ii) a positively-charged group, consisting of Lys, Arg and His, (iii) a negatively-charged group, consisting of GIu and Asp, (iv) an aromatic group,
  • 10235000_l 16 consisting of Phe, Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large aliphatic nonpolar group, consisting of VaI, Leu and He, (vii) a slightly- polar group, consisting of Met and Cys, (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, GIy, Ala, GIu, GIn and Pro, (ix) an aliphatic group consisting of VaI, Leu, lie, Met and Cys, and (x) a small hydroxyl group consisting of Ser and Thr.
  • a “structural domain” refers to a region of a polypeptide that is folded in such a way as to confer a particular secondary and/or tertiary structure, such as, for example, an alpha helix or beta sheet.
  • therapeutically effective amount refers to that amount of an antibody fusion, drug or other molecule which is sufficient to effect treatment when administered to a subject in need of such treatment.
  • the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • antibody fusions having one or more amino acid sequences incorporated into the constant region of the antibody are provided.
  • Amino acid sequences may be incorporated into the constant region of the antibody at a hydrophobic region, at a region flanked by proteasomal cleavage sites, and/or at a region having amino acid sequence similarity with the amino acid sequence.
  • an amino acid sequence is incorporated into the constant region at a location that does not affect the epitope binding activity of the antibody and/or that permits the antibody to be expressed and/or secreted at sufficient levels (e.g., levels approaching that of an unmodified antibody molecule).
  • amino acid sequence incorporated into the antibody is normative to the antibody, e.g., the sequence being incorporated into the antibody is not normally found in the antibody at that location.
  • the amino acid sequence being incorporated into the constant region of an antibody may be from a different location within the same antibody molecule, may be from a different antibody molecule, or may be from a non-antibody molecule.
  • amino acid sequences to be incorporated into an antibody may be a peptide, a polypeptide, a fragment of a polypeptide, or a fusion between two or
  • the amino acid sequence may be a naturally occurring sequence, a variant of a naturally occurring sequence, or a synthetic sequence, or combinations thereof.
  • an amino acid sequence may be incorporated into an antibody molecule by inserting the polypeptide into the antibody (e.g., the amino acid sequence is added to the sequence of the antibody).
  • an amino acid sequence may be incorporated into an antibody by replacing a portion of the antibody sequence with the introduced sequence.
  • the length of the amino acid sequence being introduced may be the same size, larger or smaller than the antibody sequence being replaced (e.g., a sequence of 10 amino acids to be incorporated may replace a region of sequence on the antibody molecule that is 5, 10, or 15 amino acids in length).
  • the amino acid sequence is the same length as the region of the antibody sequence being replaced such that the overall size of the antibody molecule is maintained.
  • amino acid sequences may be incorporated into the antibody at one or more locations.
  • the amino acid sequence may be any size, but typically is from about 4-100, 4-50, 4-25, 4-20, 5- 15, 5-10, 5-8, or 8-11 amino acids in length.
  • antibody fusions having one or more amino acid sequences incorporated into the constant region of the antibody between proteasomal cleavage sites are provided.
  • the proteasomal cleavage sites may be naturally occurring in the antibody sequence and/or may be introduced into the antibody fusion at one or more desired locations.
  • an amino acid sequence is incorporated into an antibody constant region between two naturally occurring proteasomal sequences.
  • an amino acid sequence is incorporated into an antibody constant region between proteasomal cleavage sites that have been introduced (e.g., the proteasomal cleavage sites are not naturally occurring in the antibody sequence).
  • an amino acid sequence may be incorporated into the constant region of an antibody between a combination of a naturally occurring proteasomal cleavage site and an introduced proteasomal cleavage site.
  • the proteasomal cleavage sites are located at or directly adjacent to the N-terminus and C-terminus of the amino acid sequence being incorporated into the antibody. For example, one, two, three or more consecutive residues at the N-terminus and/or C-terminus of the amino acid sequence to be
  • 10235000_l Jg incorporated into the antibody may be proteasomal cleavage sites.
  • one, two, three or more residues of the antibody sequence that are located directly next to the N-terminus and/or the C-terminus of the amino acid sequence incorporated into the antibody molecule may be proteasomal cleavage sites.
  • Various combinations thereof are also contemplated within the scope of this disclosure.
  • two or more amino acid sequences may be incorporated into the antibody at the same or different locations.
  • the antibody fusion may have the structure Ab-X-sequence 1-X-sequence 2-X-Ab, wherein Ab represents the antibody constant region that is being modified by the incorporated sequences, X represents a proteasomal cleavage site, and sequence 1 and sequence 2 represent two amino acid sequences (e.g., having the same or different sequences) being incorporated into the antibody.
  • the proteasomal cleavage sites may be naturally occurring in the antibody or normative to the antibody and may comprise one, two, three or more consecutive proteasomal cleavage sites.
  • the antibody fusions may comprise one or more amino acid residues between the proteasomal cleavage sites and the antibody constant region.
  • the antibody fusion may have the structure Ab-N-X-sequence- X-N-Ab, or Ab-N-X-sequence-X-Ab or Ab-X-sequence-X-N-Ab, wherein Ab represents the antibody constant region that is being modified by the incorporated sequences, X represents a proteasomal cleavage site, N represents one or more amino acid residues nonnative to the antibody, and sequence represents the sequence being incorporated into the antibody.
  • N may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues in length and may be located between the antibody sequence and proteasomal cleavage at one or both ends flanking the amino acid sequence being incorporated into the antibody constant region.
  • a flexible linker may be added to the antibody fusion to reduce structural constraints, increase antibody expression, facilitate proper antibody folding, and/or facilitate formation of a disulfide bond between two antibody chains.
  • flexible linkers include, for example, linkers comprising about 2 to 50, about 2 to 25, about 2 to 20, about 2 to 15, about 2 to 10, about 5 to 20, about 5 to 15, or about 5 to 10 small amino acid residues, such as alanine, glycine, serine or
  • Exemplary linkers comprise the amino acid sequence GGGAAG (SEQ ID NO: 129) or GGGAAAGAAG (SEQ ID NO: 130). Such flexible linkers may be incorporated at one or both ends flanking an amino acid sequence incorporated into the antibody.
  • the antibody fusion may have the structure Ab-X-F-sequence-F-X-Ab, Ab-X-sequence-F-X-Ab, Ab-X-F-sequence-X- Ab, Ab-F-X-sequence-X-F-Ab, Ab-X-sequence-X-F-Ab, or Ab-F-X-sequence-X-Ab, wherein Ab represents the antibody constant region that is being modified by the incorporated sequences, X represents a proteasomal cleavage site, F represents a flexible linker sequence, and sequence represents the sequence being incorporated into the antibody. Additionally, such linkers may be incorporated between the C- terminus of a heavy or light antibody chain and an amino acid sequence attached to the C-terminus.
  • the antibody fusions having an amino acid sequence incorporated into the antibody constant region may further comprise at least one amino acid sequence attached to the C-terminus of the heavy and/or light chain constant region.
  • the amino acid sequence may be attached to the C-terminus of the heavy and/or light chain constant region by a peptide or chemical linker.
  • the amino acid sequence may be a cytotoxic peptide.
  • Naturally occurring proteasomal cleavage sites may be determined using computer algorithms. For example, computer algorithms based on 2OS proteasome cleavage motifs have been used to successfully predict proteolytic hot spots in proteins of interest (Saxova, P. et al., Int Immunol 15: 781-787 (2003)). See, e.g., world wide web at cbs.dtu.dk/services/NetChop/.
  • a peptide is incorporated into an antibody constant region between two naturally occurring proteasomal cleavage sites wherein each site comprises two, three, four, or more, consecutive cleavage sites.
  • the 2OS proteasome is a 700 kDa cylindrical-shaped multicatalytic protease complex comprised of 28 subunits organized into four rings. In yeast and other eukaryotes, 7 different alpha subunits form the outer rings and 7 different beta subunits comprise the inner rings. The alpha subunits serve as binding sites for the 19S (PA700) and 1 IS (PA28) regulatory complexes, as well as a physical barrier for the inner proteolytic chamber formed by the two beta subunit rings. Thus, in vivo, the proteasome is believed to exist as a 26S particle ("the 26S proteasome"). In vivo experiments have shown that inhibition of the 2OS form of the proteasome can be
  • N-terminal nucleophile hydrolases where the nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, and other nucleophilic moieties.
  • This family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosylasparaginase.
  • PGA penicillin G acylase
  • PVA penicillin V acylase
  • GAT glutamine PRPP amidotransferase
  • bacterial glycosylasparaginase bacterial glycosylasparaginase.
  • higher vertebrates also possess three .gamma.-interferon-inducible beta subunits (LMP7, LMP2 and MECLl), which replace their normal counterparts, X, Y and Z respectively, thus altering the catalytic activities of the proteasome.
  • LMP7, LMP2 and MECLl three .gamma.-interferon-inducible beta subunits
  • proteolytic activities have been defined for the eukaryote 2OS proteasome: chymotrypsin-like activity (CT-L), which cleaves after large hydrophobic residues; trypsin-like activity (T-L), which cleaves after basic residues; and peptidylglutamyl peptide hydrolyzing activity (PGPH), which cleaves after acidic residues.
  • C-L chymotrypsin-like activity
  • T-L trypsin-like activity
  • PGPH peptidylglutamyl peptide hydrolyzing activity
  • Two additional less characterized activities have also been ascribed to the proteasome: BrAAP activity, which cleaves after branched- chain amino acids; and SNAAP activity, which cleaves after small neutral amino acids.
  • the major proteasome proteolytic activities appear to be contributed by different catalytic sites, since inhibitors, point mutations in beta subunits and the exchange of gamma interferon-inducing beta subunits
  • Introduced proteasomal cleavage sites may include one or more amino acids that lead to proteasomal cleavage, for example by the eukaryotic 2OS proteasome. Suitable amino acid residues include, for example, large hydrophobic residues (e.g., tyrosine, phenylalanine or tryptophan), basic residues (e.g. lysine, arginine, or histidine), and/or acidic residues (e.g., glutamine or asparagine). Introduced proteasomal cleavage sites may comprise at least one, two, three, four, or more, consecutive amino acids that cause proteasomal cleavage.
  • large hydrophobic residues e.g., tyrosine, phenylalanine or tryptophan
  • basic residues e.g. lysine, arginine, or histidine
  • acidic residues e.g., glutamine or asparagine
  • introduced proteasomal cleavage sites include one or more lysine and/or arginine residues, or combinations thereof (e.g., RR, KK, RK, KR, etc.). Proteasomal cleavage sites comprising the peptide sequence RRR or KKK have been shown to
  • 10235000_l 21 serve as good substrates for cleavage by cellular proteasomes (Livingston, B. D. et al., Vaccine 19: 4652-4660 (2001); Sundaram, R. et al., Vaccine 21: 2767-2781 (2003)).
  • one or more amino acid sequences may be incorporated into the constant region of the antibody at a hydrophobic region.
  • Hydrophobic regions may be determined, for example, using the Kyte-Doolittle hydrophobicity prediction algorithm (Kyte J. and Doolittle R.F., J. MoI Biol, 157: 105-31 (1982)).
  • Hydrophobic regions suitable for insertion of an amino acid sequence may be from about 4-40, 10-30, 10-20, or 10-15 amino acids in length.
  • the hydrophobic region is sufficiently large to incorporate the amino acid sequence without affecting, or without significantly affecting, proper expression and folding of the antibody molecule.
  • the hydrophobic regions may be located in a constant domain of an immunoglobulin light chain or a constant domain of an immunoglobulin heavy chain.
  • the hydrophobic region is located in the CHl, CH2 or CH3 domain of a heavy chain constant region.
  • the hydrophobic region also contains one or more proteasomal cleavage sites.
  • Exemplary hydrophobic regions that contain one or more proteasosomal cleavage sites include amino acid residues 135-146, 149-198, 243-259, 271-292, 320-329, 388-400, or 453-464 of the heavy chain constant domain based on the Kabat numbering system.
  • the antibody fusions provided herein do not contain an amino acid sequence incorporated into the CHl region. In other embodiments, the antibody fusions provided herein do not contain an amino acid sequence incorporated between residues 146-152, 178-185, and/or 213-216 (Kabat numbering scheme). In another embodiment, one or more amino acid sequences may be incorporated into the constant region of the antibody at a region having amino acid sequence similarity with the sequence being incorporated. In exemplary embodiments, an amino acid sequence may be incorporated into the constant region of the antibody at a region having at least about 40%, 42%, 45%, 47%, 50%, 55%, 60%, 70%, 75%, 80%, 90%, or greater percent similarity, with the sequence being incorporated.
  • the region of sequence similarity may be a hydrophobic region, or part of a hydrophobic region, as described above.
  • an amino acid sequence is incorporated into a hydrophobic region having the highest degree of sequence similarity with the sequence being incorporated.
  • the amino acid sequence may be incorporated into the antibody by replacing a region of the antibody having
  • 1023500CM 22 sequence similarity with the sequence to be incorporated or by changing one or more amino acid residues in the antibody sequence in order to convert a portion of the antibody sequence into the sequence to be incorporated.
  • Methods for determining sequence identity and similarity include, for example, computer programs such as the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984); BESTFIT; MegAlign (DNAstar, Madison, WI); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. MoI. Biol.
  • BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. MoI. Biol. 215:403-410 (1990)).
  • an amino acid sequence may be attached to the C- terminus of the constant region of the heavy or light chain of an antibody.
  • the amino acid sequences are attached to the antibody by a cleavable peptide linker.
  • the peptide linker may comprise for example two, three, four, five, or more, small amino acid residues, such as glycine or serine.
  • the linkers may comprise one or more amino acids that cause proteasomal cleavage (typically lysine or arginine) located near or directly adjacent to the N-terminus of the polypeptide.
  • Exemplary linkers include, for example, peptides having the sequence GGX n , GGGX n (SEQ ID NO: 51), GGGGX n (SEQ ID NO: 52), GGGSX n (SEQ ID NO: 53), GGGSGGGSX n (SEQ ID NO: 54), GGGAAGX n (SEQ ID NO: 179) or GGGAAAGAAGX n (SEQ ID NO: 180) wherein X is lysine or arginine and n is 1-5.
  • an amino acid sequence is incorporated into a constant region of the antibody at a region having structural flexibility.
  • Regions of structural flexibility include regions that have poorly defined secondary structure such as a loop, turn, or extended amino acid chains that do not fold into an alpha helix or beta sheet structure.
  • Regions of structural flexibility include regions joining two structural domains such as a region joining two domains having an alpha helix or beta sheet structure. Exemplary regions of structural flexibility contained in an antibody
  • 10235000J 23 constant region include: the region between the variable light (VL) and constant light (CL) domains of the light chain, and the regions between the variable heavy (VH) and CHl domains, CHl and CH2 domains, or CH2 and CH3 domains of the heavy chain.
  • VL variable light
  • CL constant light
  • VH variable heavy
  • CHl and CH2 domains CHl and CH2 domains
  • CH2 and CH3 domains of the heavy chain CH2 and CH3 domains of the heavy chain.
  • the hinge region of IgG, IgA and IgD isotypes is a further example of a region of structural flexibility.
  • an amino acid sequence is incorporated into or near the hinge region of an antibody.
  • the amino acid sequence may be incorporated into the hinge region itself, at the junction between the N-terminus of the hinge region and the upstream region of the heavy chain, at the junction between the C-terminus of the hinge region and the downstream region of the heavy chain, or at a location starting within about 50, 20, 10, 5, 4, 3, 2 or 1 amino acids upstream from the N- terminus, or about 50, 20, 10, 5, 4, 3, 2 or 1 amino acids downstream from the C-terminus, of the hinge region.
  • an amino acid sequence is incorporated between, or adjacent to, residues 224-251 (based on the Kabat numbering scheme) of a heavy chain or into, or adjacent to, one of the sequences shown in Figure 28.
  • an amino acid sequence is incorporated at the junction between the C-terminus of the hinge region and the downstream region of the heavy chain.
  • the amino acid sequence may optionally be flanked by proteasomal cleavage sites as described herein.
  • an amino acid sequence is incorporated into the constant domain of an antibody at a region that reduces or disrupts binding to cell surface Fc receptors.
  • One problem that may be encountered with the administration of therapeutic antibodies is the reduction of the effective dose of the antibody due to binding to Fc receptor bearing cells.
  • an antibody fusion having an amino acid sequence incorporated at or around the hinge region has reduced or completely abolished Fc receptor binding activity.
  • an antibody fusion having reduced Fc receptor binding activity means
  • the antibody fusion has less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, or less, of Fc receptor binding activity as compared to the same antibody not containing the incorporated amino acid sequence.
  • the methods described herein may be used to incorporate amino acid sequences into non-antibody proteins.
  • one or more amino acid sequences may be incorporated into a carrier protein or scaffold protein at regions that are hydrophobic, flanked by proteasomal cleavage sites, and/or that have sequence similarity with the polypeptide to be introduced.
  • Carrier proteins having multiple amino acid sequences introduced may be referred to as multi epitope carrier proteins.
  • a multi-epitope carrier protein may be attached to the C-terminus of the constant region of an antibody heavy or light chain by a cleavable peptide linker (or alternatively by chemical conjugation).
  • Specific examples of multi-epitope carrier proteins include disease specific proteins that have been modified to incorporate one or more disease specific epitopes as described further below.
  • the antibody fusions described herein may utilize a wide variety of antibodies or antibody fragments that bind to a desired target epitope.
  • the target epitope may be selected by one of skill in the art based on a desired application for the antibody fusion. Exemplary applications of the antibody fusions are described further below.
  • Nucleic acid sequences useful for production of the antibody fusions described herein may be obtained from publicly available databases or determined experimentally as described further below.
  • Antibodies for use in antibody fusions may be IgG, IgM, IgE, IgA or IgD molecules.
  • the antibody is an IgG, IgD, or IgA molecule that comprises a hinge region.
  • the antibody fusions described herein may comprise a constant region, or a portion thereof, from any type of antibody isotype, including, for example, IgG (including IgGl, IgG2, IgG3, and IgG4), IgM, IgE, IgA or IgD, or a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (see e.g., Burton DR and Woof JM, Adv. Immun.
  • the antibody fusion has a hybrid constant region wherein residues 249 and 250 (based on Kabat numbering) are glycines.
  • residues 249 and 250 are glycines.
  • the IgGl and IgG4 constant regions contain G 24 9G 2 5o residues whereas the IgG2 constant region does not
  • 10235000J 25 contain residue 249 but does contain G 250 .
  • the constant region can be further modified to introduce a glycine residue at position 249 to produce a G2/G4 fusion having G 24 g/G 25 o.
  • G2/G4 G 24 c ⁇ /G 25 o hybrid constant domain it may be desirable to introduce an amino acid sequence between the G 249 /G 25 o residues.
  • Other constant domain hybrids that contain G 249 /G 2 so may also be used in accordance with the invention.
  • the class and subclass of antibodies may be determined by any method known in the art.
  • the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially.
  • the class and subclass can be determined by ELISA or Western Blot as well as other techniques.
  • the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.
  • chimeric, humanized or primatized (CDR-grafted) antibodies, antibody fragments, as well as chimeric or CDR-grafted antibody fragments, comprising portions derived from different species may be used for construction of the antibody fusions described herein.
  • Antibody fragments useful in accordance with the antibody fusions described herein comprise at least a portion of heavy chain and/or light chain constant region.
  • Exemplary antibody fragments include, for example, Fab, Fab 2 , Fab 3 or minibodies, or Fv, scFv, diabodies and triabodies fused to at least a portion of a heavy chain and/or light chain constant region.
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S.
  • functional fragments of antibodies including fragments of chimeric, humanized, primatized or single chain antibodies comprising at least a portion of a heavy chain and/or light chain constant region, can also be used in association with the antibody fusions described herein.
  • Functional antibody fragments refer to fragments that retain at least one binding function and/or modulation function of the full-length antibody from which they are derived. Preferred functional fragments retain an antigen binding function of a corresponding full-length antibody.
  • antibody fragments capable of binding to a desired epitope including, but not limited to, Fab, Fab 1 and F(ab') 2 fragments, or Fv fragments comprising at least a portion of a heavy chain and/or light chain constant region, may be used in association with the antibody fusions.
  • Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can be used to generate Fab or F(ab') 2 fragments, respectively.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CHl domain and hinge region of the heavy chain.
  • a humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids in the framework and constant domains of the heavy and light chains have been mutated so as to avoid or abrogate an immune response in humans.
  • a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.
  • a humanized antibody may comprise portions of immunoglobulins of different origin, wherein optionally at least one portion is of human origin.
  • a humanized immunoglobulin having binding specificity for a desired epitope said immunoglobulin comprising an antigen binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region or portion thereof) may be used in association with the antibody fusions described herein.
  • the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity
  • 10235000_l 27 such as a mouse, and from immunoglobulin sequences of human origin (e.g., a chimeric immunoglobulin), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain).
  • immunoglobulin sequences of human origin e.g., a chimeric immunoglobulin
  • conventional techniques e.g., synthetic
  • genetic engineering techniques e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain.
  • a humanized immunoglobulin of the present invention is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin) and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes).
  • the humanized immunoglobulin can compete with murine monoclonal antibody for binding to a desired epitope.
  • Chimeric antibodies or CDR- grafted single chain antibodies comprising at least a portion of a heavy chain and/or light chain constant region are also encompassed by the term humanized immunoglobulin.
  • the antibody fusion molecules of the present invention are useful in a variety of applications, including research and therapeutic applications as described further below.
  • the antibody fusions described herein may be used for any method in which targeted delivery of a polypeptide to a desired cell is required.
  • the invention provides antibody fusions for modulating an immune response, including both stimulation of an immune response to a desired antigen or tolerization to an antigen.
  • the antibody fusions comprise an antibody specific for a surface protein on an immune cell and a peptide epitope for which immune modulation is desired.
  • the antibody may be an internalizing antibody such that the peptide epitope is delivered internally to the cell.
  • the specificity of the antibody directs the peptide epitope to a desired population of immune cells.
  • the peptide is then internalized along with the antibody, released inside the cell and may be displayed on the surface of the immune cell as part of an MHC complex.
  • DCs dendritic cells
  • liver sinusoidal endothelial cells LSECs
  • L-SIGN liver sinusoidal endothelial cells
  • antibody fusions for stimulating an immune response comprise an antibody that binds to a dendritic cell specific surface protein and a peptide epitope.
  • exemplary dendritic cell specific surface markers include, for example, CD83, CD205/DEC-205, CD197/CCR7, CD209/DC-SIGN.
  • exemplary peptide epitopes include, for example, anti-microbial protein epitopes such as fungal, bacterial or viral peptide epitopes that will be useful for stimulating or enhancing an immune response to a pathogen.
  • exemplary peptide epitopes include, for example, peptide epitopes useful as cancer vaccine, such as epitopes derived from the tumor associated antigens described herein.
  • antibody fusions for inducing tolerance are provided.
  • the antibody fusions comprise an antibody that binds to LSEC specific surface protein, such as L-SIGN, and a peptide epitope associated with, for example, an autoimmune disease.
  • methods of modulating an immune response include stimulation and/or enhancement of an immune response for and methods of treating an individual in need of immune stimulation, e.g., individuals suffering from a pathogen infection, cancer, or other disease state.
  • the methods also include reducing an immune response or inducing tolerance and methods for treating an individual in need of tolerization, e.g., individuals suffering
  • the methods involve administering to the individual a therapeutically effective amount of one or more antibody fusions as described herein.
  • the antibody fusion comprises an internalizing antibody that recognizes an immune cell surface protein and a peptide epitope fused to, or embedded in, a constant region of the antibody.
  • methods for modulating an immune response may involve a combination therapy with one or more other therapeutic agents such as, for example, anti-inflammatory agents, immunosuppressive agents, and/or anti-infective agents (such as for example, antibiotic, antiviral, and/or antifungal compounds, etc.).
  • anti-inflammatory drugs include, for example, steroidal (such as, for example, Cortisol, aldosterone, prednisone, methylprednisone, triamcinolone, dexamethasone, deoxycorticosterone, and fluorocortisol) and non-steroidal anti- inflammatory drugs (such as, for example, ibuprofen, naproxen, and piroxicam).
  • immunosuppressive drugs include, for example, prednisone, azathioprine (Imuran), cyclosporine (Sandimmune, Neoral), rapamycin, antithymocyte globulin, daclizumab, OKT3 and ALG, mycophenolate mofetil (Cellcept) and tacrolimus (Prograf, FK506).
  • antibiotics include, for example, sulfa drugs (e.g., sulfanilamide), folic acid analogs (e.g., trimethoprim), beta-lactams (e.g., penicillin, cephalosporins), aminoglycosides (e.g., streptomycin, kanamycin, neomycin, gentamycin), tetracyclines (e.g., chlorotetracycline, oxytetracycline, and doxycycline), macrolides (e.g., erythromycin, azithromycin, and clarithromycin), lincosamides (e.g., clindamycin), streptogramins (e.g., quinupristin and dalfopristin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, and moxifloxacin), polypeptides (e.g., polymixins,
  • antiviral agents include, for example, vidarabine, acyclovir, gancyclovir, valganciclovir, nucleoside-analog reverse transcriptase inhibitors (e.g., AZT, ddl, ddC, D4T, 3TC), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine, delavirdine), protease inhibitors (e.g., saquinavir, ritonavir, indinavir, nelfinavir), ribavirin, amantadine, rimantadine, relenza, tamiflu, pleconaril, and interferons.
  • antifungal drugs include, for example, polyene antifungals (e.g., amphotericin and nystatin), imidazole antifungals (ketoconazole and
  • methods for stimulating an immune response may involve a combination therapy with one or more immunostimulatory agents such as, for example, an adjuvant.
  • immunostimulatory agents such as, for example, an adjuvant.
  • Such combination therapies may be useful as vaccines.
  • exemplary adjuvants include, for example: synthetic imidazoquinolines such as imiquimod (S-26308, R-837) (Harrison et al., Vaccine 19: 1820-1826 (2001)) and resiquimod (S-28463, R-848) (Vasilakos et al., Cellular Immunology 204: 64-74 (2000)); Schiff bases of carbonyls and amines that are constitutively expressed on antigen presenting cells and T-cell surfaces, such as tucaresol (Rhodes et al., Nature 377: 71-75 (1995)); cytokine, chemokine and co-stimulatory molecules; ThI inducers such as interferon gamma, IL-2
  • QS21 and QS7 Aquila Biopharmaceuticals Inc., Framingham, Mass.
  • Escin Digitonin
  • the antibody fusions provided herein may be used for stimulating an immune response for treating or preventing influenza in a subject, or for treating or ameliorating symptoms associated with influenza.
  • the methods may involve administering a therapeutically effective amount of an antibody fusion as described herein comprising one or more influenza associated epitopes.
  • Exemplary influenza infections that may be treated in accordance with the methods
  • influenza types A, B and C include, for example, influenza types A, B and C.
  • influenza is influenza A, such as, for example: A/PR/8/34 or A/HK/8/68, or selected from HlNl, H2N2, H3N2, H5N1, H9N2, H2N1, H4N6, H6N2, H7N2, H7N3, H4N8, H5N2, H2N3, Hl 1N9, H3N8, H1N2, Hl 1N2, Hl 1N9, H7N7, H2N3, H6N1, H13N6, H7N1, Hl INl, H7N2 and H5N3.
  • influenza A such as, for example: A/PR/8/34 or A/HK/8/68, or selected from HlNl, H2N2, H3N2, H5N1, H9N2, H2N1, H4N6, H6N2, H7N2, H7N3, H4N8, H5N2, H2N3, Hl 1N9, H3N8, H
  • an antibody fusion may be administered substantially contemporaneously with or following infection of the subject, i.e., a therapeutic treatment.
  • the antibody fusion provides a therapeutic benefit, such as, reducing or decreasing one or more symptoms or complications of influenza infection, virus titer, virus replication or an amount of a viral protein of one or more influenza strains.
  • Symptoms or complications of influenza infection that can be reduced or decreased include, for example, chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache or death.
  • a therapeutic benefit includes hastening a subject's recovery from influenza infection.
  • an antibody fusion may be administered as part of a combination therapy with an anti-viral agent or one or more agents that inhibit one or more symptoms or complications associated with influenza infection (e.g., chills, fever, cough, sore throat, nasal congestion, body ache, head ache, fatigue, pneumonia, bronchitis, sinus infection or ear infection).
  • influenza infection e.g., chills, fever, cough, sore throat, nasal congestion, body ache, head ache, fatigue, pneumonia, bronchitis, sinus infection or ear infection.
  • the invention provides antibody fusions for growth inhibition of a targeted cell population or for targeted cell death.
  • the antibody fusions comprise an antibody specific for a surface protein on a cell population that is to be targeted for cell killing or growth inhibition and a cytotoxic peptide or growth inhibitory peptide.
  • the antibody may be an internalizing antibody such that the cytotoxic peptide or growth inhibitory peptide is delivered internally to the cell.
  • the antibody may also be targeted to an internalizing receptor on the cell surface to facilitate uptake of the antibody into the cell.
  • the specificity of the antibody directs the cytotoxic peptide or growth inhibitory peptide to the desired population of cells. The peptide is then internalized along with the antibody and released inside the cell resulting in cell death or growth inhibition.
  • antibody fusions comprising an antibody that binds to a tumor specific antigen and a cytotoxic peptide are provided. Such antibody fusions are useful for targeted destruction of tumor cells and the treatment of cancer.
  • Tumor associated antigens can be identified experimentally or may be selected from a database.
  • Databases that identify molecules that are expressed or upregulated by cancer cells include, for example, the NCI60 microarray project (see e.g., Ross et al., Nature Genetics 24: 227-34 (2000); world wide web at genome-www.stanford.edu/ nci60/), the carcinoma classification (see e.g., A.
  • Exemplary tumor associated antigens include the following: gplOO, tyrosinase, MAGE-I, MAGE-3, MART, BAGE, and TRP-I which are associated with melanoma; CEA (carcino embryonic antigen), CA 19-9, CA 50, and CA 72-4 which are associated with stomach cancer; CEA, CAl 9-9, and Muc-1 which are associated with colon cancer; CA 19-9, Ca-50, and CEA which are associated with pancreas carcinoma; CEA, NSE (neuron specific enolase), and EGF-receptor which are associated with small cell lung cancer; CEA which is associated with lung cancer; ⁇ - fetoprotein (AFP) which is associated with liver carcinoma; PSA, PMSA, CDCPl, CD26, Hepsin, HGF (hepatocyte growth factor), Met, CAIX(G250), EphhB4 (Ephrin type-B receptor 4), EGFRl, EGFR2, PDGF, VEGFR, DPP6,
  • cytotoxic peptides that may be used in association with the antibody-cytotoxic peptide fusions described herein include, for example, anthrax lethal factor, Diphtheria toxin A fragment, nonbinding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, ⁇ -sacrin, certain Aleurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phomycin, and neomycin, or fragments thereof. Yet other cytotoxic polypeptides are described in U.S. Patent Publication Nos. 2005/0191294 and 2004/0192889.
  • Peptides that cause mitochondria dependent cell-free apoptosis may also be used as cytotoxic peptides in accordance with the antibody fusions described herein.
  • a number of pro-apoptotic peptides have been described that remain relatively nontoxic outside of eukaryotic cell membranes but bind to mitochondrial membranes inside the cells and induce their swelling and cause mitochondrial dependent cell-free apoptosis (Ellerby, H. M. et al., J NeWosci 17: 6165-6178 (1997); Mehlen, P. et al., ⁇ Nature 395: 801-804 (1998)).
  • An example of a well-characterized pro-apoptotic peptide successfully utilized for selectively killing malignant hematopoietic cells and cells lining tumor blood vessels is a 14-amino-acid amphipathic peptide KLAKLAKKLAKLAK (SEQ ID NO: 55) (Ellerby, H. M. et al., Nat Med 5: 1032- 1038 (1999); Marks, AJ. et al., Cancer Res 65: 2373-2377 (2005)).
  • This peptide contains cationic lysine (K) residues on one side of the amphipathic helix and hydrophobic (Leu-Ala) residues on the other side.
  • methods of inhibiting or reducing tumor growth and methods of treating an individual suffering from cancer involve administering to the individual a therapeutically effective amount of one or more antibody fusions as described herein.
  • the antibody fusion comprises an internalizing antibody that recognizes a cancer cell surface protein and a peptide toxin fused to, embedded in, a constant region of the antibody.
  • Antibody fusions of the present invention may be useful for treating or preventing a cancer (tumor), including, but not limited to, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi's sarcoma, glioblastoma, astrocytoma, lymphoma, lung carcinoma, melanoma, renal cancer, and leukemia.
  • a cancer tumor including, but not limited to, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi's sarcoma, glioblastoma, astrocytoma, lymphoma, lung carcinoma, melanoma, renal cancer, and leukemia.
  • one or more antibody fusions can be administered together (simultaneously) or at different times (sequentially).
  • the antibody fusions can be administered with another agent for treating cancer or for inhibiting angiogenesis.
  • the subject antibody fusions can also be used with other antibody therapeutics (monoclonal or polyclonal).
  • the subject antibody fusions can be used alone.
  • the subject antibody fusions may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor).
  • proliferative disorders e.g., tumor
  • methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy.
  • conventional cancer therapies e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery
  • a wide array of conventional compounds have been shown to have antineoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies.
  • an antibody fusion may enhance the therapeutic effect of the anti-neoplastic agent or treatment with the antibody fusion may help to overcome cellular resistance to anti-neoplastic agents. This may allow a decreased dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or may restore the effectiveness of an anti-neoplastic agent in resistant cells.
  • anti-neoplastic agents may enhance the efficacy of an antibody fusion by rendering cells more susceptible to cytotoxic T cell killing or by enhancing the levels of available cancer antigens.
  • Pharmaceutical compounds that may be used for combinatory anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, geni
  • chemotherapeutic anti-tumor compounds may be categorized by their mechanism of action into groups, including, for example, the following: anti- metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin,
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • angiotensin receptor blocker vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • angiotensin receptor blocker nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone
  • pharmaceutical compounds that may be used for combinatory anti-angiogenesis therapy include: (1) inhibitors of release of "angiogenic molecules," such as bFGF (basic fibroblast growth factor); (2) neutralizers of angiogenic molecules, such as anti-jSbFGF antibodies; and (3) inhibitors of endothelial cell response to angiogenic stimuli, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D- penicillamine and gold thiomalate, vitamin D 3 analogs, alpha-interferon, and the like.
  • angiogenic molecules such as bFGF (basic fibroblast growth factor)
  • neutralizers of angiogenic molecules such as anti-jSbFGF antibodies
  • inhibitors of endothelial cell response to angiogenic stimuli including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4,
  • angiogenesis there are a wide variety of compounds that can be used to inhibit angiogenesis, for example, peptides or agents that block the VEGF- mediated angiogenesis pathway, endostatin protein or derivatives, lysine binding fragments of angiostatin, melanin or melanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen), troponin subunits, antagonists of vitronectin O!
  • peptides derived from Saposin B antibiotics or analogs (e.g., tetracycline, or neomycin), dienogest-containing compositions, compounds comprising a MetAP-2 inhibitory core coupled to a peptide, the compound EM-138, chalcone and its analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.
  • antibody fusions comprising an antibody that binds to a cell surface protein on an immune cell and a cytotoxic peptide are provided.
  • Such antibody fusions are useful for targeted destruction of immune cells involved in an unwanted immune response, such as, for example, immune responses associated with an autoimmune disorder, transplants, allergies, and inflammatory disorders.
  • Exemplary autoimmune diseases and disorders that may be treated with the antibody fusions provided herein include, for example: (1) treatment of rheumatoid arthritis by targeting dendritic cells with anti-DC-SIGN-toxin conjugates (see e.g., van Lent P.L. et al., Arthritis Rheum.
  • Antibodies directed to a desired immune cell surface protein may be produced experimentally or selected from a publicly available database as described further herein.
  • Targeted killing of certain populations of immune cells for treating or preventing autoimmune disorders, enhancing or extending transplant survival, treating or preventing allergies, or treating or preventing inflammatory disorders, may be administered as part of a combination therapy with one or more therapeutic agent such as, for example, anti-inflammatory agents, immunosuppressive agents, and/or anti-infective agents as described further herein.
  • administration of the antibody fusions of the invention may be continued while the other therapy is being administered and/or thereafter.
  • Administration of the antibody fusions may be made in a single dose, or in multiple doses.
  • administration of the antibody fusions is commenced at least several days prior to the conventional therapy, while in other instances, administration is begun either immediately before or at the time of the administration of the conventional therapy.
  • antibody fusions comprising polypeptides useful for modulating cell growth and/or differentiation and/or motility are provided.
  • Suitable polypeptides, or fragments thereof, which may be used in accordance with the antibody fusions described herein include, for example, chemotactic polypeptides, growth factors, cytokines, morphogenesis factors, cell signalling factors, cell differentiation factors, polypeptides which stimulate or suppress cell division, and polypeptides which modulate the rate of cell division. Specific examples of polypeptides may be found, for example in U.S. Patent Publication No. 2005/0136042. Also provided are methods for modulating cell growth and/or differentiation, or for treating an individual in need of modulation of cell growth and/or differentiation. The methods involve administering to the individual a therapeutically effective amount of one or more antibody fusions as described herein.
  • the antibody fusion comprises an antibody that recognizes a cell surface protein on a cell in which growth, differentiation, Or motility modulation is desired, and a polypeptide fused to, or embedded in, a constant region of the antibody.
  • the polypeptide may be a polypeptide that modulates cell growth, differentiation, and/or motility.
  • antibody fusions comprising growth inhbitory polypeptides, such as tumor suppressors, are provided.
  • tumor suppressors include, for example, p21, p53, BRCAl, BRCA2, APC, and RBl.
  • disease specific multi-epitope carrier proteins and antibody fusions comprising disease specific multi-epitope carrier proteins are provided.
  • Disease specific carrier proteins refer to proteins that are associated with a disease state and which may be used for incorporating other disease epitopes into the protein. Methods for designing and constructing disease specific carrier proteins are described in detail in the exemplification.
  • disease specific carrier proteins include, for example, polypeptides associated with either Type 1 diabetes mellitus (TlDM) or insulin dependent diabetes mellitus (IDDM) such as glutamic- acid decarboxylase 65 (GAD65), heat shock protein 60 (HSP60), insulinoma associated protein 2 (IA-2) and proinsulin (PI)) or polypeptides associated with cancer such as: gplOO and Tyrosinase associated with Melanoma, Late Membrane Protein 2 (LMP-2) associated with Lymphoma and Carcinoembryonic Antigen associated with various types of adenocarcinomas.
  • TlDM Type 1 diabetes mellitus
  • IDDM insulin dependent diabetes mellitus
  • GAD65 glutamic- acid decarboxylase 65
  • HSP60 heat shock protein 60
  • IA-2 insulinoma associated protein 2
  • PI proinsulin
  • the carrier proteins may be modified to incorporate antigens from other disease specific proteins such that a strong, multifactorial immune response or tolerization effect may be achieved.
  • the invention also provides methods for modulating an immune response, or treating an individual in need of immune response modulation by administering to a patient a disease specific multi-epitope carrier protein, optionally fused to, or embedded in, an antibody.
  • the disease specific multi-epitope carrier proteins may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disease specific epitopes from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, disease specific proteins.
  • a disease specific multi-epitope carrier protein may comprise two or more disease specific epitopes from a single protein incorporated into a carrier protein that does not naturally contain the epitopes.
  • the antibody fusions described herein may comprise an amino acid sequence that increases the serum half life of the antibody fusion.
  • polypeptides that may extend the serum-half life of the antibody fusions include, for example, albumin (see e.g., U.S. Patent Nos. 5,876,969 and 5,766,88) and transferrin (see e.g., U.S. Patent Publication No. 2003/0226155), or functional fragments thereof.
  • the antibody fusions described herein may comprise an amino acid sequence that increases or enhances transport across a cellular membrane.
  • a number of peptide based cellular transporters have been developed by several research groups. These peptides are capable of crossing cellular membranes in vitro and in vivo with high efficiency. Examples of such fusogenic peptides include a 16-amino acid fragment of the homeodomain of ANTENNAPEDIA, a Drosophila transcription factor (Wang et al., PNAS USA.
  • nucleic acids encoding antibody fusions are also encompassed within the scope of the invention.
  • expression vectors comprising nucleic acids encoding the antibody fusions, and host cells comprising expression vectors for producing the antibody fusions.
  • Antibodies useful for production of the antibody fusions described herein may be designed to bind to a desired epitope or may be selected from publicly available sources of known antibodies. For example, databases of antibody sequences may be found on the world wide web at imgt.cines.fr. Nucleic acid sequences encoding an antibody may be manipulated to incorporate one or more sequences encoding a polypeptide using standard recombinant DNA techniques. The nucleic acid sequences encoding the antibody fusions may be introduced into an expression vector and a suitable host cell for expression of the antibody fusion molecule as described further below.
  • a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells.
  • the antibody producing cells preferably those of the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest.
  • the fused cells can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • immunogens derived from a polypeptide of interest can be used to immunize a mammal, such as a mouse, a hamster or rabbit. See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of a polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera Following immunization of an animal with an antigenic preparation of a polypeptide, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells
  • 10235000_1 43 can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • immortalizing cells such as myeloma cells.
  • Such techniques are well known in the art and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96).
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a desired polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies.
  • F(ab') 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab') 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • antibodies described herein are further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for a polypeptide of interest conferred by at least one CDR region of the antibody.
  • Techniques for the production of a light chain or heavy chain dimers, or any minimal fragment thereof such as an Fv or a single chain (scFv) construct are described, for example, in US Patent No. 4,946,778.
  • transgenic mice or other organisms including other mammals may be used to express humanized antibodies. Methods of generating these antibodies are known in the art. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.
  • Such humanized immunoglobulins can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain.
  • genes e.g., cDNA
  • nucleic acid e.g., DNA sequences coding for
  • 1O 235 OOOj 44 humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced.
  • cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).
  • a method for generating a monoclonal antibody that binds specifically to a desired polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the polypeptide in an amount effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody- producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the polypeptide.
  • antibody-producing cells e.g., cells from the spleen
  • a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma- derived cells produce the monoclonal antibody that binds specifically to polypeptide.
  • the monoclonal antibody may be purified from the cell culture.
  • an antibody to be used for certain therapeutic purposes will preferably be able to target a particular cell type. Accordingly, to obtain antibodies of this type, it may be desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g., by fluorescence activated cell sorting). Likewise, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing antibody: antigen interactions to identify particularly desirable antibodies.
  • Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays and immunohistochemistry.
  • the hybridoma cell lines as well as the monoclonal antibodies produced by these hybridoma cell lines, are provided.
  • the cell lines have uses other than for the production of the monoclonal antibodies.
  • the cell lines can be fused with other cells (such as suitably drug-marked human myeloma, mouse myeloma, human-mouse heteromyeloma or human lymphoblastoid cells) to produce additional hybridomas, and thus provide for the transfer of the genes encoding the monoclonal antibodies.
  • hybridoma cell lines can be used as a source of nucleic acids encoding the immunoglobulin chains, which can be isolated and expressed (e.g., upon transfer to other cells using any suitable technique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter, U.S. Pat. No. 5,225,539)).
  • clones comprising a rearranged light or heavy chain can be isolated (e.g., by PCR) or cDNA libraries can be prepared from mRNA isolated from the cell lines, and cDNA clones encoding a desired immunoglobulin chain can be isolated.
  • nucleic acids encoding the heavy and/or light chains of the antibodies, or portions thereof can be obtained and used in accordance with recombinant DNA techniques for the production of the specific immunoglobulin, immunoglobulin chain, or variants thereof (e.g., humanized immunoglobulins) in a variety of host cells or in an in vitro translation system.
  • the nucleic acids including cDNAs, or derivatives thereof encoding variants such as a humanized immunoglobulin or immunoglobulin chain
  • suitable prokaryotic or eukaryotic vectors e.g., expression vectors
  • suitable host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the host cell genome).
  • host cells can be maintained under conditions suitable for expression (e.g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.), whereby the encoded polypeptide is produced.
  • the encoded protein can be recovered and/or isolated (e.g., from the host cells or medium). It will be appreciated that the method of production encompasses expression in a host cell of a transgenic animal (see e.g., WO 92/03918, GenPharm International, published Mar. 19, 1992).
  • Antibodies can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • such phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage, including fd and Ml 3.
  • the antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein.
  • phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24: 952-958 (1994); Persic et al., Gene 187: 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • techniques to recombinantly produce Fab, Fab 1 and F(ab') 2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
  • Polynucleotides encoding antibodies having a desired binding specificity may be obtained by any method known in the art.
  • 10235000_l 47 immunospecific for a desired antigen can be obtained, for example, as described above, from the literature or from a database such as GenBank.
  • Polynucleotides encoding an antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17: 242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may be produced from a cDNA library obtained from a tissue or cell expressing the antibody such as a hybridoma cell line selected to express an antibody.
  • the desired antibody genes may be isolated from the library by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
  • standard recombinant DNA techniques may be used to incorporate a nucleic acid sequence encoding a polypeptide into the antibody sequence at a desired location.
  • the vector for the production of the antibody fusion may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the antibody fusion coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. and Ausubel et al. eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
  • An expression vector comprising the nucleotide sequence of an antibody fusion can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody fusion.
  • the expression of the antibody fusion is regulated by a constitutive promoter, an inducible promoter, or a tissue specific promoter.
  • the host cells used to express the recombinant antibody fusions may be either bacterial cells (such as Escherichia coli) or eukaryotic cells. Eukaryotic cells may be particularly useful for the expression of antibody fusion comprising a whole recombinant immunoglobulin molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 1998, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
  • host-expression vector systems may be utilized to express the antibody fusions described herein.
  • Such host-expression systems represent vehicles by which the coding sequences of the antibody fusions may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody fusions in situ.
  • These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces or Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphatic cells (see U.S.
  • mammalian cell systems e.g., COS
  • Per C.6 cells rat retinal cells developed by Crucell
  • recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody fusion being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), in which the antibody fusion coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody fusion coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • the antibody fusion coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (see e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody fusion coding sequences.
  • These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeIa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
  • cell lines which stably express an antibody fusion may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody fusions described herein.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 : 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77: 357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
  • an antibody fusion described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing an antibody fusion is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody fusion, production of the antibody fusion will also increase (Crouse et al., 1983, MoI. Cell. Biol. 3:25
  • the host cell may be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • the antibody fusion of the invention may be purified by any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the invention provides methods and pharmaceutical compositions comprising antibody fusions of the invention.
  • the invention also provides methods of treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of an antibody fusion, or a pharmaceutical composition comprising an antibody fusion.
  • an antibody fusion is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects).
  • Subjects that may be treated with an antibody fusion described herein include, for example, an animal, such as a mammal including non-primates (e.g.
  • the antibody fusion molecules described may be formulated with a pharmaceutically acceptable carrier. Such antibody fusions can be administered alone or as a component of a pharmaceutical formulation (composition). The antibody fusions may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the subject antibody fusions include those suitable for oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), rectal, and/or intravaginal administration.
  • parenteral e.g., intravenous, intraarterial, intramuscular, subcutaneous injection
  • inhalation e.g., intrabronchial, intranasal or oral inhalation, intranasal drops
  • rectal e.g., rectal, and/or intravaginal administration.
  • Other suitable methods of administration can also include rechargeable or biodegradable devices and slow release polymeric devices.
  • the pharmaceutical compositions described herein can be any suitable for oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), inhalation (e.g.
  • 10235000_l 53 also be administered as part of a combinatorial therapy with other agents (either in the same formulation or in a separate formulation).
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • methods of preparing these formulations or compositions include combining another type of anti-tumor or anti-angiogenesis agent and a carrier and, optionally, one or more accessory ingredients.
  • the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of one or more subject antibody fusions as an active ingredient.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • one or more antibody fusions of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents,
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsif ⁇ ers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Topical formulations may further include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.
  • 10235000_l 55 subject antibody fusions may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an antibody fusion, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an antibody fusion, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • compositions suitable for parenteral administration may comprise one or more antibody fusions in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium
  • Injectable depot forms are made by forming microencapsule matrices of one or more antibody fusions in biodegradable polymers such as polylactide- polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • Formulations for intravaginal or rectal administration may be presented as a suppository, which may be prepared by mixing one or more antibody fusions of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • EXAMPLE 1 Computer aided analysis of immunoglobulin sequences for the identification of proteasomal cleavage hot spots and hydrophobic areas
  • peptide grafted antibodies having a peptide incorporated into a hydrophobic region and flanked by proteasomal cleavage sites were constructed and tested to determine the expression profile, antigen binding activity, activation of T- cell response and other characteristics.
  • proteasome cleavage hot spot These five areas are spread over the entire protein, one in the constant heavy (CH) 1 domain and two each in the CH2 and CH3 domains. Insertion of the hydrophobic peptide antigens into one of these five sites may permit efficient processing and productive presentation in the context of MHC molecules on the cell surface. In addition, as these same five sites are also strongly hydrophobic, an antibody-peptide construct with an inserted hydrophobic peptide epitope may be less likely to fold improperly and may be expected to produce well. To further decrease the structure perturbing effects of introduced hydrophobic peptide epitopes, the hydrophobic patch that most closely matches with the peptide epitope sequence may be selected for grafting the peptide into the antibody constant region.
  • Arginines and lysines have been shown to serve as very efficient substrates for proteasomal cleavage and have been used to facilitate release of MHC class I peptide antigens from multi- epitope vaccines (Livingston, B. D. et al., Vaccine 19: 4652-4660 (2001); Sundaram, R. et al., Vaccine 21 : 2767-2781 (2003)).
  • EXAMPLE 2 Screening for cellular responses to tetanus toxin peptides to address the possibility of delivering peptide antigens to antigen presenting cells via antibodies.
  • TT tetanus toxin
  • Peptides outlined in Table 2 were first screened for immunological responses in humans.
  • Four volunteers underwent vaccination with tetanus toxoid, their antibody responses to tetanus toxoid protein and cellular responses to HLA-DR binding helper T-cell epitopes (listed in Table 2) were evaluated. Two weeks after the vaccination, all four donors exhibited substantial levels of antibodies to TT protein (see Figure 4A). In contrast, only two of the four donors (#5 and #13) showed significant levels (P ⁇ 0.05 vs. medium) of T-cell
  • EXAMPLE 3 Design, expression and binding properties of peptide embedded antibodies produced by insertion and sequence similarity replacement methods
  • peptide 632DR had the closest sequence similarity to a portion of the human IgGl amino acid sequence, which allows for sequence similarity replacement of the peptide into the immunoglobulin sequence. Therefore, peptide 632DR was grafted into a DC-SIGN/L- SIGN reactive antibody (clone ElO) using two different strategies, namely, sequence similarity replacement (described further below) and insertion with flanking proteasomal cleavage sites (described further below) as shown in Figures 5A-B. The 632DR peptide was inserted into the hydrophobic patch right after the hinge region
  • Peptide sequences were compared with the amino acid sequences of various antibodies using the MegAlign program from DNAstar, which includes a number of different alignment programs and algorithms.
  • the dotplot option for aligning pairs of sequences was used in a first examination of the sequences.
  • the percent match may be set, for example, at 30% or greater.
  • HLA supertype epitopes of TT which are peptides of 9 amino acids, identities of 3, 4, and sometimes 5 amino acids were found.
  • Two separate clones were constructed using overlap PCR to embed the TT helper epitope 632DR into different locations in the L-SIGN/DC-SIGN reactive chimeric antibody ElO-IgG.
  • the TT epitope was inserted into the CH2 domain between Gycines 249 and 250 (Kabat numbering).
  • Two arginine residues were placed upstream of the 632DR epitope and three arginine residues were placed downstream of the 632DR epitope to give a final insertion
  • fragment A was amplified using the forward primer (E10Age5For: 5' TTC CCC
  • the ElOinsertionRev primer contained a tail, which encoded part of the peptide 632DR insertion. Fragment B was generated using the forward primer (ElOinsertionFor: 5' GTG AGC ACC ATC GTG CCC TAC ATC GGC CCC GCC CTG AAC ATC AGA AGA GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 66) which annealed to a portion of the ElO DNA sequence encoding glycine 250 and the down stream amino acids (250-258), and the reverse primer (ElOEcoRBRev 5' G ATT ATG ATC AAT GAA TTC TGG CCG TCG CAC TCA T 3') (SEQ ID NO: 67) which annealed to a region spanning the stop codon and a unique EcoRI site within the vector at the end of the CH3 region.
  • the ElOinsertionFor primer contained a tail, which encoded part of the peptide 632DR insertion and hybridized
  • the expand high fidelity PCR system (Roche) was used with the following program: 96°C for 5 minutes followed by 30 cycles of: 96°C for 30 seconds, 56°C for 30 seconds, 72°C for 2 minutes, with a final extension period of 10 minutes at 72°C and a hold at 4 0 C. Fragments A and B were then gel purified and combined for an overlap extension PCR.
  • the expand high fidelity PCR system was again used running the above program for 10 cycles then adding the E10Age5For and ElOEcoRDRev primers and running the above program for an additional 30 cycles.
  • the overlap extension PCR product was then gel isolated and digested with restriction endonucleases Age I and Eco RI.
  • the digested fragment was again gel isolated and cloned by ligating it back into the plasmid vector piece of the ElO-IgG parental clone, which had been digested with Age I and Eco RI followed by gel isolation.
  • the replacement clone was constructed using a fragment of TT peptide 632DR having the amino acid sequence ISDVSTIVPYIGPALNI (SEQ ID NO: 5).
  • the TT peptide 632DR fragment was used to replace the region of the CH2 domain beginning with Valine 276 and ending with Valine 292. No additional arginines were added to the epitope in the replacement clone.
  • the replacement clone was constructed using PCR overlap extension as described above for the insertion clone but using the following primers.
  • the ElOreplacementRev primer (5' GCC GAT GTA GGG CAC GAT GGT GCT CAC GTC GCT GAT CAC GCA TGT GAC CTC AGG GGT CCG GGA 3') (SEQ ID NO: 68) was used in place of the ElOinsertionRev primer in the creation of Fragment A, and the ElOreplacementFor primer (5' GTG AGC ACC ATC GTG CCC TAC ATC GGC CCC GCC CTG AAC ATC GAC GGC GTG GAG GTG CAT AAT GCC AAG 3') (SEQ ID NO: 69) was used in place of the ElOinsertionFor primer in the creation of Fragment B.
  • the ElOreplacementRev primer anneals to the DNA region encoding amino acids 267 to 275 within the CH2 domain and has a tail encoding part of the TT 632DR peptide fragment.
  • ElOinsertionFor primer anneals to the region of the CH2 domain encoding amino acids 295 to 305 and has a tail encoding part of the TT 632DR peptide fragment and which hybridizes to the tail of the ElOreplacementRev primer tail. Ligation and cloning into the vector was conducted as described above for the insertion clone. The sequences of the final cloned products were confirmed by DNA sequencing.
  • the 947DR peptide (FNNFTVSFWLRVPKVSASHLE, SEQ ID NO: 62) was inserted into the CH2 region of ElO-IgG between glycines 249 and 250 to create the clone ElOchGl- HC947tt. Three flanking arginine residues were added to each end of the inserted peptide.
  • fragment A was amplified using the reverse primer 947E10 REV (5' TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA GCG GCG GCC CAG TTC TGG TGC TGA 3') (SEQ ID NO: 115) and fragment B was amplified using the forward primer 947FOR (5' GTG TCC TTC
  • the 947DR peptide was inserted into clone E10chGl-HC632tt downstream of the 632DR peptide after the second of the three flanking arginine residues. Additional arginine residues were placed after the inserted 947 peptide to create an insert with two upstream arginines, followed by the 632DR peptide, followed by two more arginines, then the 947DR peptide and lastly three more arginines (e.g., RR-632peptide-RR-947peptide- RRR).
  • fragment A was amplified using the reverse primer 632ING 1 REV (5' CAC TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA TCT TCT GAT GTT CAG GGC GGG GCC 3') (SEQ ID NO: 117) and fragment B was amplified using the forward primer 632ING1 FOR (5' TCC TTC TGG CTG CGC GTG CCA AAA GTG TCC GCT TCC CAT CTG GAG AGA AGA AGA GGA CCG TCA GTC TTC TTC CCC CCA 3') (SEQ ID NO: 118).
  • the 632DR peptide was inserted into clone El OchGl -HC947tt downstream of the 947DR peptide after the second of the three flanking arginine residues. Additional arginine residues were placed after the inserted 632DR peptide to create an insert with three upstream arginines, followed by the 947DR peptide, followed by two more arginines, the 632DR peptide and lastly three more arginines (e.g., RR-947peptide-RR-632peptide-RRR).
  • fragment A was amplified using the reverse primer 947IN REV (5' GAT GTA TGG CAC GAT GGT GGA CAC GTC GGA GAT TTT GTC GAT GCG GCG CTC CAG ATG GGA AGC GGA 3') (SEQ ID NO: 119) and fragment B was amplified using the forward primer 947IN FOR (5' GTG TCC ACC ATC GTG CCA TAC ATC GGC CCA GCT CTG AAC ATC CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 120). Construction of clones in the L-SIGN specific antibody Cl
  • 10235000J 53 C7 (e.g., inserts of 632DR, 947DR, 632/947, and 947/632).
  • the C7 antibody contains a G2G4 constant region. Both single peptide insertions and double peptide tandem insertions were constructed using overlap PCR methods similar to those described above.
  • the peptide 632DR was inserted into antibody C7 to create the clone C7chG2G4-HC632tt. Fragment A was amplified using the forward primer
  • E10Age5For 5' TTC CCC GAA CCG GTG ACG GTG TCG T 3'
  • SEQ ID NO: 64 which anneals to C7, in combination with the backward primer PVA 632 REV
  • PVA 632 REV 5' GAT GTA TGG CAC GAT GGT GGA CAC GTC GGA GAT TTT GTC GAT GCG GCG GCG TGC CAC AGG TGG TGC TGA GGA AGA GAT GGA GGT GGA 3'
  • Fragment B was generated using the forward primer 632 FOR (5' GTG TCC ACC ATC GTG CCA TAC ATC GGC CCA GCT CTG AAC ATC CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 122) and the reverse primer EcoRIG2G4REV (5' CTG ATT ATG ATC AAT GAA TTC TCA TCA TTT 3') (SEQ ID NO: 123). Fragments A and B were then gel purified and combined into an overlap extension PCR reaction. As above, the overlap extension PCR product was gel isolated and digested with Age I and Eco RI.
  • the digested fragment was again gel isolated and cloned by ligation into the plasmid vector piece of the C7 parental clone, which had been digested with Age I and Eco RI and gel isolated. Clones obtained were sequenced to confirm proper construction. The peptide 947DR was inserted into antibody C7 to create the clone C7chG2G4- HC947tt.
  • fragment A was amplified using the backward primer PVA 947 REV (5' TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA GCG GCG GCG TGC CAC AGG TGG TGC TGA GGA AGA GAT GGA GGT GGA 3') (SEQ ID NO: 124) and fragment B was generated using the forward primer 947 FOR (5' GTG TCC TTC TGG CTG CGC GTG CCA AAA GTG TCC GCT TCC CAT CTG GAG CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 116).
  • the overlap extension PCR product was gel isolated and digested with Age I and Eco RI. The digested fragment was again gel isolated cloned by ligation into the plasmid vector piece of the C7 parental clone. Clones were sequenced to insure proper construction. Clones containing double peptide tandem insertions were constructed using the above clones (C7chG2G4-HC632tt and C7chG2G4-HC947tt) and inserting the second peptide downstream using overlap PCR as detailed in the above examples. Briefly, to construct clone C7chG2G4-HC632tt/947tt, the 947DR peptide was
  • fragment A was amplified using the reverse primer 632G2G4IN REV (5' CAC TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA GCG GCG GAT GTT CAG AGC TGG GCC 3') (SEQ ID NO: 125) and fragment B was amplified using the forward primer 632G2G4IN FOR (5' TCC TTC TGG CTG CGC GTG CCA AAA GTG TCC GCT TCC CAT CTG GAG CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 126).
  • Clone C7chG2G4- HC947tt/632tt was similarly constructed by inserting the 632DR peptide into clone C7chG2G4-HC947tt just downstream of the 947DR peptide. Three arginine residues flanked either side of the insertion while two arginine residues were placed between the peptides (e.g., RRR-947peptide-RR-632peptide-RRR).
  • Overlap PCR was used, as detailed above, with the exception that for the construction of clone C7chG2G4- HC947tt/632tt, fragment A was amplified using the reverse primer 947IN REV (described above) and fragment B was amplified using the forward primer 947IN FOR (described above). Overlap extension PCR products were then cloned into the vector C7 and sequenced as described above.
  • Fragment A was amplified using the forward primer E10Age5For in combination with the backward primer ELLG 632 REV (5' GAT GTA TGG CAC GAT GGT GGA CAC GTC GGA GAT TTT GTC GAT GCG GCG GCG GCC CAG CAG TTC TGG TGC TGA GGA AGA GAT GGA GGT GGA 3 ') (SEQ ID NO: 127).
  • Fragment B was generated using the forward primer 632 and the reverse primer EcoRIG2G4REV as above.
  • the peptide 947DR was inserted into the C7 antibody to create the clone C7chG2G4-HC- ELLG947tt using methods similar to those described above except that fragment A was amplified using the backward primer ELLG 947 REV (5' TTT TGG CAC GCG
  • ElO G2G4 clones the heavy chain constant region of clone ElOchGl was converted from a Gl to a G2G4 constant region. This was accomplished by partially digesting the ElOchGl clone with the restriction enzyme Apa I followed by complete digestion with EcoRI. An Apa I site is located near the start of the heavy chain constant region, whereas the EcoRI site is located past the terminal stop codon. This digestion effectively removes all but the first four amino acids of the heavy chain constant region. It should be noted that these first four amino acids are the same in both the Gl and G2G4 constant regions. This fragment was then replaced with the corresponding Apa I to EcoRI fragment from the G2G4 clone
  • C7chG2G4-HC632tt C7chG2G4-HC947tt, C7chG2G4-HC632tt/947tt, and C7chG2G4-HC947tt/632tt.
  • E10chG2G4-HC632tt E10chG2G4-HC947tt
  • E10chG2G4-HC632tt/947tt E10chG2G4-HC947tt/632tt.
  • plasmids were transiently transfected into 293 EBNA cells using Effectine (Qiagen). Briefly, 1.2 x 10 7 293 cells were seeded in 15OmM tissue culture dishes in DMEM + 10% FBS. The following day, each dish was transfected with 16 ug of the IgG expression plasmid along with 4 ug of pAdVAntage and 800 ng of pE-GFP-1, using 1 ml of EC Buffer, 160 ul of Enhancer and 200 ul of Effectine according to the manufacturer's instructions. At twenty-four hours post transfection, the media was changed to serum free media conditions (IS PRO media from Irvine Scientific). After an additional 24 hours, 2.5 mis of 20 X TC Sugar Rush reagent (0.5M HEPES, 20% glucose) was added. Cells were incubated for an additional 4 days and the media supernatant was harvested and purified by protein A affinity chromatography.
  • Effectine Qiagen
  • the affinity purified peptide-inserted antibody migrates at a molecular weight greater than that of native antibody commensurate with the ⁇ 2kDa size difference. Furthermore, the relative affinities of the purified peptide-inserted antibody to both the DC-SIGN and L-SIGN receptors on the cell are very similar to that of the native antibody (see Figure 7B).
  • EXAMPLE 4 Activation ofT-cell responses by antibody mediated delivery of peptide antigen to DCs
  • E10-632DR insertion clone
  • E10-632DR insertion clone
  • iDCs immature dendritic cells
  • the iDCs were differentiated from blood monocytes obtained from vaccinated donors by incubating with cytokines (IL-4 and GM-CSF) for six to nine days.
  • the iDCs were treated with the native (ElO) and peptide inserted antibodies for one hour, washed and mixed with autologous T-cells and left for five days to proliferate. As shown in Figure 8, targeting with E10-632DR, elicited a significant level (p ⁇ 0.005 vs.
  • E10-632DR peptide-inserted antibody was used to test whether inserting a peptide into the junction between the hinge and CH2 domain prevents binding to the Fc receptors.
  • the relative binding of the native antibody (ElO) and peptide inserted antibody (E10-632DR) to Fc receptor bearing monocytic cell line U937 was tested by a competition flow cytometry analysis (Figure 11). As shown in Figure 11, the native antibody (ElO) competes away the binding of a biotinylated human IgGl in a dose dependent manner similar to the unbiotinylated to IgGl.
  • the peptide inserted antibody (E 10-632DR) does not compete for the binding at any dose tested similar to IgG2G4, an antibody genetically mutated not to bind the Fc receptor. Disruption of Fc receptor binding may be beneficial in a variety of applications because it prevents non-specific binding of the antibody fusion construct by Fc
  • EXAMPLE 7 Design of peptide inserted antibody using sequence similarity replacement in the CH3 domain
  • This hydrophobic patch is the most distant site from the antigen reactive portion of the antibody. Therefore, structural perturbances in this area may be less likely to affect the antigen binding properties of the antibody.
  • the design for this antibody construct is schematically outlined in Figure 12.
  • EXAMPLE 8 Design of peptide inserted antibody by insertion into the CHl domain
  • Figure 14B shows the sequence of the G2G4 constant region from a region within the CH3 region to the carboxy terminus of the protein and the symbol "S" underneath the sequence denotes regions that are predicted to be potential 2OS proteasome cleavage sites.
  • the inserted peptide (underlined) may be processed by endogenous sites at the start and end of the peptide. However, in order to ensure that the peptide would be processed appropriately, it may be necessary to introduce strong proteasome cleavage signals, such as RR or KK residues, flanking one or both ends of the peptide. Examples of such insertions and the expected cleavage patterns resulting from them are shown in Figure 14C. For ease of viewing the inserted cleavage signals are written in lower case letters.
  • RR or KK could be used at either position or in combination (e.g., RR-peptide, KK-peptide, RK-peptide, KR- peptide, peptide-RR, peptide-KK, peptide-RK, peptide-KR, RR-peptide-RR, KK- peptide-KK, RR-peptide-KK, KK-peptide-RR, RK-peptide-RK, KR-peptide-KR, RK-peptide-KR, KR-peptide-RK, RR-peptide-RK, RR-peptide-KR, KK-peptide-RK, KK-peptide-KR, KK- peptide-KR, RK-peptide-RR, RK-peptide-KK, KR-peptide-RR., or KR-peptide-KK).
  • the two lysines, KK could replace either the histidine (H) and/or the tyrosine (Y) residues immediately following the TT 230 peptide.
  • the histidine, tyrosine, threonine and glutamine residues following the location of introduction of the TT230 peptide could be replaced or deleted in order to accommodate the four residues introduced in the antibody fusion shown in the lower portion of Figure 14C (e.g., the two arginine and two lysine residues flanking the TT 230 peptide).
  • the cysteine adjacent to the amino end of the introduced peptide is important to antibody structure and would not be removed or replaced with cleavage signal peptides.
  • the sequence from tetanus toxoid peptide 702 can be aligned with the G2G4 sequence at the carboxy terminus. As this peptide aligns with and effectively replaces the carboxy terminus, a cleavage signal after the terminal lysine would not be necessary. Proteasomal cleavage prediction suggests that the terminal peptide should be available as a cleavage product ( Figure 14E). Should the cleavable region "QK" that includes the first lysine of the desired peptide epitope be insufficient for proper processing, the glutamine (Q) could be replaced with another lysine to generate the cleavage signal without altering the length of the carboxy terminus.
  • the examples illustrated in Figures 14A-E utilize class I epitopes, however, the approach may also be possible with class II epitopes.
  • the tetanus toxoid class II epitope that begins at amino acid 632 can be aligned with the G2G4 sequence as shown in Figure 14F.
  • the period symbol indicates amino acids that are in a similar group, e.g. hydrophilic neutral, while the colon again represents conservative amino acid changes.
  • the cysteine residue located within the aligned region would not be altered as it may be important in antibody structure.
  • Analysis of the class II epitope shown has indicated that the first 7 amino acids can be removed and still permit recognition of the peptide as a class II epitope (Reece, J. C.
  • class I epitopes have more constraints in terms of the size of the epitope, such that efficient processing sites must surround the introduced peptide.
  • class II epitopes are more flexible in terms of the size of the epitope.
  • the antibody- class II epitope fusion described above may be processed efficiently by the naturally occurring surrounding proteasome sites without the necessity of introducing additional lysine or arginine residues. However, addition of lysine and/or arginine residues may be added if the processing observed is insufficient.
  • the same strategy of replacement following alignment can be performed with other IgG heavy chain constant regions such as, for example, the Gl heavy chain.
  • Gl and G2G4 heavy chains have fairly similar amino acid sequences, some of the regions identified in the G2G4 region for sequence similarity replacement would be similar or identical to the location for placement in the Gl heavy chain.
  • EXAMPLE 10 Design of antibody peptide fusion having peptide fused to the C- terminus of the antibody We designed an antibody peptide fusion having the peptide antigen fused to the carboxy-terminus of the antibody by a flexible spacer composed of two tandem repeats of four glycines and one serine. This design allows for independent folding of the antibody and the attached peptide. This strategy has been successfully used in the past by (Peschen, D.
  • a disease specific protein e.g., a tumor antigen (or autoantigen) and then attaching the epitope grafted carrier protein to a targeting antibody.
  • Design of such antibody peptide fusions first involves identification of the endogenous epitopes in the carrier protein.
  • the carrier protein is also analyzed to identify the proteasomal cleavage sites for grafting of other disease specific epitopes into the carrier protein.
  • various disease antigens responsible for the autoimmune form of diabetes and human cancers were analyzed for the presence of 2OS proteasomal cleavage hotspots and endogenous epitopes that have been shown to be the most active in human patients.
  • a total of four human autoantigens namely, glutamic-acid decarboxylase 65 (GAD65), heat shock protein 60 (HSP60), insulinoma associated protein 2 (IA-2) and proinsulin (PI) responsible for causing either Type 1 diabetes mellitus (TlDM) or insulin dependent diabetes mellitus (IDDM) and four human tumor antigens (namely, gplOO and Tyrosinase associated with Melanoma, Late Membrane Protein 2 (LMP-2) associated with Lymphoma and Carcinoembryonic Antigen associated with various types of adenocarcinomas) were analyzed by computer-aided algorithms to locate hydrophobic patches and proteasomal cleavage hot spots. Additionally, endogenous epitopes that
  • Table 3 Ranking of hydrophobic sequences of autoantigens containing most proteasomal cleavage sites (hotspots) for epitope grafting.
  • Cleavage hot spot refers to a hydrophobic patch having a higher cleavage score than the entire protein score (shown in bold).
  • Cleavage score is determined as number of individual cleavage sites (S)/number of amino acid residues.
  • T-cell epitopes of autoantigens found to be most active in TlDM or IDDM patients.
  • Cleavage hot spot refers to a hydrophobic patch having a higher cleavage score than the entire protein score (shown in bold).
  • Cleavage score is determined as number of individual cleavage sites (S)/number of amino acid residues.
  • EXAMPLE 12 Selection of a carrier protein for grafting of multiple disease specific epitopes
  • the disease being targeted is considered such as, for example, diabetes or cancer, or a particular type of cancer, such as, skin cancer or blood cancer.
  • the selection of an autoantigen as a carrier protein is based on the presence of a "maximum number of 2OS proteasomal hotspots.” This method will allow for the incorporation of the greatest number of epitopes from other autoantigens responsible for the disease, e.g., diabetes in this example. For example, IA-2 has the greatest number of proteasomal cleavage hotspots when compared to the other potential carrier proteins that were analyzed.
  • Figures 16-19 show the proteasomal cleavage hot spots for the diabetes carrier proteins indicated by underlined regions.
  • Figure 18 shows the sequence for IA-2 which has six proteasomal cleavage hot spots as compared to four, four, and two, hot spots for GAD65 ( Figure 16), HSP60 (Figure 17), and PI ( Figure 19), respectively. Therefore, each of the clinically relevant diabetic epitopes listed in Table 4 may be incorporated into the IA-2 carrier protein at a proteasomal cleavage hotspot.
  • selection of an optimal location for grafting of epitopes into a proteasomal cleavage hot spot of a carrier protein may be based on sequence similarity between the epitope and a region of the carrier protein having a proteasomal cleavage hotspot. Additionally, when selecting a carrier protein, consideration will also be given to the size of the carrier protein. A smaller carrier protein will facilitate production and subsequent conjugation to an antibody. Accordingly, the smallest disease protein that contains enough proteasomal hot spots to accommodate all of the disease epitopes desired to be delivered may be selected.
  • Carrier proteins comprising multiple disease specific epitopes may be associated with an antibody by expressing the carrier protein as a fusion with the antibody molecule, e.g., embedded into the constant region or attached to the C-terminus of the heavy or light chain constant domain.
  • the fusion may additionally comprise a peptide linker between the antibody and the carrier protein, such as a cleavable peptide linker as described below.
  • the carrier protein may be conjugated to the antibody, for example, using glutaraldehyde (see e.g., Reichlin M. Methods Enzymol. 70(A): 159-65 (1980); Yao TJ et al., Clin Cancer Res. 5: 77-81 (1999); Mittelman A et al, Clin Cancer Res. 1: 705-13 (1995)).
  • the selected antigenic peptides from disease proteins can be directly grafted onto the targeting antibody using the strategies described in the examples above.
  • An anti-cancer antibody having a toxin polypeptide embedded in the Fc region may be designed for use in selectively targeting and destroying cancer cells.
  • An exemplary pro-apoptotic polypeptide that may be used for targeted cytotoxicity of cancer cells is a 14-amino-acid amphipathic peptide KLAKLAKKLAKLAK (SEQ ID NO: 55) that has been successfully used for selectively killing malignant hematopoietic cells and cells lining tumor blood vessels (Ellerby, H. M. et al., Nat Med 5: 1032-1038 (1999); Marks, AJ. et al., Cancer Res 65: 2373-2377 (2005)).
  • This polypeptide, or another cytotoxic polypeptide may be introduced into the constant region of a cancer specific antibody using the methods described above for introduction of an antigenic polypeptide (e.g., insertion or sequence similarity replacement into a region flanked by naturally occurring proteasomal cleavage sites and/or by introduction of flanking proteasomal cleavage sites).
  • the resulting peptide toxin embedded antibodies may be screened for target cell killing using a MTT or MTS dye reduction assay (Marks, A. J. et al., Cancer Res 65: 2373-2377 (2005); Perchellet, E. M.
  • EXAMPLE 14 Screening method for identifying tumor internalizing antibodies Peptide toxins mediate their toxic effects upon internalization into the cells.
  • a peptide toxin may be embedded in or attached to the C-terminus of a library of antibodies, such as, for example, a library of tumor cell reactive antibodies.
  • the antibody fusions are then mixed with cells and screened for cell proliferation or cell death using an assay having an easy read out, such as, for example, an assay based on dye reduction methods (e.g., MTT).
  • an assay having an easy read out such as, for example, an assay based on dye reduction methods (e.g., MTT).
  • a library of peptides may be screened for toxic activity by fusing the library of peptides to an internalizing antibody, such as, for example, a known tumor cell internalizing antibody (e.g., growth receptor antibody, 4D5).
  • an internalizing antibody such as, for example, a known tumor cell internalizing antibody (e.g., growth receptor antibody, 4D5).
  • the peptide antibody fusions are then mixed with cells and screened for cell proliferation or cell death as described above. Peptides having toxic activity may thus be identified.
  • EXAMPLE 16 Design ofcleavable linkers for peptide embedded toxins into the DC-SIGN/L-SIGN antibody (clone ElO)
  • Figure 25A An exemplary cleavable linker for attachment of the peptide toxin to the Fab heavy chain of clone ElO is illustrated in Figure 25A.
  • Figure 25B shows a table indicating the 2OS proteasome cleavage scores for the sequence shown in Figure 25A. The site with highest cleavage score is highlighted.
  • Figure 26A An exemplary cleavable linker for attachment of the peptide toxin to the C- terminus of Fab light chain of clone ElO is illustrated in Figure 26A.
  • Figure 26B shows a table indicating the 2OS proteasome cleavage scores for the sequence shown in Figure 26A. The site with highest cleavage score is highlighted.
  • FIG. 27A An exemplary cleavable linker for inserting the peptide toxin into the hinge region of chimeric IgGl (clone ElO) is illustrated in Figure 27A.
  • Figure 27A shows a table indicating the 2OS proteasome cleavage scores for the sequence shown in Figure 27A. The site with highest cleavage score is highlighted.
  • EXAMPLE 17 Design of peptide antibody fusions utilizing peptide linkers to increase proper expression
  • the proportion of Hab can vary; this may reflect the length of the peptides inserted or their composition. This effect appears to be independent of whether Gl or G2G4 heavy chains are used.
  • a linker peptide is added to the end of the light chain and in front of the arginine stretch preceding the inserted peptide, so that steric hindrance between inserted peptide regions in the light and heavy chains is alleviated.
  • These linkers are composed of amino acids that promote flexibility of the protein backbone, such as glycine, and additional small amino acids with limited reactivity, such as alanine.
  • linkers Two examples of such linkers would be the amino acid sequence GGGAAG (SEQ ID NO: 129), a six amino acid linker, or the sequence GGGAAAGAAG (SEQ ID NO: 130), a ten amino acid linker. Other sequences and lengths of linkers may be envisioned by those skilled in the art. Two examples of the use of such linkers in the presentation of the 947tt peptide are shown in Table 8. Such linkers may also improve the ability of cells to express peptides at the C terminus of the light chain in general.
  • EXAMPLE 18 Engrafting peptides with surrounding proteasomal cleavage sites at the C-terminal end of the ElO light chain
  • the 632DR and 947DR peptides were chosen for insertion, either singly or in tandem, at the C-terminal end of the ElO light chain.
  • three arginines (RRR) were designed into the engrafted constructs between the C-terminal end of the light chain and either 632DR or 947DR.
  • RRR arginines
  • Table 9 shows the final sequences from the end of the light chain through the engrafted peptides for all the light chains generated.
  • the above light chain constructs were generated by overlap PCR using the primers listed in Table 10. Primers were synthesized by Operon.
  • the ElO light chain construct with the 632DR peptide engrafted was designated E10chGl-LC632tt.
  • a PCR fragment of 586 bp was first generated by pairing primers 060427 UP FOR and 060427 UP REV and using the ElO plasmid DNA as a template.
  • the fragment was gel purified and used in a PCR reaction with fragments 632DR for and 632DR rev (which overlap each other), along with additional 060427 UP FOR and 632 s rev, to generate a 670 bp fragment. After gel purification, this fragment and ElO were digested with Acc65l andNotl and subjected to gel electrophoresis.
  • this fragment was subjected to gel electrophoresis and a 615 bp fragment was isolated and ligated to the 11542 bp Acc65VNotl ElO fragment (above) to generate E10chGl-LC947tt.
  • the tandem epitope insertions were generated using the single epitope insertion plasmids as PCR templates.
  • Construct E10chGl-LC947tt/632tt was generated using E10chGl-LC632tt as template, pairing primers 060427 UP FOR with 060616 947 IR to generate a 636 bp fragment, and primer 060616 947 IF with 632DR s rev to generate a 130 bp fragment, which were gel purified.
  • These purified fragments were then subjected to PCR with added 060427 UP FOR and 632DR s rev to generate a 742 bp fragment, which was digested with Acc65I and Notl along with E10chGl-LC632tt. Fragments of 684 and 11542 bp were gel purified from the PCR fragment digest and plasmid digest, respectively, and were ligated together to generate E 1 OchG 1 -LC947tt/632tt.
  • E10chGl-LC632tt/947tt was generated using E10chGl-LC947tt as template, pairing primers 060427 UP FOR with 060724 632 IR to generate a 637 bp fragment, and primer 060724 632 IF with 947DR s rev to generate a 128 bp fragment, which were gel purified. These purified fragments were then subjected to PCR with added 060427 UP FOR and 947DR s rev to generate a 742 bp fragment, which was digested with Acc65I and Notl to generate a 684 bp fragment.
  • the light chain variants developed above are shown in the first four rows of Table 11. These light chains were transferred to a number of other constructs bearing alternate heavy chains with or without inserted peptides.
  • the heavy chain constructs are described above.
  • the light chain variants were transferred into different heavy chain contexts by digestion of the constructs above with- ⁇ l and Notl, isolation of the light chain containing fragment, and replacement of the corresponding light chain containing XballNotl region in the heavy chain construct of interest. In a few cases, the replacement was done with Acc65llNot ⁇ fragments. In this way, the remaining constructs of Table 11 were generated.
  • primer 060925 LL 947 (long linker) was used with primer 947 s rev and ElO chGl LC947 as a template in order to generate a 140 bp PCR fragment. This was used in overlap PCR with the 586 bp PCR fragment previously generated with primers 060427 UP FOR and 060427 UP REV described above to generate a 703 bp fragment. Digestion of this fragment
  • Custom oligonucleotide primers were purchased from Sigma-Genosys. All PCR steps were performed using the Expand High Fidelity PCR System (Roche Applied Science). PinAI restriction enzyme was obtained from Roche Applied Science. All other restriction enzymes and T4 DNA Ligase were obtained from New England Biolabs. Conversion ofscFv-2G12 into a rabbit-human chimeric IgGl
  • the rabbit anti-human CD 19 single-chain antibody scFv-2G12 was converted to a rabbit-human chimeric whole IgGl by overlap extension PCR using the following primers:
  • the scFv-2G12 kappa light chain V region was amplified from vector PAX243-scFv-2G12 using primers R2G12VK-F1 and R2G12VK-hCK-R.
  • the human kappa constant region was amplified from a human kappa light
  • the scFv-2G12 heavy chain V region was amplified from vector PAX243-scFv-2G12 using primers R2G12VH-F1 and R2G12VH-hCG-R.
  • the human IgGl heavy chain CHl region was amplified from a rabbit-human chimeric IgGl Fab expression vector using primers R2G12VH-hCG-F and hCG-Rl .
  • Overlap extension PCR was performed on the two products using primers R2G12VH-F1 and hCG-Rl to make the rabbit-human chimeric Fab heavy chain.
  • the PCR product was digested with XhoI/PinAI and cloned into PAX243-2G12VK-hCK.
  • the resulting chimeric Fab construct was designated PAX243-2G12/cFab.
  • KLAKLAKKLAKLAK RRR KLAKLAKKLAKLAK (SEQ ID NO: 159) were added to the end of the 2G12/cIgGl light chain by overlap extension PCR using the following primers:
  • the 2G12 light chain was amplified from plasmid 3B.1BB- 2G12/cIgGl using primers R2G12VK-F1 and hCK[KLAK]x2-R2.
  • the resulting product was then subjected to a second round of PCR with primers R2G12VK-F1 and hCK[KLAK]x2-R3.
  • the final PCR product was digested with Xbal/Notl and cloned into the Xbal/Notl sites of vector 3B. IBB- 2G12/cIgGl .
  • the resulting construct was designated 3B.1BB-2G12/LC- KLAKx2/cIgGl.
  • Sequences encoding three copies of the p21 peptide toxin flanked by proteasomal cleavage sites were inserted between Gly249 and Gly250 of the 2G12/cIgGl heavy chain (amino acid sequence PAPELLG 2 49G 250 PSVFLFPPK (SEQ ID NO: 163)) by overlap extension PCR using the following primers:
  • the 5' segment of the human IgGl heavy chain ending with codon Gly249 was amplified from plasmid 3B.lBB-2G12/cIgGl using primers E10Age5For and hCH2[p21]x3-R2. The resulting product was then subjected to a second round of PCR with primers E10Age5For and hCH2[p21]x3-R3. The 3 'segment of the human IgGl heavy chain beginning with codon Gly250
  • 10235000 1 86 was amplified from plasmid 3B. lBB-2G12/dgGl using primers hCH2[p21]x3-F2 and 3B. IBBSaB-R. The resulting product was then subjected to a second round of PCR with primers hCH2[p21]x3-F3 and 3B. IBBSaB-R. A third round of overlap extension PCR was then performed on the two second round PCR products using primers E10Age5For and 3B. IBBSaB-R. The final product was digested with PinAI/Sall and cloned into the PinAI /Sail sites of vector 3B. lBB-2G12/dgGl . The resulting construct was designated 3B.lBB-2G12/HC-p21x3/cIgGl.
  • the 2G12 light chain containing the KLAK peptide sequences was combined with the 2Gl 2 heavy chain containing the p21 peptide sequences by digesting plasmid 3B.lBB-2G12/LC-KLAKx2/cIgGl with Xbal/NotI and cloning the light chain insert into the Xbal/NotI sites of plasmid 3B.1BB- 2G12/HC-p21x3/cIgGl .
  • the resulting construct was designated 3B. IBB- 2G12/LC-KLAKx2/HC-p21x3/cIgGl .
  • the 2G12/cIgGl and 2G12/LC-KLAKx2/HC-p21x3/cIgGl antibodies were expressed in 293 -EBNA cells by transient transfection using Effectene reagent (Qiagen) and purified by protein A affinity chromatograph.
  • EXAMPLE 21 Evaluation of immune responses to a large panel of peptide embedded antibodies targeting antigen-presenting cells in an autologous T-cell assay system
  • a panel of peptide embedded antibodies was successfully produced. We evaluated whether these antibodies successfully deliver the embedded peptides to the antigen processing and presentation machinery of antigen presenting cells, resulting in stimulatory T cell responses to these peptides. To evaluate T cell responses, sixteen human donors were first screened for their T cell response to three universal tetanus toxoid HLA-DR binding peptides (described earlier). One hundred thousand to
  • peripheral blood lymphocytes 10235000 1 87 200,000 peripheral blood lymphocytes (PBL) were incubated with 500 nM peptide for five days. Proliferation responses were determined by 3 H-thymidine incorporation in the last 18 hrs. Statistically significant immune responses (P ⁇ 0.01) were induced in 25% (4 of 15) of the donors by peptides 632DR and 947DR (see Table 13). Based on this result, these two peptides were selected for embedding into DC-SIGN/L-SIGN reactive antibody clone ElO (described earlier). A large panel of peptide-embedded antibodies was then evaluated for the elicitation of immune responses in an autologous DC-T cell assay system.
  • Immature DCs >90% DC-SIGN positive
  • Immature DCs from three donors were incubated with antibodies at 10 ⁇ g/mL ( Figure 3 IA and Figure 32A) or 0.1 ⁇ g/mL ( Figure 3 IB and 32B), free peptide (1 ⁇ g/mL) and TT protein (100 ng/mL) for 2 hrs at 37 0 C, unbound antibody removed and co-cultured with 100,000 purified autologous T cells (>95% CD3 positive) and incubated for five days. Proliferation was assessed by 3 H Thymidine incorporation in the last 24 hrs of the assay. The assay was performed in four replicate wells for each treatment.
  • Table 14 Statistical analysis of T-cell proliferation responses shown in Figure 31. Significant differences were analyzed by comparing T-cell responses induced by peptide grafted antibodies with ungrafted native antibody using two-tailed unpaired Student's t test. Significance was accepted when/? ⁇ 0.01.
  • EXAMPLE 22 Assessment of cell growth inhibition by peptide toxin embedded antibodies targeting B-cell leukemic cells
  • growth inhibitory Peptides As a first step towards evaluating the utility of tumor targeting antibodies for delivering growth inhibitory peptides to cancer cells, four growth-inhibitory peptides were selected based on their ability to interfere with the function of key cellular proteins, e.g., p53, CDK etc. (Chen, Y. N., et al. 1999. Proc Natl Acad Sci U S A 96, 4325-4329; Datta, K., et al. 2001. Cancer Res 61, 1768-1775; Kim, A. L., et al. 1999. J Biol Chem 274, 34924-34931; and Marks, A. J., et al. 2005.
  • key cellular proteins e.g., p53, CDK etc.
  • Each of these four peptides was then chemically synthesized collinearly with additional sequences (e.g., HIV TAT peptide) capable of naturally traversing the mammalian cell membrane and facilitate transport of the attached growth inhibitory peptide into the cytoplasm (see Table 16 for the sequence design). All four growth inhibitory peptides were then evaluated on a panel of eleven tumor cell lines in a cell growth inhibition assay. Various tumor cell lines were incubated with a 2-fold titrated dose (range 100 ⁇ M to 1.5 ⁇ M) of growth inhibitory peptides for two days. Growth inhibition was assessed by MTS bioreduction in the last two hours of the assay.
  • additional sequences e.g., HIV TAT peptide
  • B-cells and B-cell leukemic cells e.g., Raji or Namalwa was selected for exploring the utility of embedding peptides p21 and KLAK on the inhibition of RAJI cell growth.
  • a prerequisite for efficient delivery of peptides into the cytoplasm of cells is its ability to undergo internalization by the targeted tumor cells.
  • the internalization of 2Gl 2 was therefore determined on RAJI cells.
  • RAJI cells were incubated with rabbit single chain antibody, clone 2G12 or a known internalizing CD19 mouse niAb, clone BC3 at 4 0 C and 37 0 C. The level of cell surface antibody remaining after 2 hrs was measured by flow cytometry.
  • Binding and growth inhibition of the peptide embedded antibodies was performed on RAJI tumor cells.
  • One million Raji cells were incubated with 10 ⁇ g/mL of full length IgGl antibodies embedded with three copies of p21 peptide (in the hinge) and one copy of the KLAK peptide (at the C-terminal end of light chain) antibodies for 30 min at 4C in FACS buffer, washed with ImL buffer and incubated with goat anti-human PE (1/100 dilution) for 30 min at 4 0 C, washed and binding level analyzed on a FACScalibur.
  • the results of this study illustrated in Figure 34 indicated a similar level of binding for both the native and peptide toxin embedded full length
  • the present invention provides among other things antibody-peptide fusion proteins and methods for producing and using antibody-peptide fusions proteins. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will be discussed.
  • any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) (www.tigr.org) and/or the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov).
  • TIGR The Institute for Genomic Research
  • NCBI National Center for Biotechnology Information

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Abstract

Provided are antibody fusions wherein a peptide has been incorporated into the constant domain of an antibody. The peptides may be incorporated at hydrophobic regions within the constant domain and/or between proteasomal cleavage sites that are either naturally occurring or introduced. Also provided are antibody fusions wherein a peptide has been attached to the C-terminus of the constant domain of an antibody heavy or light chain through a cleavable linker. Methods for using the antibody fusions include therapeutic applications, such as immune modulation and selective cell killing, and research applications, such as screening assays to identify internalizing antibodies or cytotoxic agents.

Description

ANTIBODY-POLYPEPTIDE FUSION PROTEINS AND METHODS
FOR PRODUCING AND USING SAME RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 60/729,404, filed October 21, 2005, which application is hereby incorporated by reference in its entirety.
BACKGROUND When a healthy host (human or animal) encounters an antigen, normally the host initiates an immune response. This immune response can be a humoral response and/or a cellular response. In the humoral response, antibodies are produced by B- cells and are secreted into the blood and/or lymph in response to an antigenic stimulus. The antibody then neutralizes the antigen by binding specifically to epitopes on its surface, marking it for destruction by phagocytic cells and/or complement- mediated mechanisms. The cellular response is characterized by the selection and expansion of specific helper and cytotoxic T-lymphocytes capable of directly eliminating the cells which contain the antigen.
Antigen Presenting Cells (APCs) process the encountered antigens differently. Exogenous antigens are typically processed within the endosomes of the APC and the generated peptide fragments are presented on the surface of the cell complexed with Major Histocompatibility Complex (MHC) Class II. The presentation of this complex to CD4+ T cells stimulates the CD4+ T helper cells. As a result, cytokines secreted by the helper cells stimulate B cells to produce antibodies against the exogenous antigen (humoral response). Immunizations using antigens typically generate antibody response through this endosomal antigen processing pathway.
On the other hand, intracellular antigens, as well as some exogenous antigens, are processed in the proteasome and the resulting peptide fragments are presented as complexes with MHC Class I on the surface of APCs. Following binding of this complex to the co-receptor CD8 molecule, antigen presentation to CD8+ T cells occurs which results in cytotoxic T cell (CTL) immune response to remove the host cells that carry the antigen.
An effective therapeutic vaccine should be able to induce strong T helper cell and CTL responses against an intracellular antigen or an antigen delivered into the
10235000J 1 appropriate cellular compartment so as to activate the MHC Class I processing pathway. This suggests that a therapeutic vaccine that can induce a strong CTL response should be processed through the proteasomal pathway and presented via the MHC Class I. This can be achieved either by producing the antigen within the host cell, or it can be delivered to the appropriate cellular compartment so that it gets processed and presented so as to elicit a cellular response. Several approaches have been documented in the literature for the intracellular delivery of the antigen. Among these, viral vectors (Lorenz et al. (1999) Hum Gene Ther 10(7): 1095-103), the use of cDNA-transfected cells (Donnelly et al. (1997) Annu Rev Immunol 15: 617-48) as well as the expression of the antigen through injected cDNA vectors (Lai and Bennett
(1998) Crit Rev Immunol 18(5): 449-84), have been documented.
Dendritic cells derived from blood monocytes, by virtue of their capability as professional antigen presenting cells have been shown to have great potential as immune modulators which stimulate primary T cell response (Steinman et al. (1999) Hum Immunol 60(7): 562-7; Banchereau and Steinman (1998) Nature 392(6673): 245-52). This unique property of the DCs to capture, process, present the antigen and stimulate naive T cells has made them very important tools for therapeutic vaccine development (Laupeze et al. (1999) Hum Immunol 60(7): 591-7). Targeting of the antigen to the DCs is a crucial step in the antigen presentation and the presence of several receptors on the DCs for the Fc region of monoclonal antibodies has been exploited for this purpose (Regnault et al. (1999) J Exp Med 189(2): 371-80). Examples of this approach include ovarian cancer Mab-B43.13, Anti-PSA antibody as well as Anti-HBV antibody antigen complexes (Wen et al. (1999) Int Rev Immunol 18(3): 251-8). Cancer immunotherapy using DCs exposed to or pulsed with tumor associated antigens have been shown to produce tumor-specific immune responses and anti-tumor activity (Campton et al. (2000) J Invest Dermatol 115(1): 57-61; Fong and Engleman (2000) Annu Rev Immunol 18: 245-73). Promising results were obtained in clinical trials in vivo using tumor-antigen-pulsed DCs (Tarte and Klein
(1999) Leukemia 13(5): 653-63). These studies clearly demonstrate the efficacy of using DCs to generate immune responses against cancer antigens.
Development of safe and effective vaccines for protecting against infection and disease is of great importance. There is still a great need for vaccines that elicit the induction of a strong and long-lasting immunity characterized by both a humoral and cell-mediated immune response for treating infections and diseases such as
10235000 1 9 cancer. In particular, a need exists for methods to selectively targeting peptides to specific cell populations and release the peptides by proteasomal cleavage inside the targeted cells.
SUMMARY
Provided herein are novel antibody fusion constructs for selective delivery of an amino acid sequence (e.g., a peptide or polypeptide) to cells. The novel constructs are useful as vaccines, e.g., for targeted delivery of antigen to antigen presenting cells for the treatment of cancer, autoimmune diseases, inflammation or infectious diseases. Also provided are methods for using the antibody fusion constructs for a variety of therapeutic applications, such as elimination of cancer or other unwanted cells using antibody fusions comprising cytotoxic peptides incorporated into internalizing antibodies specific for receptors on cancer cells or other unwanted cells in a disease state. Other therapeutic applications include targeted delivery of chemotactic peptides or other therapeutic peptides, such as growth factors or fragments thereof. The antibody fusion constructs described herein may also be used as a screening tool for identifying internalizing antibodies or cytotoxic peptides.
For antibody based epitope delivery, receptor specific antibodies may be used. In exemplary embodiments, the peptide epitope may be grafted into a location in the antibody that does not disrupt protein folding and allows for robust production of the antibody fusion proteins. When delivered to a cell, the antibody fusion will be taken up by the target cell and processed correctly to release the appropriate peptide epitope. In certain embodiments, selection of an antibody constant region in which Fc Receptor binding is minimized (e.g., immunoglobulin (Ig) G2G4) may be desirable to avoid or reduce non-specific cellular uptake by Fc receptor bearing cells.
Controlled intracellular release of a peptide from the antibody fusion construct may be achieved by incorporating the peptide into the antibody between proteasomal cleavage sites. The proteasomal cleavage sites may be naturally occurring in the antibody or may be introduced into the antibody. Exemplary proteasomal cleavage sites comprise two or more lysine and/or arginine residues. Incorporation of a polypeptide into an antibody between flanking proteasomal cleavage sites permits insertion into any region of the antibody and allows maximal flexibility in selecting a site of incorporation that favors efficient folding and expression of the protein.
10235000 1 In certain embodiments, peptides may be incorporated into the constant domain of an antibody at a region exhibiting sequence similarity with the peptide and/or at a hydrophobic region within the constant domain. As many peptide epitopes are hydrophobic, a region of the antibody constant domain that is both hydrophobic and has sequence similarity with the peptide may be selected for incorporation. Introduction of a polypeptide into a region of hydrophobicity and/or sequence similarity may facilitate efficient expression and proper folding of the antibody fusion construct.
In an exemplary embodiment, an antibody fusion is provided wherein the antibody comprises a polypeptide incorporated into the constant region within the hinge region or close to the hinge region. The polypeptide may be flanked at one or both ends by proteasomal cleavage sites, such as two or more lysine and/or arginine residues.
In certain embodiments, the antibody fusions provided herein may comprise antibody fragments such as, for example, a Fab or a single chain antibody (ScFv) fused to at least a portion of a heavy and/or light chain constant region.
In certain embodiments, the antibody fusions provided herein may comprise an amino acid sequence attached to the C-terminus of the heavy and/or light chain of the antibody. Antibody fusions that have at least one amino acid sequence incorporated into the constant region and at least one polypeptide attached to the C- terminus of the heavy and/or light chain are also provided.
In certain embodiments, the antibody fusions may have a peptide incorporated into the constant domain and/or fused to the C-terminus of the constant domain in association with a cleavable peptide linker. In exemplary embodiments, such cleavable linkers are also flexible.
In various embodiments, the antibody fusions may comprise one or more polypeptides incorporated into the same or different locations in the antibody molecule.
In one embodiment, an antibody fusion comprising a disease specific carrier protein having multiple disease specific epitopes incorporated into the carrier protein is provided. The disease specific carrier protein may have one or more disease specific peptide epitopes incorporated at regions of sequence similarity and/or hydrophobicity and/or between proteasomal cleavage sites. The carrier protein may be linked to an antibody having a desired specificity by chemical conjugation or
10235000 1 Λ through a cleavable peptide linker. The specificity of the antibody may be selected for targeted delivery of the disease specific carrier protein to a desired cell type.
Also provided is a method for targeted destruction of a population of cells. The method involves contacting the cells with an antibody fusion comprising an antibody and a cytotoxic peptide. The antibody is specific for a protein expressed on the surface of a cell or cell population that is to be targeted for destruction. Upon , binding and internalization of the antibody, the cytotoxic peptide is released inside the cell resulting in cell death. In an exemplary embodiment, the antibody fusions comprising a cytotoxic peptide may be used for treating cancer. The antibody fusions useful for treating cancer comprise an antibody specific for one or more tumor associated antigens and/or an antibody that binds to a cell surface protein involved in undesirable immune suppression, such as, for example, regulatory T-cells.
In yet other embodiments, the antibody fusions described herein may be used for targeted delivery of a variety of polypeptides including, for example, growth factors or fragments thereof, and chemotactic agents. Targeted delivery of such polypeptides is useful for controlled stimulation of cell growth, cell differentiation, or other cellular functions.
In one embodiment, screening methods for identifying internalizing antibodies or cytotoxic peptides are provided. For example, libraries of antibodies comprising a peptide toxin linked to a plurality of antibodies may be constructed. Such libraries can be screened against cancer cells, or any other cell target of interest and internalizing antibodies may be identified using cell death as a read out. In another example, a library of potential cytotoxic peptides may be attached or incorporated into an internalizing antibody. The antibody fusion library is mixed with cells and cytotoxic peptides may be identified using cell death as a read out.
The practice of the present invention may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S.
10235000 1 ς J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (J. Woodward ed., IRL Press, 1985); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology , Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Hogan, B., Costantini, F. and Lacy, E., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The appended claims are incorporated into this section by reference.
BRIEF DESCRIPTION OF THE FIGURES
The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which: FIGURE 1 shows the amino acid sequence of immunoglobulin G2G4 fused constant region (SEQ ID NO: 1) showing 2OS proteasome cleavage sites and hydrophobic areas. The letter "S" underneath the sequence indicates 2OS proteasome cleavage sites predicted by the algorithm "NetChop." Highlighted areas indicate hydrophobic patches predicted by the Kyte-Doolittle algorithm. FIGURE 2 shows the amino acid sequence of immunoglobulin Gl constant region (SEQ ID NO: 2) showing 2OS proteasome cleavage sites and hydrophobic areas. The letter "S" underneath the sequence indicates 2OS proteasome cleavage sites predicted by the algorithm "NetChop." Highlighted areas indicate hydrophobic patches predicted by the Kyte-Doolittle algorithm. FIGURE 3 shows an alignment of the amino acid sequences for the immunoglobulin Gl constant region (SEQ ID NO: 2) and the immunoglobulin G2G4 fused constant region (SEQ ID NO: 1). Sequence differences are indicated asterisks. Hydrophobic patches are highlighted.
10235000 1 FIGURES 4A-B illustrate immunological responses to tetanus toxin (TT). Figure 4A shows the antibody responses to TT protein. Serum from all donors except donor 14 was collected two weeks after vaccination. Donor 14 was vaccinated in the year 1997, but had equivalent levels of antibody responses (see donor 14 - prevaccine 2) as the vaccinated donors. Figure 4B shows the T-cell responses to TT peptides two weeks after vaccination. 100,000 PBL were incubated with lug/ml peptide and 25ng/ml TT protein for five days. Proliferation was enumerated by 3H-Thymidine incorporation during the last 18 hours. Highly significant values (e.g., PO.005) when compared with medium treated samples are indicated by asterisks. FIGURES 5A-B illustrate antibody fusions that were constructed using sequence similarity and insertion strategies.- Polypeptide TT (632DR) was introduced into the constant region of the DC-SIGN/L-SIGN antibody (clone ElO) by insertion and sequence similarity replacement. Figure 5 A shows a schematic of the sites of introduction of the TT polypeptide (632DR) into the constant region of the antibody and the local sequences involved in each construct. The local sequence for insertion construct is SEQ ID NO: 3, the local sequence on the Ig constant region (ElO) for the sequence similarity replacement construct is SEQ ID NO: 4, and the TT polypeptide sequence incorporated into the constant region (632DR) is SEQ ID NO: 5. Figure 5B shows the amino acid sequence of the IgGl constant region (SEQ ID NO: 2) of the antibody. The sites for incorporation of the TT polypeptide (632DR) by insertion and replacement are indicated by arrows.
FIGURES 6A-B show expression and binding characteristics of antibody fusions illustrated in Figures 5A-B. Panel A is a Western blot showing the expression of antibody fusions generated by insertion and replacement (as described in Figure 5) as compared to native antibody. 20 ul of supernatant from each clone was run on a native SDS-PAGE gradient gel (4%-15%), transferred to nitrocellulose and detected with anti-light chain-HRP conjugate. The peptide fusion generated by insertion shows a size difference of about ~2kDa as compared to the native antibody. Figure 6B illustrates the binding of the insertion and replacement antibody fusions to DC- SIGN receptors on cells (0.5 million/sample) as compared to native antibody. The experiments were conducted using K562 cells (K562) or K562 cells overexpressing the DC-SIGN receptor (K562/DC-SIGN) or the L-SIGN receptor (K562/L-SIGN).
FIGURES 7A-B show affinity purified antibody fusions (insertion clone) and the binding affinity of the antibody fusion insertion clone as compared to the native
1023S000_l η antibody. Figure 7A shows a native SDS-PAGE gradient gel (4%-15%) of affinity purified peptide inserted antibodies. As shown on the gel, the affinity purification eliminated partially formed antibodies (e.g., as compared to supernatant fraction shown in Figure 6A). Figure 7B shows the relative binding affinity of the peptide inserted antibody as compared to the native antibody. Binding affinities were determined using K562 cells overexpressing DC-SIGN or L-SIGN receptors.
FIGURE 8 provides the results of T-cell activation after targeted delivery of TT peptide to immature dendritic cells (iDCs). T-cells from vaccinated donors were used. 10,000 iDCs were incubated with 10 ug/ml (66 nM) of native antibody (ElO), 10 ug/ml (66 nM) of peptide inserted antibody (E10-632DR), lug/ml (500 nM) of the 632DR fragment of TT protein, or medium, for lhr at 370C. The iDCs were then washed and added to 100,000 T-cells and incubated for five days. As a positive control, 10,000 iDCs were included with TT protein (100 ng/ml) and added to T-cells without washing. Proliferation was enumerated by 3H-Thymidine incorporation. Highly significant values (e.g., PO.005), when compared to medium or native antibody (ElO), are indicated by asterisks.
FIGURES 9A-B illustrate the dose dependent response of native and peptide inserted antibodies for T-cell activation and iDC binding. Figure 9A illustrates the dose dependent response of T-cell activation to the targeting antibody (E10-632DR) and blocking of T-cell activation by competition with native antibody (ElO). 10,000 iDC were incubated with antibodies and free peptide for 1 hour at 370C, washed and added to 100,000 T-cells and incubated for five days. The T-cell stimulation of the peptide inserted antibody (E10-632DR) alone was blocked upon competition with native antibody (E10-632DR + ElO) but was not blocked by competition with a control antibody (E10-632DR + control), e.g., an antibody not specific for a SIGN receptor. Proliferation was enumerated by 3H-Thymidine incorporation. Highly significant values (e.g., PO.00005), when compared to medium or native antibody (ElO), are indicated by an asterisk. Figure 9B illustrates dose dependent binding of native antibody (ElO) and peptide inserted antibody (E10-632DR) to the immature dendritic cells used in Figure 9 A.
FIGURE 10 shows a graph illustrating that antibody targeting produces a sustained immune response. The cells were treated as described in Figure 9 A, except that T cells were added 2 and 4 days after antibody targeting. Highly significant
1O235OOO 1 values (e.g., P<0.05), when compared to medium at 4 days, are indicated by an asterisk.
FIGURE 11 shows the competitive binding of native antibody (ElO) and peptide inserted antibody (E10-632DR) to the Fc receptors. U937 cells were incubated with 15 ng/ml biotin-hlgGl together with various concentrations of competing unbiotinylated antibodies. The percentage of cells bound to biotin-hlgGl was determined using streptavidin-phycoerythrin by flow cytometry analysis. FIGURE 12 is a schematic of a peptide inserted antibody designed by sequence similarity replacement in the CH3 domain of the heavy chain constant region. The local amino acid sequence at the site of peptide introduction is shown (SEQ ID NO: 6).
FIGURE 13 is a schematic of a peptide inserted antibody having a peptide sequence inserted into the CHl domain of the heavy chain constant region and flanked by arginine (top) or lysine (bottom) residues. The local amino acid sequence at the site of peptide introduction is shown for both the top (SEQ ID NO: 7) and bottom constructs (SEQ ID NO: 8).
FIGURES 14A-G show several embodiments of antibody peptide fusions having a tetanus toxin (TT) peptide introduced into the G2G4 constant region. Figure 14A shows the sequence similarity between a region of the CH3 domain of the G2G4 constant region (SEQ ID NO: 9) and the TT 230 peptide (SEQ ID NO: 10). Figure 14B shows the local sequence and proteasomal cleavage sites (S) for the TT 230 peptide embedded into the CH3 domain of the G2G4 constant region (SEQ ID NO: 11). Figure 14C illustrates two variations of the embedded TT 230 peptide having proteasomal cleavage sites introduced at the N-terminus (top; SEQ ID NO: 12) or at the N-terminus and C-terminus (bottom; SEQ ID NO: 13). Figure 14D shows the sequence similarity between a region of the CH3 domain of the G2G4 constant region (SEQ ID NO: 14) and the TT 702 peptide (SEQ ID NO: 15). Figure 14E shows the local sequence and proteasomal cleavage sites (S) for the TT 702 peptide embedded into the CH3 domain of the G2G4 constant region (SEQ ID NO: 16). Figure 14F shows the sequence similarity between a region of the CH2 domain of the G2G4 constant region (SEQ ID NO: 17) and the TT 632-651 peptide (SEQ ID NO: 18). Figure 14G shows the local sequence and proteasomal cleavage sites (S) for the TT 632-651 peptide embedded into the CH2 domain of the G2G4 constant region (SEQ ID NO: 19).
10235000 1 Q FIGURE 15 shows the local sequence alignment for two antibody peptide fusions having the TT 830-844 peptide attached to the C-terminus of the antibody heavy chain via a cleavable linker having two arginine (top; SEQ ID NO: 20) or two lysine (bottom; SEQ ID NO: 21) residues. FIGURE 16 shows the amino acid sequence for human Glutamic-Acid
Decarboxylase 65kDa protein (GAD65) (SEQ ID NO: 22). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
FIGURE 17 shows the amino acid sequence for human Heat Shock Protein 6OkDa (HSP60) (SEQ ID NO: 23). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
FIGURE 18 shows the amino acid sequence for Human Insulinoma Associated protein (IA-2) (SEQ ID NO: 24). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
FIGURE 19 shows the amino acid sequence for Human Proinsulin (PI) (SEQ ID NO: 25). Hydrophobic patches are highlighted, 20S proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case. FIGURE 20 shows the amino acid sequence for Melanocyte Lineage-Specific
Antigen gplOO (SEQ ID NO: 26). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitopes are boxed and lower case.
FIGURE 21 shows the amino acid sequence for Melanocyte Associated Tumor Antigen, Tyrosinase (SEQ ID NO: 27). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
FIGURE 22 shows the amino acid sequence for LMP-2 Membrane Protein (SEQ ID NO: 28). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
FIGURE 23 shows the amino acid sequence for Carcinoembryonic Antigen (CEA) (SEQ ID NO: 29). Hydrophobic patches are highlighted, 2OS proteasomal cleavage hotspots are underlined, and the most active disease epitope is boxed and lower case.
10235000J 10 FIGURE 24 shows a flow diagram illustrating a method for designing disease specific carrier proteins with multiple embedded disease specific epitopes.
FIGURE 25A shows the local sequence and proteasomal cleavage sites (S) for a peptide toxin attached to the Fab heavy chain of clone ElO (SEQ ID NO: 30). Figure 25B is a table showing the proteasomal cleavage scores for the sequence shown in Figure 25A. The site with the highest cleavage score is highlighted.
FIGURE 26A shows the local sequence and proteasomal cleavage sites (S) for a peptide toxin attached to the C-terminus of the Fab light chain of clone ElO (SEQ ID NO: 31). Figure 26B is a table showing the proteasomal cleavage scores for the sequence shown in Figure 26A. The site with the highest cleavage score is highlighted.
FIGURE 27A shows the local sequence and proteasomal cleavage sites (S) for a peptide toxin embedded into the hinge region of chimeric IgGl clone ElO (SEQ ID NO: 32). Figure 27B is a table showing the proteasomal cleavage scores for the sequence shown in Figure 27A. The site with the highest cleavage score is highlighted.
FIGURE 28 shows a table providing the sequences of the hinge region from various human, mouse, rat, guinea pig and rabbit antibodies (SEQ ID NOs: 33-50).
FIGURE 29 shows a variety of goat anti-Fab purified chimeric antibodies that were run on an SDS-PAGE gel and stained with Coomassie blue. Lane a:
E10chG2G4, lane b: E10chG2G4-LC947tt, lane c: E10chG2G4-LC632tt/947tt, lane d: E10chGl-HC947tt, lane e: Molecular weight marker, lane f: ElOchGl- HC632tt/947tt, lane g: Molecular weight marker, lane h: E10chG2G4-HC947tt/632tt, lane i: E10chG2G4-HC632tt/LC947tt, and lane j: E10chGl-HC632tt/LC947tt. FIGURE 30 shows a half antibody (Hab) that was run on an SDS-PAGE gel and stained with Coomassie blue or western blotted. Lane a: Molecular weight marker, lane b: E10chGl-HC632tt/LC947, Coomassie blue stain, lane c: ElOchGl- HC632tt/LC947, Western with goat anti-human kappa chain antibody, and lane d: E10chGl-HC632tt/LC947, Western with goat anti-human gamma chain antibody. FIGURE 31 shows immune responses elicited by DC-SIGN antibody (clone
ElOchlgGl) grafted with TT epitopes, 632DR and 947DR. Panel A: 10 μg/mL antibody and Panel B: 0.1 μg/mL antibody.
10235000J 1 1 FIGURE 32 shows the immune responses elicited by DC-SIGN antibody (clone E10chIgG2G4) grafted with TT epitopes, 632DR and 947DR. Panel A: 10 μg/mL antibody and Panel B: 0.1 μg/mL antibody.
FIGURE 33 shows internalization of CD19 single chain antibody, clone 2G12 on RAJI cells. Percentage of internalization indicated on top of the bars was determined as (Geo. mean fluorescence at 4°C - Geo. mean fluorescence at 37°C)/(Geo. mean fluorescence at 4°C) X 100.
FIGURE 34 shows binding of peptide toxin embedded full length IgGl antibodies (clone 2G12) to RAJI cells. FIGURE 35 shows binding of peptide toxin embedded single chain antibodies
(clone 2G12) to RAJI cells.
FIGURE 36 shows inhibition of RAJI cell growth by peptide toxin embedded CDl 9 antibody, clone 2G12. Panel A: High Dose Full length Toxin Embedded Antibodies; Panel B: Low Dose Full length Toxin Embedded Antibodies; Panel C: High Dose Single Chain Toxin Embedded Antibodies; Panel D: Low Dose Single Chain Toxin Embedded Antibodies; and Panel E: Synthetic Free Peptide Toxins.
DETAILED DESCRIPTION 1. Definitions As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art. The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included.
The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.
An "immune cell" refers to those cells critical for immune response in an individual and which are commonly found in the lymphatic system, and in particular, in lymph nodes. Such cells include T cells (or T lymphocytes), B cells (or B lymphocytes), natural killer (NK) cells, macrophages and dendritic cells.
1C235000J 12 An "immunoglobulin" is a tetrameric molecule. In a naturally-occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50- 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)) (incorporated by reference in its entirety for all purposes). IgG, IgA and IgD isotypes have a "hinge region" which is an amino acid sequence of from about 10-60 amino acids that confers flexibility on the immunoglobulin molecule. The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites. Immunoglobulins may be organized into higher order structures. IgA is generally a dimer of two tetramers. IgM is generally a pentamer of five tetramers.
Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. MoI. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
An "antibody" refers to an intact immunoglobulin, or to an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen- binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter
1Q235000J 12 alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
An Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CHl domains; a F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of the VH and CHl domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment (Ward et al., Nature 341 : 544-546, 1989) consists of a VH domain.
A single-chain antibody (scFv) is an antibody in which VL and VH regions are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Diabodies are bivalent or bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak, R. J., et al., Structure 2:1121-1123, 1994). One or more CDRs may be incorporated into a molecule either covalently or noncovalently. A minibody is a bivalent or bispecific antibody in which two scFv monomers are joined by two constant domains (see e.g., Hudson, PJ. and Sourisu, C, Nature Medicine 9: 129-134 (2003)). An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites (e.g., bivalent), a single-chain antibody or Fab fragment may have one or two binding sites, while a "bispecific" or "bifunctional" antibody has two different binding sites. The term "human antibody" includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In a preferred embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). These antibodies may be prepared in a variety of ways, as described below.
10235000J 14 The term "hinge" or "hinge region," as used herein, refers to a region of the heavy chain that comprises amino acid residues 224 to 251 (Kabat numbering scheme). This region encompasses the genetic hinge (e.g., amino acid residues 224- 243 using the Kabat numbering scheme) as well as amino acid residues C-terminal to the genetic hinge that are structurally flexible. Exemplary hinge region sequences include, for example, the hinge regions for human IgG, IgA and IgD isotypes and mouse, rat, guinea pig and rabbit IgG isotypes that are provided in Figure 28 (see also, Burton DR, Molecular Genetics of Immunoglobulin, Chapter 1, Calabi, F. and Neuberger, M.S., eds; Elsevier Science Publishers B.V. (1987)). The hinge region may be divided into three subregions referred to as the upper hinge, the middle hinge, and the lower hinge (Figure 28).
The term "chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. The term "tumor-associated antigen" refers to a polypeptide which is preferably presented by tumor cells and thus allows a distinction between tumor cells and non-tumor cells. Tumor associated antigens are proteins expressed inside or on the surface of tumor cells which are putative targets for immune responses. They often differ from normal cellular counterparts by mutations, deletions, different levels of expression, changes in secondary modifications or expression in other stages of development. The proteins are preferably expressed on the cellular surface and, in addition, presented as processed peptides on the tumor cell surface by MHC class I molecules. Examples of tumor-associated antigens include, for example, CAl 25, CA19-9, CA15-3, D97, gplOO, CD20, CD21, TAG-72, EGF receptor, Epithelial cell adhesion molecule (Ep-CAM), Carcino-embryonic antigen (CEA), Prostate specific antigen (PSA), PMSA, CDCPl, CD26, Hepsin, HGF (hepatocyte growth factor), Met, CAIX(G250), EphhB4 (Ephrin type-B receptor 4), EGFRl, EGFR2, PDGF, VEGFR, DPP6, syndecan 1, IGFBP2 (Human insulin-like growth factor binding protein 2), CD3, CD28, CTL4, VEGF, Her2/Neu receptor, tyrosinase, MAGE 1, MAGE 3, MART, BAGE, TRP-I, CA 50, CA 72-4, MUC 1, NSE (neuron specific enolase), α- fetoprotein (AFP), SSC (squamous cell carcinoma antigen), BRCA-I, BRCA-2 and hCG.
10235000J 25 The term "sequence similarity," with reference to an amino acid sequence, refers to the proportion of amino acid matches plus conservative amino acid substitutions between two amino acid sequences over a window of comparison. The term "percentage of sequence similarity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of matched positions by adding (i) the number of positions at which the identical amino acid occurs in both sequences and (ii) the number of positions at which the sequences contain conservative amino acid substitutions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence similarity.
The term "sequence identity" means that sequences are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino acid basis for polypeptides) over a window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. The term "conserved residue" refers to an amino acid that is a member of a group of amino acids having certain common properties. The term "conservative amino acid substitution" refers to the substitution (conceptually or otherwise) of an amino acid from one such group with a different amino acid from the same group. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure, Springer- Verlag 1990). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of
Protein Structure, Springer- Verlag 1990). One example of a set of amino acid groups defined in this manner include: (i) a charged group, consisting of GIu and Asp, Lys, Arg and His, (ii) a positively-charged group, consisting of Lys, Arg and His, (iii) a negatively-charged group, consisting of GIu and Asp, (iv) an aromatic group,
10235000_l 16 consisting of Phe, Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large aliphatic nonpolar group, consisting of VaI, Leu and He, (vii) a slightly- polar group, consisting of Met and Cys, (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, GIy, Ala, GIu, GIn and Pro, (ix) an aliphatic group consisting of VaI, Leu, lie, Met and Cys, and (x) a small hydroxyl group consisting of Ser and Thr.
A "structural domain" refers to a region of a polypeptide that is folded in such a way as to confer a particular secondary and/or tertiary structure, such as, for example, an alpha helix or beta sheet.
The term "therapeutically effective amount" refers to that amount of an antibody fusion, drug or other molecule which is sufficient to effect treatment when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
2. Antibody Fusion Molecules
In one embodiment, antibody fusions having one or more amino acid sequences incorporated into the constant region of the antibody are provided. Amino acid sequences may be incorporated into the constant region of the antibody at a hydrophobic region, at a region flanked by proteasomal cleavage sites, and/or at a region having amino acid sequence similarity with the amino acid sequence. In exemplary embodiments, an amino acid sequence is incorporated into the constant region at a location that does not affect the epitope binding activity of the antibody and/or that permits the antibody to be expressed and/or secreted at sufficient levels (e.g., levels approaching that of an unmodified antibody molecule). The amino acid sequence incorporated into the antibody is normative to the antibody, e.g., the sequence being incorporated into the antibody is not normally found in the antibody at that location. The amino acid sequence being incorporated into the constant region of an antibody may be from a different location within the same antibody molecule, may be from a different antibody molecule, or may be from a non-antibody molecule. In various embodiments, amino acid sequences to be incorporated into an antibody may be a peptide, a polypeptide, a fragment of a polypeptide, or a fusion between two or
10235000J 17 more peptide sequences. The amino acid sequence may be a naturally occurring sequence, a variant of a naturally occurring sequence, or a synthetic sequence, or combinations thereof.
An amino acid sequence may be incorporated into an antibody molecule by inserting the polypeptide into the antibody (e.g., the amino acid sequence is added to the sequence of the antibody). Alternatively, an amino acid sequence may be incorporated into an antibody by replacing a portion of the antibody sequence with the introduced sequence. When incorporating an amino acid sequence by replacement, the length of the amino acid sequence being introduced may be the same size, larger or smaller than the antibody sequence being replaced (e.g., a sequence of 10 amino acids to be incorporated may replace a region of sequence on the antibody molecule that is 5, 10, or 15 amino acids in length). In an exemplary embodiment, the amino acid sequence is the same length as the region of the antibody sequence being replaced such that the overall size of the antibody molecule is maintained. In certain embodiments, two, three, four, five, six, or more, amino acid sequences may be incorporated into the antibody at one or more locations. The amino acid sequence may be any size, but typically is from about 4-100, 4-50, 4-25, 4-20, 5- 15, 5-10, 5-8, or 8-11 amino acids in length.
In one embodiment, antibody fusions having one or more amino acid sequences incorporated into the constant region of the antibody between proteasomal cleavage sites are provided. The proteasomal cleavage sites may be naturally occurring in the antibody sequence and/or may be introduced into the antibody fusion at one or more desired locations. In one embodiment, an amino acid sequence is incorporated into an antibody constant region between two naturally occurring proteasomal sequences. In another embodiment, an amino acid sequence is incorporated into an antibody constant region between proteasomal cleavage sites that have been introduced (e.g., the proteasomal cleavage sites are not naturally occurring in the antibody sequence). In yet another embodiment, an amino acid sequence may be incorporated into the constant region of an antibody between a combination of a naturally occurring proteasomal cleavage site and an introduced proteasomal cleavage site. In an exemplary embodiment, the proteasomal cleavage sites are located at or directly adjacent to the N-terminus and C-terminus of the amino acid sequence being incorporated into the antibody. For example, one, two, three or more consecutive residues at the N-terminus and/or C-terminus of the amino acid sequence to be
10235000_l Jg incorporated into the antibody may be proteasomal cleavage sites. Alternatively, one, two, three or more residues of the antibody sequence that are located directly next to the N-terminus and/or the C-terminus of the amino acid sequence incorporated into the antibody molecule may be proteasomal cleavage sites. Various combinations thereof are also contemplated within the scope of this disclosure.
In certain embodiments, two or more amino acid sequences may be incorporated into the antibody at the same or different locations. When incorporating more than one amino acid sequence into the antibody at the same location, it may be desirable to have proteasomal cleavage sites flanking each of the incorporated sequences. For example, the antibody fusion may have the structure Ab-X-sequence 1-X-sequence 2-X-Ab, wherein Ab represents the antibody constant region that is being modified by the incorporated sequences, X represents a proteasomal cleavage site, and sequence 1 and sequence 2 represent two amino acid sequences (e.g., having the same or different sequences) being incorporated into the antibody. The proteasomal cleavage sites may be naturally occurring in the antibody or normative to the antibody and may comprise one, two, three or more consecutive proteasomal cleavage sites.
In another embodiment, the antibody fusions may comprise one or more amino acid residues between the proteasomal cleavage sites and the antibody constant region. For example, the antibody fusion may have the structure Ab-N-X-sequence- X-N-Ab, or Ab-N-X-sequence-X-Ab or Ab-X-sequence-X-N-Ab, wherein Ab represents the antibody constant region that is being modified by the incorporated sequences, X represents a proteasomal cleavage site, N represents one or more amino acid residues nonnative to the antibody, and sequence represents the sequence being incorporated into the antibody. In various embodiments, N may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues in length and may be located between the antibody sequence and proteasomal cleavage at one or both ends flanking the amino acid sequence being incorporated into the antibody constant region.
In certain embodiments, a flexible linker may be added to the antibody fusion to reduce structural constraints, increase antibody expression, facilitate proper antibody folding, and/or facilitate formation of a disulfide bond between two antibody chains. Examples of flexible linkers include, for example, linkers comprising about 2 to 50, about 2 to 25, about 2 to 20, about 2 to 15, about 2 to 10, about 5 to 20, about 5 to 15, or about 5 to 10 small amino acid residues, such as alanine, glycine, serine or
10235000_l 19 combinations thereof. Exemplary linkers comprise the amino acid sequence GGGAAG (SEQ ID NO: 129) or GGGAAAGAAG (SEQ ID NO: 130). Such flexible linkers may be incorporated at one or both ends flanking an amino acid sequence incorporated into the antibody. For example, the antibody fusion may have the structure Ab-X-F-sequence-F-X-Ab, Ab-X-sequence-F-X-Ab, Ab-X-F-sequence-X- Ab, Ab-F-X-sequence-X-F-Ab, Ab-X-sequence-X-F-Ab, or Ab-F-X-sequence-X-Ab, wherein Ab represents the antibody constant region that is being modified by the incorporated sequences, X represents a proteasomal cleavage site, F represents a flexible linker sequence, and sequence represents the sequence being incorporated into the antibody. Additionally, such linkers may be incorporated between the C- terminus of a heavy or light antibody chain and an amino acid sequence attached to the C-terminus.
In certain embodiments, the antibody fusions having an amino acid sequence incorporated into the antibody constant region may further comprise at least one amino acid sequence attached to the C-terminus of the heavy and/or light chain constant region. The amino acid sequence may be attached to the C-terminus of the heavy and/or light chain constant region by a peptide or chemical linker. In an exemplary embodiment, the amino acid sequence may be a cytotoxic peptide.
Naturally occurring proteasomal cleavage sites may be determined using computer algorithms. For example, computer algorithms based on 2OS proteasome cleavage motifs have been used to successfully predict proteolytic hot spots in proteins of interest (Saxova, P. et al., Int Immunol 15: 781-787 (2003)). See, e.g., world wide web at cbs.dtu.dk/services/NetChop/. In exemplary embodiments, a peptide is incorporated into an antibody constant region between two naturally occurring proteasomal cleavage sites wherein each site comprises two, three, four, or more, consecutive cleavage sites.
The 2OS proteasome is a 700 kDa cylindrical-shaped multicatalytic protease complex comprised of 28 subunits organized into four rings. In yeast and other eukaryotes, 7 different alpha subunits form the outer rings and 7 different beta subunits comprise the inner rings. The alpha subunits serve as binding sites for the 19S (PA700) and 1 IS (PA28) regulatory complexes, as well as a physical barrier for the inner proteolytic chamber formed by the two beta subunit rings. Thus, in vivo, the proteasome is believed to exist as a 26S particle ("the 26S proteasome"). In vivo experiments have shown that inhibition of the 2OS form of the proteasome can be
10235000J 20 readily correlated to inhibition of 26S proteasome. Cleavage of amino-terminal prosequences of beta subunits during particle formation expose amino-terminal threonine residues, which serve as the catalytic nucleophiles. The subunits responsible for catalytic activity in proteaseome thus possess an amino terminal nucleophilic residue, and these subunits belong to the family of N-terminal nucleophile (Ntn) hydrolases (where the nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, and other nucleophilic moieties). This family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosylasparaginase. In addition to the ubiquitously expressed beta subunits, higher vertebrates also possess three .gamma.-interferon-inducible beta subunits (LMP7, LMP2 and MECLl), which replace their normal counterparts, X, Y and Z respectively, thus altering the catalytic activities of the proteasome. Through the use of different peptide substrates, three major proteolytic activities have been defined for the eukaryote 2OS proteasome: chymotrypsin-like activity (CT-L), which cleaves after large hydrophobic residues; trypsin-like activity (T-L), which cleaves after basic residues; and peptidylglutamyl peptide hydrolyzing activity (PGPH), which cleaves after acidic residues. Two additional less characterized activities have also been ascribed to the proteasome: BrAAP activity, which cleaves after branched- chain amino acids; and SNAAP activity, which cleaves after small neutral amino acids. The major proteasome proteolytic activities appear to be contributed by different catalytic sites, since inhibitors, point mutations in beta subunits and the exchange of gamma interferon-inducing beta subunits alter these activities to various degrees.
Introduced proteasomal cleavage sites may include one or more amino acids that lead to proteasomal cleavage, for example by the eukaryotic 2OS proteasome. Suitable amino acid residues include, for example, large hydrophobic residues (e.g., tyrosine, phenylalanine or tryptophan), basic residues (e.g. lysine, arginine, or histidine), and/or acidic residues (e.g., glutamine or asparagine). Introduced proteasomal cleavage sites may comprise at least one, two, three, four, or more, consecutive amino acids that cause proteasomal cleavage. In an exemplary embodiment, introduced proteasomal cleavage sites include one or more lysine and/or arginine residues, or combinations thereof (e.g., RR, KK, RK, KR, etc.). Proteasomal cleavage sites comprising the peptide sequence RRR or KKK have been shown to
10235000_l 21 serve as good substrates for cleavage by cellular proteasomes (Livingston, B. D. et al., Vaccine 19: 4652-4660 (2001); Sundaram, R. et al., Vaccine 21: 2767-2781 (2003)).
In another embodiment, one or more amino acid sequences may be incorporated into the constant region of the antibody at a hydrophobic region. Hydrophobic regions may be determined, for example, using the Kyte-Doolittle hydrophobicity prediction algorithm (Kyte J. and Doolittle R.F., J. MoI Biol, 157: 105-31 (1982)). Hydrophobic regions suitable for insertion of an amino acid sequence may be from about 4-40, 10-30, 10-20, or 10-15 amino acids in length. In exemplary embodiments, the hydrophobic region is sufficiently large to incorporate the amino acid sequence without affecting, or without significantly affecting, proper expression and folding of the antibody molecule. The hydrophobic regions may be located in a constant domain of an immunoglobulin light chain or a constant domain of an immunoglobulin heavy chain. In one embodiment, the hydrophobic region is located in the CHl, CH2 or CH3 domain of a heavy chain constant region. In an exemplary embodiment, the hydrophobic region also contains one or more proteasomal cleavage sites. Exemplary hydrophobic regions that contain one or more proteasosomal cleavage sites include amino acid residues 135-146, 149-198, 243-259, 271-292, 320-329, 388-400, or 453-464 of the heavy chain constant domain based on the Kabat numbering system. In certain embodiments, the antibody fusions provided herein do not contain an amino acid sequence incorporated into the CHl region. In other embodiments, the antibody fusions provided herein do not contain an amino acid sequence incorporated between residues 146-152, 178-185, and/or 213-216 (Kabat numbering scheme). In another embodiment, one or more amino acid sequences may be incorporated into the constant region of the antibody at a region having amino acid sequence similarity with the sequence being incorporated. In exemplary embodiments, an amino acid sequence may be incorporated into the constant region of the antibody at a region having at least about 40%, 42%, 45%, 47%, 50%, 55%, 60%, 70%, 75%, 80%, 90%, or greater percent similarity, with the sequence being incorporated. The region of sequence similarity may be a hydrophobic region, or part of a hydrophobic region, as described above. In an exemplary embodiment, an amino acid sequence is incorporated into a hydrophobic region having the highest degree of sequence similarity with the sequence being incorporated. The amino acid sequence may be incorporated into the antibody by replacing a region of the antibody having
1023500CM 22 sequence similarity with the sequence to be incorporated or by changing one or more amino acid residues in the antibody sequence in order to convert a portion of the antibody sequence into the sequence to be incorporated. Methods for determining sequence identity and similarity include, for example, computer programs such as the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984); BESTFIT; MegAlign (DNAstar, Madison, WI); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. MoI. Biol. 215: 403-410 (1990), PSI- BLAST (Altschul S. F. et al., Nucleic Acids Res. 25: 389-3402 (1997)), eMatrix software (Wu et al., J. Comp. Biol. 6: 219-235 (1999)), eMotif software (Nevill-
Manning et al, Proc. Int. Conf. Intell. Syst. MoI. Biol. 4: 202-209 (1997)), and pFam software (Sonnhammer et al., Nucleic Acids Res. 26(1): 320-322 (1998)). The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. MoI. Biol. 215:403-410 (1990)).
In certain embodiments, an amino acid sequence may be attached to the C- terminus of the constant region of the heavy or light chain of an antibody. The amino acid sequences are attached to the antibody by a cleavable peptide linker. The peptide linker may comprise for example two, three, four, five, or more, small amino acid residues, such as glycine or serine. Additionally, the linkers may comprise one or more amino acids that cause proteasomal cleavage (typically lysine or arginine) located near or directly adjacent to the N-terminus of the polypeptide. Exemplary linkers include, for example, peptides having the sequence GGXn, GGGXn (SEQ ID NO: 51), GGGGXn (SEQ ID NO: 52), GGGSXn (SEQ ID NO: 53), GGGSGGGSXn (SEQ ID NO: 54), GGGAAGXn (SEQ ID NO: 179) or GGGAAAGAAGXn (SEQ ID NO: 180) wherein X is lysine or arginine and n is 1-5.
In certain embodiments, an amino acid sequence is incorporated into a constant region of the antibody at a region having structural flexibility. Regions of structural flexibility include regions that have poorly defined secondary structure such as a loop, turn, or extended amino acid chains that do not fold into an alpha helix or beta sheet structure. Regions of structural flexibility include regions joining two structural domains such as a region joining two domains having an alpha helix or beta sheet structure. Exemplary regions of structural flexibility contained in an antibody
10235000J 23 constant region include: the region between the variable light (VL) and constant light (CL) domains of the light chain, and the regions between the variable heavy (VH) and CHl domains, CHl and CH2 domains, or CH2 and CH3 domains of the heavy chain. The hinge region of IgG, IgA and IgD isotypes is a further example of a region of structural flexibility.
In an exemplary embodiment, an amino acid sequence is incorporated into or near the hinge region of an antibody. The amino acid sequence may be incorporated into the hinge region itself, at the junction between the N-terminus of the hinge region and the upstream region of the heavy chain, at the junction between the C-terminus of the hinge region and the downstream region of the heavy chain, or at a location starting within about 50, 20, 10, 5, 4, 3, 2 or 1 amino acids upstream from the N- terminus, or about 50, 20, 10, 5, 4, 3, 2 or 1 amino acids downstream from the C-terminus, of the hinge region. In an exemplary embodiment, an amino acid sequence is incorporated between, or adjacent to, residues 224-251 (based on the Kabat numbering scheme) of a heavy chain or into, or adjacent to, one of the sequences shown in Figure 28. In one embodiment, an amino acid sequence is incorporated at the junction between the C-terminus of the hinge region and the downstream region of the heavy chain. The amino acid sequence may optionally be flanked by proteasomal cleavage sites as described herein. In another embodiment, an amino acid sequence is incorporated into the constant domain of an antibody at a region that reduces or disrupts binding to cell surface Fc receptors. One problem that may be encountered with the administration of therapeutic antibodies is the reduction of the effective dose of the antibody due to binding to Fc receptor bearing cells. A significant number of blood cells and a variety of tissues express Fc receptors. As a result, it may be necessary to administer higher doses of an antibody therapeutic leading to higher costs associated with the treatment. Additionally, when the therapeutic antibody relies on the binding specificity of the variable region of the antibody for selective delivery, binding of the antibody nonspecifically to cells expressing Fc receptors may result in an undesired response. Methods for assaying Fc receptor binding are know in the art and are described further in the Exemplification section. In an exemplary embodiment, an antibody fusion having an amino acid sequence incorporated at or around the hinge region has reduced or completely abolished Fc receptor binding activity. In certain embodiments, an antibody fusion having reduced Fc receptor binding activity means
10235000J 24 that the antibody fusion has less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, or less, of Fc receptor binding activity as compared to the same antibody not containing the incorporated amino acid sequence.
In other embodiments, the methods described herein may be used to incorporate amino acid sequences into non-antibody proteins. For example, one or more amino acid sequences may be incorporated into a carrier protein or scaffold protein at regions that are hydrophobic, flanked by proteasomal cleavage sites, and/or that have sequence similarity with the polypeptide to be introduced. Carrier proteins having multiple amino acid sequences introduced may be referred to as multi epitope carrier proteins. In certain embodiments, a multi-epitope carrier protein may be attached to the C-terminus of the constant region of an antibody heavy or light chain by a cleavable peptide linker (or alternatively by chemical conjugation). Specific examples of multi-epitope carrier proteins include disease specific proteins that have been modified to incorporate one or more disease specific epitopes as described further below.
The antibody fusions described herein may utilize a wide variety of antibodies or antibody fragments that bind to a desired target epitope. The target epitope may be selected by one of skill in the art based on a desired application for the antibody fusion. Exemplary applications of the antibody fusions are described further below. Nucleic acid sequences useful for production of the antibody fusions described herein may be obtained from publicly available databases or determined experimentally as described further below.
Antibodies for use in antibody fusions may be IgG, IgM, IgE, IgA or IgD molecules. In a preferred embodiment, the antibody is an IgG, IgD, or IgA molecule that comprises a hinge region. In exemplary embodiments, the antibody fusions described herein may comprise a constant region, or a portion thereof, from any type of antibody isotype, including, for example, IgG (including IgGl, IgG2, IgG3, and IgG4), IgM, IgE, IgA or IgD, or a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (see e.g., Burton DR and Woof JM, Adv. Immun. 51 : 1-18 (1992); Canfield SM and Morrison SL, J. Exp. Med. 173: 1483-1491 (1991); Mueller JP, et al., MoI. Immunol. 34(6): 441-452 (1997)). In an exemplary embodiment, the antibody fusion has a hybrid constant region wherein residues 249 and 250 (based on Kabat numbering) are glycines. For example, the IgGl and IgG4 constant regions contain G249G25o residues whereas the IgG2 constant region does not
10235000J 25 contain residue 249 but does contain G250. In a G2/G4 hybrid constant region, wherein the 249-250 region comes from the G2 sequence, the constant region can be further modified to introduce a glycine residue at position 249 to produce a G2/G4 fusion having G24g/G25o. When using the G2/G4 G24cι/G25o hybrid constant domain, it may be desirable to introduce an amino acid sequence between the G249/G25o residues. Other constant domain hybrids that contain G249/G2so may also be used in accordance with the invention. The class and subclass of antibodies may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA or Western Blot as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.
In certain embodiments, chimeric, humanized or primatized (CDR-grafted) antibodies, antibody fragments, as well as chimeric or CDR-grafted antibody fragments, comprising portions derived from different species, may be used for construction of the antibody fusions described herein. Antibody fragments useful in accordance with the antibody fusions described herein comprise at least a portion of heavy chain and/or light chain constant region. Exemplary antibody fragments include, for example, Fab, Fab2, Fab3 or minibodies, or Fv, scFv, diabodies and triabodies fused to at least a portion of a heavy chain and/or light chain constant region. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; and Winter, European Patent No. 0,239,400 Bl. See also, Newman, R. et al., BioTechnology 10: 1455-1460 (1992), regarding primatized antibody. See, e.g., Ladner et al., U.S. Pat. No. 4,946,778; and Bird, R. E. et al., Science 242: 423-426 (1988)), regarding single chain antibodies.
1023S00Q I 26 In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies comprising at least a portion of a heavy chain and/or light chain constant region, can also be used in association with the antibody fusions described herein. Functional antibody fragments refer to fragments that retain at least one binding function and/or modulation function of the full-length antibody from which they are derived. Preferred functional fragments retain an antigen binding function of a corresponding full-length antibody.
For example, antibody fragments capable of binding to a desired epitope including, but not limited to, Fab, Fab1 and F(ab')2 fragments, or Fv fragments comprising at least a portion of a heavy chain and/or light chain constant region, may be used in association with the antibody fusions. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can be used to generate Fab or F(ab')2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHl domain and hinge region of the heavy chain. A humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids in the framework and constant domains of the heavy and light chains have been mutated so as to avoid or abrogate an immune response in humans. Alternatively, a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293. A humanized antibody may comprise portions of immunoglobulins of different origin, wherein optionally at least one portion is of human origin. Accordingly, a humanized immunoglobulin having binding specificity for a desired epitope, said immunoglobulin comprising an antigen binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region or portion thereof) may be used in association with the antibody fusions described herein. For example, the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity,
10235000_l 27 such as a mouse, and from immunoglobulin sequences of human origin (e.g., a chimeric immunoglobulin), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain).
Another example of a humanized immunoglobulin of the present invention is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin) and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). In one embodiment, the humanized immunoglobulin can compete with murine monoclonal antibody for binding to a desired epitope. Chimeric antibodies or CDR- grafted single chain antibodies comprising at least a portion of a heavy chain and/or light chain constant region are also encompassed by the term humanized immunoglobulin.
The antibody fusion molecules of the present invention are useful in a variety of applications, including research and therapeutic applications as described further below.
3. Applications for Antibody Fusions
The antibody fusions described herein may be used for any method in which targeted delivery of a polypeptide to a desired cell is required.
In one embodiment, the invention provides antibody fusions for modulating an immune response, including both stimulation of an immune response to a desired antigen or tolerization to an antigen. The antibody fusions comprise an antibody specific for a surface protein on an immune cell and a peptide epitope for which immune modulation is desired. The antibody may be an internalizing antibody such that the peptide epitope is delivered internally to the cell. The specificity of the antibody directs the peptide epitope to a desired population of immune cells. The peptide is then internalized along with the antibody, released inside the cell and may be displayed on the surface of the immune cell as part of an MHC complex.
Selective targeting of immune cell modulating peptides to antigen presenting cells of interest can result in either robust activation or suppression of the immune response depending on the cell population being targeted. For example, presentation
10235000_1 28 of peptides by activated dendritic cells (DCs) has been shown to stimulate an immune response (Bonifaz, L. C. et al., J Exp Med 199: 815-824 (2004)). Identification of cell surface receptors, which are expressed exclusively on DCs (e.g., DC-SIGN, DEC205) allows for selective targeting of these specialized antigen-presenting cells via receptor specific antibodies. In cancer or infectious disease, it would be advantageous to deliver peptides from known tumor antigens e.g. PSMA, gplOO, or others described above, to stimulate antigen presenting cells to enhance the immune response against the cancer cells or infectious agents. In contrast, antigen presentation by liver sinusoidal endothelial cells (LSECs) is known to suppress the immune response (Knolle, P. A. et al., Gastroenterology 116: 1428-1440 (1999); Limmer, A. et al., Nat Med 6: 1348-1354 (2000)). Specific receptors such as L-SIGN allow for selective targeting of these specialized tolerizing antigen-presenting cells via receptor-specific antibodies. In the autoimmune setting, it would be advantageous to deliver peptides that are known to be associated with autoimmune responses such as GAD or insulin peptides in diabetes to a tolerizing antigen-presenting cell.
In an exemplary embodiment, antibody fusions for stimulating an immune response are provided. The antibody fusions comprise an antibody that binds to a dendritic cell specific surface protein and a peptide epitope. Exemplary dendritic cell specific surface markers include, for example, CD83, CD205/DEC-205, CD197/CCR7, CD209/DC-SIGN. Exemplary peptide epitopes include, for example, anti-microbial protein epitopes such as fungal, bacterial or viral peptide epitopes that will be useful for stimulating or enhancing an immune response to a pathogen. Other exemplary peptide epitopes include, for example, peptide epitopes useful as cancer vaccine, such as epitopes derived from the tumor associated antigens described herein. In another embodiment, antibody fusions for inducing tolerance are provided. The antibody fusions comprise an antibody that binds to LSEC specific surface protein, such as L-SIGN, and a peptide epitope associated with, for example, an autoimmune disease.
In certain embodiments, methods of modulating an immune response are provided. The methods include stimulation and/or enhancement of an immune response for and methods of treating an individual in need of immune stimulation, e.g., individuals suffering from a pathogen infection, cancer, or other disease state. The methods also include reducing an immune response or inducing tolerance and methods for treating an individual in need of tolerization, e.g., individuals suffering
10235000_l 29 from an autoimmune disorder, transplant patients, patients suffering from allergies, etc. The methods involve administering to the individual a therapeutically effective amount of one or more antibody fusions as described herein. In an exemplary embodiment, the antibody fusion comprises an internalizing antibody that recognizes an immune cell surface protein and a peptide epitope fused to, or embedded in, a constant region of the antibody. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.
In certain embodiments, methods for modulating an immune response may involve a combination therapy with one or more other therapeutic agents such as, for example, anti-inflammatory agents, immunosuppressive agents, and/or anti-infective agents (such as for example, antibiotic, antiviral, and/or antifungal compounds, etc.). Exemplary anti-inflammatory drugs include, for example, steroidal (such as, for example, Cortisol, aldosterone, prednisone, methylprednisone, triamcinolone, dexamethasone, deoxycorticosterone, and fluorocortisol) and non-steroidal anti- inflammatory drugs (such as, for example, ibuprofen, naproxen, and piroxicam).
Exemplary immunosuppressive drugs include, for example, prednisone, azathioprine (Imuran), cyclosporine (Sandimmune, Neoral), rapamycin, antithymocyte globulin, daclizumab, OKT3 and ALG, mycophenolate mofetil (Cellcept) and tacrolimus (Prograf, FK506). Exemplary antibiotics include, for example, sulfa drugs (e.g., sulfanilamide), folic acid analogs (e.g., trimethoprim), beta-lactams (e.g., penicillin, cephalosporins), aminoglycosides (e.g., streptomycin, kanamycin, neomycin, gentamycin), tetracyclines (e.g., chlorotetracycline, oxytetracycline, and doxycycline), macrolides (e.g., erythromycin, azithromycin, and clarithromycin), lincosamides (e.g., clindamycin), streptogramins (e.g., quinupristin and dalfopristin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, and moxifloxacin), polypeptides (e.g., polymixins), rifampin, mupirocin, cycloserine, aminocyclitol (e.g., spectinomycin), glycopeptides (e.g., vancomycin), and oxazolidinones (e.g., linezolid). Exemplary antiviral agents include, for example, vidarabine, acyclovir, gancyclovir, valganciclovir, nucleoside-analog reverse transcriptase inhibitors (e.g., AZT, ddl, ddC, D4T, 3TC), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine, delavirdine), protease inhibitors (e.g., saquinavir, ritonavir, indinavir, nelfinavir), ribavirin, amantadine, rimantadine, relenza, tamiflu, pleconaril, and interferons. Exemplary antifungal drugs include, for example, polyene antifungals (e.g., amphotericin and nystatin), imidazole antifungals (ketoconazole and
10235000_l 30 miconazole), triazole antifungals (e.g., fluconazole and itraconazole), flucytosine, griseofulvin, and terbinafine.
In certain embodiments, methods for stimulating an immune response may involve a combination therapy with one or more immunostimulatory agents such as, for example, an adjuvant. Such combination therapies may be useful as vaccines. Exemplary adjuvants include, for example: synthetic imidazoquinolines such as imiquimod (S-26308, R-837) (Harrison et al., Vaccine 19: 1820-1826 (2001)) and resiquimod (S-28463, R-848) (Vasilakos et al., Cellular Immunology 204: 64-74 (2000)); Schiff bases of carbonyls and amines that are constitutively expressed on antigen presenting cells and T-cell surfaces, such as tucaresol (Rhodes et al., Nature 377: 71-75 (1995)); cytokine, chemokine and co-stimulatory molecules; ThI inducers such as interferon gamma, IL-2, IL-12, IL-15 and IL-18; Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13; other chemokine and co-stimulatory genes such as MCP- 1, MIP-I alpha, MIP-I beta, RANTES, TCA-3, CD80, CD86 and CD40L; other immunostimulatory targeting ligands such as CTLA-4 and L-selectin; apoptosis stimulating proteins and peptides such as Fas; synthetic lipid based adjuvants, such as vaxfectin, (Reyes et al., Vaccine 19: 3778-3786 (2001)), squalene, alpha-tocopherol, polysorbate 80, DOPC and cholesterol; endotoxin (e.g., LPS) (Beutler, B., Current Opinion in Microbiology 3: 23-30 (2000)); CpG oligo- and di-nucleotides (Sato et al., Science 273: 352-354 (1996); Hemmi et al., Nature 408: 740-745, (2000); WO
96/02555; WO 99/33488; U.S. Patent No. 6,008,200; and U.S. Patent No. 5,856,462); other potential ligands that trigger Toll receptors to produce ThI -inducing cytokines, such as synthetic Mycobacterial lipoproteins, Mycobacterial protein pi 9, peptidoglycan, teichoic acid, lipid A, and lipid A derivatives such as monophosphoryl lipid A or 3-de-O-acylated monophosphoryl lipid A; and saponins, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.
In an exemplary embodiment, the antibody fusions provided herein may be used for stimulating an immune response for treating or preventing influenza in a subject, or for treating or ameliorating symptoms associated with influenza. The methods may involve administering a therapeutically effective amount of an antibody fusion as described herein comprising one or more influenza associated epitopes. Exemplary influenza infections that may be treated in accordance with the methods
10235000J 31 provided herein include, for example, influenza types A, B and C. In an exemplary embodiment, the influenza is influenza A, such as, for example: A/PR/8/34 or A/HK/8/68, or selected from HlNl, H2N2, H3N2, H5N1, H9N2, H2N1, H4N6, H6N2, H7N2, H7N3, H4N8, H5N2, H2N3, Hl 1N9, H3N8, H1N2, Hl 1N2, Hl 1N9, H7N7, H2N3, H6N1, H13N6, H7N1, Hl INl, H7N2 and H5N3. In various embodiments, an antibody fusion may be administered substantially contemporaneously with or following infection of the subject, i.e., a therapeutic treatment. In other embodiments, the antibody fusion provides a therapeutic benefit, such as, reducing or decreasing one or more symptoms or complications of influenza infection, virus titer, virus replication or an amount of a viral protein of one or more influenza strains. Symptoms or complications of influenza infection that can be reduced or decreased include, for example, chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache or death. In still another embodiment, a therapeutic benefit includes hastening a subject's recovery from influenza infection. In still other embodiments, an antibody fusion may be administered as part of a combination therapy with an anti-viral agent or one or more agents that inhibit one or more symptoms or complications associated with influenza infection (e.g., chills, fever, cough, sore throat, nasal congestion, body ache, head ache, fatigue, pneumonia, bronchitis, sinus infection or ear infection).
In another embodiment, the invention provides antibody fusions for growth inhibition of a targeted cell population or for targeted cell death. The antibody fusions comprise an antibody specific for a surface protein on a cell population that is to be targeted for cell killing or growth inhibition and a cytotoxic peptide or growth inhibitory peptide. The antibody may be an internalizing antibody such that the cytotoxic peptide or growth inhibitory peptide is delivered internally to the cell. The antibody may also be targeted to an internalizing receptor on the cell surface to facilitate uptake of the antibody into the cell. The specificity of the antibody directs the cytotoxic peptide or growth inhibitory peptide to the desired population of cells. The peptide is then internalized along with the antibody and released inside the cell resulting in cell death or growth inhibition.
In one embodiment, antibody fusions comprising an antibody that binds to a tumor specific antigen and a cytotoxic peptide are provided. Such antibody fusions are useful for targeted destruction of tumor cells and the treatment of cancer.
1Q235000 1 32 Tumor associated antigens can be identified experimentally or may be selected from a database. Databases that identify molecules that are expressed or upregulated by cancer cells include, for example, the NCI60 microarray project (see e.g., Ross et al., Nature Genetics 24: 227-34 (2000); world wide web at genome-www.stanford.edu/ nci60/), the carcinoma classification (see e.g., A. Su et al., Cancer Research 61: 7388-7393 (2001); world wide web at gnf.org/cancer/epican), and the Lymphoma/Leukemia molecular profiling project (see e.g., Alizadeh et al., Nature 403: 503-11 (2000); world wide web at llmpp.nih.gov/lymphoma/). Experimental methods useful for identifying molecules that are expressed or upregulated by cancer cells include, for example, microarray experiments, quantitative PCR, FACS and Northern analysis.
Exemplary tumor associated antigens include the following: gplOO, tyrosinase, MAGE-I, MAGE-3, MART, BAGE, and TRP-I which are associated with melanoma; CEA (carcino embryonic antigen), CA 19-9, CA 50, and CA 72-4 which are associated with stomach cancer; CEA, CAl 9-9, and Muc-1 which are associated with colon cancer; CA 19-9, Ca-50, and CEA which are associated with pancreas carcinoma; CEA, NSE (neuron specific enolase), and EGF-receptor which are associated with small cell lung cancer; CEA which is associated with lung cancer; α- fetoprotein (AFP) which is associated with liver carcinoma; PSA, PMSA, CDCPl, CD26, Hepsin, HGF (hepatocyte growth factor), Met, CAIX(G250), EphhB4 (Ephrin type-B receptor 4), EGFRl, EGFR2, PDGF, VEGFR, DPP6, syndecan 1, IGFBP2 (Human insulin-like growth factor binding protein 2), CD3, CD28, CTL4, and VEGF which are associated with prostate cancer; CA 19-9 which is associated with gall bladder cancer; SSC (squamous cell carcinoma antigen) which is associated with squamous cell carcinoma; CEA CA 15-3, CEA, BRCA-I, BRCA-2, Muc-1, and Her2/Neu receptor which are associated with mammary carcinoma; AFP and hCG which are associated with testes cancer; CA-125, CEA, CA 15-3, AFP, and TAG-72 which are associated with ovarial carcinoma; and CD 19, CD20 and CD21 which are associated with B cell lymphoma. Antibodies directed to a desired tumor associated antigen may be produced experimentally or selected from a publicly available database as described further herein.
10235000_l 33 Exemplary cytotoxic peptides that may be used in association with the antibody-cytotoxic peptide fusions described herein include, for example, anthrax lethal factor, Diphtheria toxin A fragment, nonbinding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sacrin, certain Aleurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phomycin, and neomycin, or fragments thereof. Yet other cytotoxic polypeptides are described in U.S. Patent Publication Nos. 2005/0191294 and 2004/0192889.
Peptides that cause mitochondria dependent cell-free apoptosis may also be used as cytotoxic peptides in accordance with the antibody fusions described herein. A number of pro-apoptotic peptides have been described that remain relatively nontoxic outside of eukaryotic cell membranes but bind to mitochondrial membranes inside the cells and induce their swelling and cause mitochondrial dependent cell-free apoptosis (Ellerby, H. M. et al., J NeWosci 17: 6165-6178 (1997); Mehlen, P. et al., ■ Nature 395: 801-804 (1998)). An example of a well-characterized pro-apoptotic peptide successfully utilized for selectively killing malignant hematopoietic cells and cells lining tumor blood vessels is a 14-amino-acid amphipathic peptide KLAKLAKKLAKLAK (SEQ ID NO: 55) (Ellerby, H. M. et al., Nat Med 5: 1032- 1038 (1999); Marks, AJ. et al., Cancer Res 65: 2373-2377 (2005)). This peptide contains cationic lysine (K) residues on one side of the amphipathic helix and hydrophobic (Leu-Ala) residues on the other side. This design allows the cationic amino acids to react with the head groups of the anionic phospholipids on the outer leaflet of mitochondrial membranes and the amphipathic helices distort the lipid matrix resulting in the loss of membrane barrier function (de Kroon, A. I. et al., Biochim Biophys Acta 1325: 108-116 (1997)). In contrast, this amphipathic peptide has no toxic effect on the outer plasma membranes of the cells as eukaryotic cell membranes generally have low membrane potentials and are almost exclusively composed of zwitterionic phospholipids (de Kroon, A. I. et al., Biochim Biophys Acta 1325: 108-116 (1997); Hovius, R. et al., FEBS Lett 330: 71-76 (1993)). There are a number of naturally occurring antibacterial peptides that are toxic to bacteria and mitochondria due to their common ancestral membrane structures but have no effect
iO235ooo_i 34 on eukaryotic cell membranes (Bessalle, R. et al., FEBS Lett 274: 151-155 (1990); Blondelle, S.E., and Houghten, R.A. Biochemistry 31: 12688-12694 (1992); Javadpour, M. M. et al., J Med Chem 39: 3107-3113 (1996)).
In certain embodiments, methods of inhibiting or reducing tumor growth and methods of treating an individual suffering from cancer are provided. The methods involve administering to the individual a therapeutically effective amount of one or more antibody fusions as described herein. In an exemplary embodiment, the antibody fusion comprises an internalizing antibody that recognizes a cancer cell surface protein and a peptide toxin fused to, embedded in, a constant region of the antibody. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.
Antibody fusions of the present invention may be useful for treating or preventing a cancer (tumor), including, but not limited to, colon carcinoma, breast cancer, mesothelioma, prostate cancer, bladder cancer, squamous cell carcinoma of the head and neck (HNSCC), Kaposi's sarcoma, glioblastoma, astrocytoma, lymphoma, lung carcinoma, melanoma, renal cancer, and leukemia.
In certain embodiments, one or more antibody fusions can be administered together (simultaneously) or at different times (sequentially). In addition, the antibody fusions can be administered with another agent for treating cancer or for inhibiting angiogenesis. In a specific embodiment, the subject antibody fusions can also be used with other antibody therapeutics (monoclonal or polyclonal).
In certain embodiments, the subject antibody fusions can be used alone. Alternatively, the subject antibody fusions may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present invention recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of one or more antibody fusions of the invention.
A wide array of conventional compounds have been shown to have antineoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies.
10235000_l 35 Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.
When a subject antibody fusion is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such combination therapy may be shown to enhance the therapeutic effect of either agent alone. For example, an antibody fusion may enhance the therapeutic effect of the anti-neoplastic agent or treatment with the antibody fusion may help to overcome cellular resistance to anti-neoplastic agents. This may allow a decreased dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or may restore the effectiveness of an anti-neoplastic agent in resistant cells. Similarly, anti-neoplastic agents may enhance the efficacy of an antibody fusion by rendering cells more susceptible to cytotoxic T cell killing or by enhancing the levels of available cancer antigens.
Pharmaceutical compounds that may be used for combinatory anti-tumor therapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.
10235000_1 3g These chemotherapeutic anti-tumor compounds may be categorized by their mechanism of action into groups, including, for example, the following: anti- metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes - dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); immunomodulatory agents (thalidomide and analogs thereof such as lenalidomide (Revlimid, CC-5013) and CC-
10235000_1 37 4047 (Actimid)), cyclophosphamide; anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disrupters.
In certain embodiments, pharmaceutical compounds that may be used for combinatory anti-angiogenesis therapy include: (1) inhibitors of release of "angiogenic molecules," such as bFGF (basic fibroblast growth factor); (2) neutralizers of angiogenic molecules, such as anti-jSbFGF antibodies; and (3) inhibitors of endothelial cell response to angiogenic stimuli, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D- penicillamine and gold thiomalate, vitamin D3 analogs, alpha-interferon, and the like. For additional proposed inhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab. Invest, 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6573256. In addition, there are a wide variety of compounds that can be used to inhibit angiogenesis, for example, peptides or agents that block the VEGF- mediated angiogenesis pathway, endostatin protein or derivatives, lysine binding fragments of angiostatin, melanin or melanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen), troponin subunits, antagonists of vitronectin O!v/33, peptides derived from Saposin B, antibiotics or analogs (e.g., tetracycline, or neomycin), dienogest-containing compositions, compounds comprising a MetAP-2 inhibitory core coupled to a peptide, the compound EM-138, chalcone and its analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos. 6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.
1023500O-1 3g In another embodiment, antibody fusions comprising an antibody that binds to a cell surface protein on an immune cell and a cytotoxic peptide are provided. Such antibody fusions are useful for targeted destruction of immune cells involved in an unwanted immune response, such as, for example, immune responses associated with an autoimmune disorder, transplants, allergies, and inflammatory disorders.
Exemplary autoimmune diseases and disorders that may be treated with the antibody fusions provided herein include, for example: (1) treatment of rheumatoid arthritis by targeting dendritic cells with anti-DC-SIGN-toxin conjugates (see e.g., van Lent P.L. et al., Arthritis Rheum. 48: 360-9 (2003)); (2) treatment of type 1 diabetes, rheumatoid arthritis, autoimmune thyroid diseases, Graves disease, Hashimoto's thyroiditis, systemic lupus erythematosus, multiple sclerosis, Crohn's disease, psoriasis, psoriatic arthritis, sympathetic ophthalmitis, autoimmune neuropathies, autoimmune oophoritis, autoimmune orchitis, autoimmune lymphoproliferative syndrome, antiphospholipid syndrome, Sjogren's syndrome, scleroderma, lupus, Addison's disease, polyendocrine deficiency syndrome (types 1 and 2), Guillain-Barre syndrome, immune thrombocytopenic purpura, pernicious anemia, myasthenia gravis, primary biliary cirrhosis, mixed connective tissue disease, primary glomerulonephritis, vitiligo, autoimmune uveitis, autoimmune hemolytica, autoimmune thrombocytopenia, celiac disease, dermatitis herpetiformis, autoimmune hepatitis, pemphigus (including pemphigus vulgaris and pemphigus foliaceus), bullous pemphigoid, autoimmune myocarditis, autoimmune vasculitis, autoimmune eye diseases, alopecia areata, autoimmune atherosclerosis, Behcet's disease, autoimmune myelopathy, autoimmune hemophilia, autoimmune interstitial cystitis, autoimmune diabetes insipidus, autoimmune endometriosis, relapsing polychondritis, ankylosing spondylitis, autoimmune urticaria, paraneoplastic autoimmune syndromes, dermatomyositis, Miller Fisher syndrome, IgA nephropathy, Goodpasture syndrome, and herpes gestationis, by targeting B cells with anti-CD32-toxin conjugates; (3) treatment of the autoimmune diseases listed above as well as inflammatory conditions by targeting T cells with anti-CD3 -toxin conjugates; and (4) treatment or prevention of allergies by targeting mast cells with anti-FcεRl -toxin conjugates.
Antibodies directed to a desired immune cell surface protein may be produced experimentally or selected from a publicly available database as described further herein. Exemplary cytotoxic peptides that may be used in association with the
10235000 1 39 antibody-cytotoxic peptide fusions for treatment of autoimmune disorders are described above. Targeted killing of certain populations of immune cells for treating or preventing autoimmune disorders, enhancing or extending transplant survival, treating or preventing allergies, or treating or preventing inflammatory disorders, may be administered as part of a combination therapy with one or more therapeutic agent such as, for example, anti-inflammatory agents, immunosuppressive agents, and/or anti-infective agents as described further herein.
Depending on the nature of the combinatory therapy, administration of the antibody fusions of the invention may be continued while the other therapy is being administered and/or thereafter. Administration of the antibody fusions may be made in a single dose, or in multiple doses. In some instances, administration of the antibody fusions is commenced at least several days prior to the conventional therapy, while in other instances, administration is begun either immediately before or at the time of the administration of the conventional therapy. In other embodiments, antibody fusions comprising polypeptides useful for modulating cell growth and/or differentiation and/or motility are provided. Suitable polypeptides, or fragments thereof, which may be used in accordance with the antibody fusions described herein include, for example, chemotactic polypeptides, growth factors, cytokines, morphogenesis factors, cell signalling factors, cell differentiation factors, polypeptides which stimulate or suppress cell division, and polypeptides which modulate the rate of cell division. Specific examples of polypeptides may be found, for example in U.S. Patent Publication No. 2005/0136042. Also provided are methods for modulating cell growth and/or differentiation, or for treating an individual in need of modulation of cell growth and/or differentiation. The methods involve administering to the individual a therapeutically effective amount of one or more antibody fusions as described herein. In an exemplary embodiment, the antibody fusion comprises an antibody that recognizes a cell surface protein on a cell in which growth, differentiation, Or motility modulation is desired, and a polypeptide fused to, or embedded in, a constant region of the antibody. The polypeptide may be a polypeptide that modulates cell growth, differentiation, and/or motility. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.
102350Q0_l 40 In an exemplary embodiment, antibody fusions comprising growth inhbitory polypeptides, such as tumor suppressors, are provided. Examples of tumor suppressors include, for example, p21, p53, BRCAl, BRCA2, APC, and RBl.
In another embodiment, disease specific multi-epitope carrier proteins and antibody fusions comprising disease specific multi-epitope carrier proteins are provided. Disease specific carrier proteins refer to proteins that are associated with a disease state and which may be used for incorporating other disease epitopes into the protein. Methods for designing and constructing disease specific carrier proteins are described in detail in the exemplification. Examples of disease specific carrier proteins include, for example, polypeptides associated with either Type 1 diabetes mellitus (TlDM) or insulin dependent diabetes mellitus (IDDM) such as glutamic- acid decarboxylase 65 (GAD65), heat shock protein 60 (HSP60), insulinoma associated protein 2 (IA-2) and proinsulin (PI)) or polypeptides associated with cancer such as: gplOO and Tyrosinase associated with Melanoma, Late Membrane Protein 2 (LMP-2) associated with Lymphoma and Carcinoembryonic Antigen associated with various types of adenocarcinomas. The carrier proteins may be modified to incorporate antigens from other disease specific proteins such that a strong, multifactorial immune response or tolerization effect may be achieved. The invention also provides methods for modulating an immune response, or treating an individual in need of immune response modulation by administering to a patient a disease specific multi-epitope carrier protein, optionally fused to, or embedded in, an antibody. In various embodiments, the disease specific multi-epitope carrier proteins may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disease specific epitopes from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, disease specific proteins. In one embodiment, a disease specific multi-epitope carrier protein may comprise two or more disease specific epitopes from a single protein incorporated into a carrier protein that does not naturally contain the epitopes.
In certain embodiments, the antibody fusions described herein may comprise an amino acid sequence that increases the serum half life of the antibody fusion. Examples of polypeptides that may extend the serum-half life of the antibody fusions include, for example, albumin (see e.g., U.S. Patent Nos. 5,876,969 and 5,766,88) and transferrin (see e.g., U.S. Patent Publication No. 2003/0226155), or functional fragments thereof.
!0235000_1 41 In certain embodiments, the antibody fusions described herein may comprise an amino acid sequence that increases or enhances transport across a cellular membrane. A number of peptide based cellular transporters have been developed by several research groups. These peptides are capable of crossing cellular membranes in vitro and in vivo with high efficiency. Examples of such fusogenic peptides include a 16-amino acid fragment of the homeodomain of ANTENNAPEDIA, a Drosophila transcription factor (Wang et al., PNAS USA. 92: 3318-3322 (1995)); a 17-mer fragment representing the hydrophobic region of the signal sequence of Kaposi fibroblast growth factor with or without NLS domain (Antopolsky et al., Bioconj. Chem. 10: 598-606 (1999)); a 17-mer signal peptide sequence of caiman crocodylus Ig(5) light chain (Chaloin et al., Biochem. Biophys. Res. Comm. 243: 601-608 (1997)); a 17-amino acid fusion sequence of HIV envelope glycoprotein gp4114 (Morris et al., Nucleic Acids Res. 25: 2730-2736 (1997)); the HIV-I Tat49-57 fragment (Schwarze et al., Science 285: 1569-1572 (1999)); a transportan A- achimeric 27-mer consisting of N-terminal fragment of neuropeptide galanine and membrane interacting wasp venom peptide mastoporan (Lindgren et al., Bioconjugate Chem. 11 : 619-626 (2000)); and a 24-mer derived from influenza virus hemagglutinin envelop glycoprotein (Bongartz et al., Nucleic Acids Res. 22: 4681-4688 (1994)). Also encompassed within the scope of the invention are nucleic acids encoding antibody fusions, expression vectors comprising nucleic acids encoding the antibody fusions, and host cells comprising expression vectors for producing the antibody fusions.
4. Methods of Antibody Production Antibodies useful for production of the antibody fusions described herein may be designed to bind to a desired epitope or may be selected from publicly available sources of known antibodies. For example, databases of antibody sequences may be found on the world wide web at imgt.cines.fr. Nucleic acid sequences encoding an antibody may be manipulated to incorporate one or more sequences encoding a polypeptide using standard recombinant DNA techniques. The nucleic acid sequences encoding the antibody fusions may be introduced into an expression vector and a suitable host cell for expression of the antibody fusion molecule as described further below.
1Q235OOO_1 42 Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed as described herein, or using other suitable techniques. A variety of methods have been described. See e.g., Kohler et al., Nature, 256: 495- 497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550- 552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991). Generally, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells. The antibody producing cells, preferably those of the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select recombinant antibody from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies. See e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani et al., U.S. Pat. No. 5,545,807.
To illustrate, immunogens derived from a polypeptide of interest can be used to immunize a mammal, such as a mouse, a hamster or rabbit. See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of a polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
Following immunization of an animal with an antigenic preparation of a polypeptide, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells
10235000_1 43 (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a desired polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
In certain embodiments, antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
In certain embodiments, antibodies described herein are further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for a polypeptide of interest conferred by at least one CDR region of the antibody. Techniques for the production of a light chain or heavy chain dimers, or any minimal fragment thereof such as an Fv or a single chain (scFv) construct are described, for example, in US Patent No. 4,946,778. Also, transgenic mice or other organisms including other mammals may be used to express humanized antibodies. Methods of generating these antibodies are known in the art. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023; Queen et al., European Patent No. 0,451,216; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400; Padlan, E. A. et al., European Patent Application No. 0,519,596 Al. See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)).
Such humanized immunoglobulins can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain. For example, nucleic acid (e.g., DNA) sequences coding for
1O235OOOj 44 humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).
A method for generating a monoclonal antibody that binds specifically to a desired polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the polypeptide in an amount effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody- producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the polypeptide. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma- derived cells produce the monoclonal antibody that binds specifically to polypeptide. The monoclonal antibody may be purified from the cell culture.
In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, an antibody to be used for certain therapeutic purposes will preferably be able to target a particular cell type. Accordingly, to obtain antibodies of this type, it may be desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g., by fluorescence activated cell sorting). Likewise, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding. A variety of different techniques are available for testing antibody: antigen interactions to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays and immunohistochemistry.
10235000J 45 In certain embodiments, the hybridoma cell lines, as well as the monoclonal antibodies produced by these hybridoma cell lines, are provided. The cell lines have uses other than for the production of the monoclonal antibodies. For example, the cell lines can be fused with other cells (such as suitably drug-marked human myeloma, mouse myeloma, human-mouse heteromyeloma or human lymphoblastoid cells) to produce additional hybridomas, and thus provide for the transfer of the genes encoding the monoclonal antibodies.
In addition, the hybridoma cell lines can be used as a source of nucleic acids encoding the immunoglobulin chains, which can be isolated and expressed (e.g., upon transfer to other cells using any suitable technique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter, U.S. Pat. No. 5,225,539)). For instance, clones comprising a rearranged light or heavy chain can be isolated (e.g., by PCR) or cDNA libraries can be prepared from mRNA isolated from the cell lines, and cDNA clones encoding a desired immunoglobulin chain can be isolated. Thus, nucleic acids encoding the heavy and/or light chains of the antibodies, or portions thereof, can be obtained and used in accordance with recombinant DNA techniques for the production of the specific immunoglobulin, immunoglobulin chain, or variants thereof (e.g., humanized immunoglobulins) in a variety of host cells or in an in vitro translation system. For example, the nucleic acids, including cDNAs, or derivatives thereof encoding variants such as a humanized immunoglobulin or immunoglobulin chain, can be placed into suitable prokaryotic or eukaryotic vectors (e.g., expression vectors) and introduced into a suitable host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the host cell genome). For production, host cells can be maintained under conditions suitable for expression (e.g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.), whereby the encoded polypeptide is produced. If desired, the encoded protein can be recovered and/or isolated (e.g., from the host cells or medium). It will be appreciated that the method of production encompasses expression in a host cell of a transgenic animal (see e.g., WO 92/03918, GenPharm International, published Mar. 19, 1992).
Antibodies can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
10235000_l 46 In a particular embodiment, such phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and Ml 3. The antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24: 952-958 (1994); Persic et al., Gene 187: 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab1 and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques, 12(6):864-869, 1992; and Sawai et al., Am. J. Reprod. Immunol., 34:26-34, 1995; and Better et al., Science, 240:1041-1043, 1988 (each of which is incorporated by reference in its entirety). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999, 1993; and Skerra et al., Science, 240:1038-1040, 1988.
Polynucleotides encoding antibodies having a desired binding specificity may be obtained by any method known in the art. The nucleotide sequence of antibodies
10235000_l 47 immunospecific for a desired antigen can be obtained, for example, as described above, from the literature or from a database such as GenBank. Polynucleotides encoding an antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17: 242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR. Alternatively, a polynucleotide encoding an antibody may be produced from a cDNA library obtained from a tissue or cell expressing the antibody such as a hybridoma cell line selected to express an antibody. The desired antibody genes may be isolated from the library by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art. Once a polynucleotide encoding an antibody has been obtained, standard recombinant DNA techniques may be used to incorporate a nucleic acid sequence encoding a polypeptide into the antibody sequence at a desired location.
Once a nucleic acid sequence encoding an antibody fusion has been obtained, the vector for the production of the antibody fusion may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the antibody fusion coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. and Ausubel et al. eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
An expression vector comprising the nucleotide sequence of an antibody fusion can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody fusion. In specific embodiments, the expression of the antibody fusion is regulated by a constitutive promoter, an inducible promoter, or a tissue specific promoter.
10235000 1 48 The host cells used to express the recombinant antibody fusions may be either bacterial cells (such as Escherichia coli) or eukaryotic cells. Eukaryotic cells may be particularly useful for the expression of antibody fusion comprising a whole recombinant immunoglobulin molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 1998, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
A variety of host-expression vector systems may be utilized to express the antibody fusions described herein. Such host-expression systems represent vehicles by which the coding sequences of the antibody fusions may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody fusions in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces or Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphatic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (rat retinal cells developed by Crucell)) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Methods for making antibodies in plants, yeast or fungi/algae that are applicable to the production of the antibody fusions described herein are described, for example, in U.S. Patent Nos. 6,046,037 and 5,959,177 and U.S. Patent Publication Nos. 2005/0037420 and 2005/0138692.
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody fusion being expressed. For
10235000J 49 example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody fusion, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), in which the antibody fusion coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody fusion coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody fusion coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (see e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody fusion coding sequences.
These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and
10235000_l 50 synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153: 51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeIa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express an antibody fusion may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody fusions described herein. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 : 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48: 202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
10235000J 51 Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77: 357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIB TECH 11: 155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. MoI. Biol. 150:1.
The expression levels of an antibody fusion described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody fusion is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody fusion, production of the antibody fusion will also increase (Crouse et al., 1983, MoI. Cell. Biol. 3:257). The host cell may be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
10235000_l 52 Once the antibody fusion of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
5. Pharmaceutical Compositions
The invention provides methods and pharmaceutical compositions comprising antibody fusions of the invention. The invention also provides methods of treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of an antibody fusion, or a pharmaceutical composition comprising an antibody fusion. In one embodiment, an antibody fusion is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects). Subjects that may be treated with an antibody fusion described herein include, for example, an animal, such as a mammal including non-primates (e.g. cows, pigs, horses, cats, dogs, rats etc.) and primates (e.g., monkey, such as a cynomolgous monkey, and a human). In an exemplary embodiment, the subject is a human. In certain embodiments, the antibody fusion molecules described may be formulated with a pharmaceutically acceptable carrier. Such antibody fusions can be administered alone or as a component of a pharmaceutical formulation (composition). The antibody fusions may be formulated for administration in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Formulations of the subject antibody fusions include those suitable for oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), rectal, and/or intravaginal administration. Other suitable methods of administration can also include rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions described herein can
10235000_l 53 also be administered as part of a combinatorial therapy with other agents (either in the same formulation or in a separate formulation).
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. In certain embodiments, methods of preparing these formulations or compositions include combining another type of anti-tumor or anti-angiogenesis agent and a carrier and, optionally, one or more accessory ingredients. In general, the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product. Formulations for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of one or more subject antibody fusions as an active ingredient.
In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more antibody fusions of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl
1023500O-1 54 sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifϊers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Methods of the invention can be administered topically, either to skin or to mucosal membranes such as those on the cervix and vagina. This offers the greatest opportunity for direct delivery to the tumor with the lowest chance of inducing side effects. The topical formulations may further include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The
10235000_l 55 subject antibody fusions may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an antibody fusion, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to an antibody fusion, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Pharmaceutical compositions suitable for parenteral administration may comprise one or more antibody fusions in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
10235000J 56 Injectable depot forms are made by forming microencapsule matrices of one or more antibody fusions in biodegradable polymers such as polylactide- polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
Formulations for intravaginal or rectal administration may be presented as a suppository, which may be prepared by mixing one or more antibody fusions of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
EXEMPLIFICATION
The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention in any way. EXAMPLE 1: Computer aided analysis of immunoglobulin sequences for the identification of proteasomal cleavage hot spots and hydrophobic areas
Several peptide grafted antibodies having a peptide incorporated into a hydrophobic region and flanked by proteasomal cleavage sites were constructed and tested to determine the expression profile, antigen binding activity, activation of T- cell response and other characteristics.
To select suitable sites for the insertion of peptide antigens into the antibody constant region, computer-aided analysis of the constant region of IgG2G4 and IgGl were carried out to identify 2OS proteasome cleavage hot spots. Additionally, as many peptide epitopes are very hydrophobic, the immunoglobulin sequences were also analyzed to locate areas of strong hydrophobicity. The results of this analysis are summarized in Figures 1 and 2. A total of seven areas were found to be strongly hydrophobic (highlighted in grey) in each protein. Five of the seven hydrophobic patches contain multiple cleavage sites (designated by the letter "S" under the Ig sequence) for the 2OS proteasome with at least 2 adjacent cleavage sites (e.g., a 2OS
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proteasome cleavage hot spot). These five areas are spread over the entire protein, one in the constant heavy (CH) 1 domain and two each in the CH2 and CH3 domains. Insertion of the hydrophobic peptide antigens into one of these five sites may permit efficient processing and productive presentation in the context of MHC molecules on the cell surface. In addition, as these same five sites are also strongly hydrophobic, an antibody-peptide construct with an inserted hydrophobic peptide epitope may be less likely to fold improperly and may be expected to produce well. To further decrease the structure perturbing effects of introduced hydrophobic peptide epitopes, the hydrophobic patch that most closely matches with the peptide epitope sequence may be selected for grafting the peptide into the antibody constant region. Likewise, if the sequence of the peptide epitope matches with a hydrophobic patch that does not contain proteasomal cleavage sites, it is possible to artificially insert such sites in the form of arginines or lysines on either end of the peptide epitopes. Arginines and lysines have been shown to serve as very efficient substrates for proteasomal cleavage and have been used to facilitate release of MHC class I peptide antigens from multi- epitope vaccines (Livingston, B. D. et al., Vaccine 19: 4652-4660 (2001); Sundaram, R. et al., Vaccine 21 : 2767-2781 (2003)). Based on these design principles, various examples are outlined below for the production of peptide epitope grafted antibodies using the IgG2G4 constant region as an example. These design principles are more generally applicable for inserting peptide epitopes into constant regions of other human immunoglobulins as the amino acid sequences of the various antibody isotypes resemble each other very closely. As an example, sequence similarities between constant regions of IgG2G4 and IgGl are presented in Figure 3. EXAMPLE 2: Screening for cellular responses to tetanus toxin peptides To address the possibility of delivering peptide antigens to antigen presenting cells via antibodies, proof of principle studies were performed using peptides from tetanus toxin (TT) (outlined in table 1 and table 2 below) and an antibody cross- reactive with both DC-SIGN and L-SIGN receptors. Peptides outlined in Table 2 were first screened for immunological responses in humans. Four volunteers underwent vaccination with tetanus toxoid, their antibody responses to tetanus toxoid protein and cellular responses to HLA-DR binding helper T-cell epitopes (listed in Table 2) were evaluated. Two weeks after the vaccination, all four donors exhibited substantial levels of antibodies to TT protein (see Figure 4A). In contrast, only two of the four donors (#5 and #13) showed significant levels (P<0.05 vs. medium) of T-cell
10235000J 53 T7US2006/041215 responses to the TT protein as measured by a five-day H-thymidine based proliferation assay (see Figure 4B). Furthermore, the same two donors demonstrated proliferative responses to TT peptides as well. While peptide 947DR activated T-cell responses in both donor 13 and donor 5 (P< 0.01 vs. medium), peptide 632DR activated T-cell proliferation in donor 13 alone (P< 0.01 vs. medium, see Figure 4B). Table 1. Human Class I binding peptide epitopes selected based on computer- aided analysis of tetanus toxin (TT) protein sequence (Parker, K.C. et al., J Immunol 152: 163-175 (1994)).
Figure imgf000060_0001
Table 2. Universal tetanus toxin peptide epitopes known to bind human class II DRB allele (Diethelm-Okita, B. M. et al., J Infect Dis 181 : 1001-1009 (2000); Valmori, D. et al., J Immunol 152: 2921-2929 (1994)).
Figure imgf000060_0002
EXAMPLE 3: Design, expression and binding properties of peptide embedded antibodies produced by insertion and sequence similarity replacement methods
Of the three HLA-DR binding TT peptides shown in Table 2, peptide 632DR had the closest sequence similarity to a portion of the human IgGl amino acid sequence, which allows for sequence similarity replacement of the peptide into the immunoglobulin sequence. Therefore, peptide 632DR was grafted into a DC-SIGN/L- SIGN reactive antibody (clone ElO) using two different strategies, namely, sequence similarity replacement (described further below) and insertion with flanking proteasomal cleavage sites (described further below) as shown in Figures 5A-B. The 632DR peptide was inserted into the hydrophobic patch right after the hinge region
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(see Figure 5B) based on the flexibility of this region. As hinge regions are considered to be highly flexible, insertion of peptides into this region may not disrupt the tertiary structure significantly, thereby allowing for good expression of the peptide-inserted antibody. Determination of sequence similarity
Peptide sequences were compared with the amino acid sequences of various antibodies using the MegAlign program from DNAstar, which includes a number of different alignment programs and algorithms. The dotplot option for aligning pairs of sequences was used in a first examination of the sequences. By setting the window size to equal that of the peptide, for small peptides, and the percent match at various values, a number of matches were obtained that had identical amino acids aligned within the comparison. The percent match may be set, for example, at 30% or greater. For a variety of HLA supertype epitopes of TT, which are peptides of 9 amino acids, identities of 3, 4, and sometimes 5 amino acids were found. These were then further examined using a knowledge of the similarities among various amino acids in terms of polar, non-polar, acidic, basic, hydrophobic, and hydrophilic properties to identify amino acids that appeared to be conserved residues. Alternatively, alignment of a small peptide with the Clustal W algorithm in MegAlign can be performed by setting the gap penalty on the pairwise alignment parameters to 100 and the gap length to 0. Alternatively, Clustal V can also be set with a gap penalty of 100 and used for analysis. Both of these programs identify one best alignment, whereas the DotPlot approach may identify several possible alignments, including the best from the other two programs, that bear further examination for conserved amino acids. The Lipman Pearson algorithm in MegAlign can also be used by adjusting the K-tuple to 1, and increasing the gap length penalty to 50. This particular algorithm also denotes conserved amino acids in its output. Incorporation of the peptide epitopes into the antibody by overlap extension method
Two separate clones were constructed using overlap PCR to embed the TT helper epitope 632DR into different locations in the L-SIGN/DC-SIGN reactive chimeric antibody ElO-IgG. In the first clone (the insertion clone), the TT epitope was inserted into the CH2 domain between Gycines 249 and 250 (Kabat numbering). Two arginine residues were placed upstream of the 632DR epitope and three arginine residues were placed downstream of the 632DR epitope to give a final insertion
10235000_l gQ peptide of 25 total amino acids residues having the sequence: RRIDKISDVSTIVPYIGPALNIRRR (SEQ ID NO: 63).
For construction of the insertion clone by overlap PCR, the segment of the ElO antibody containing the insertion was amplified as two fragments (A and B). Fragment A was amplified using the forward primer (E10Age5For: 5' TTC CCC
GAA CCG GTG ACG GTG TCG T 3') (SEQ ID NO: 64) which annealed to a region spanning a unique Age I restriction endonuclease site upstream of the hinge region (DNA encoding amino acids 148-157 of the CHl domain), in combination with the backward primer (ElOinsertionRev: 5' GCC GAT GTA GGG CAC GAT GGT GCT CAC GTC GCT GAT CTT GTC GAT TCT TCT CCC CAG GAG TTC AGG TGC TGA GGA AGA 3') (SEQ ID NO: 65) that annealed to 9 bases of the intron and the ElO DNA sequence encoding amino acids 244-249 of the CH2 domain. The ElOinsertionRev primer contained a tail, which encoded part of the peptide 632DR insertion. Fragment B was generated using the forward primer (ElOinsertionFor: 5' GTG AGC ACC ATC GTG CCC TAC ATC GGC CCC GCC CTG AAC ATC AGA AGA AGA GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 66) which annealed to a portion of the ElO DNA sequence encoding glycine 250 and the down stream amino acids (250-258), and the reverse primer (ElOEcoRBRev 5' G ATT ATG ATC AAT GAA TTC TGG CCG TCG CAC TCA T 3') (SEQ ID NO: 67) which annealed to a region spanning the stop codon and a unique EcoRI site within the vector at the end of the CH3 region. The ElOinsertionFor primer contained a tail, which encoded part of the peptide 632DR insertion and hybridized to the tail of the ElOinsertionRev primer.
For PCR reactions, the expand high fidelity PCR system (Roche) was used with the following program: 96°C for 5 minutes followed by 30 cycles of: 96°C for 30 seconds, 56°C for 30 seconds, 72°C for 2 minutes, with a final extension period of 10 minutes at 72°C and a hold at 40C. Fragments A and B were then gel purified and combined for an overlap extension PCR. The expand high fidelity PCR system was again used running the above program for 10 cycles then adding the E10Age5For and ElOEcoRDRev primers and running the above program for an additional 30 cycles. The overlap extension PCR product was then gel isolated and digested with restriction endonucleases Age I and Eco RI. The digested fragment was again gel isolated and cloned by ligating it back into the plasmid vector piece of the ElO-IgG parental clone, which had been digested with Age I and Eco RI followed by gel isolation.
10235000J gl The replacement clone was constructed using a fragment of TT peptide 632DR having the amino acid sequence ISDVSTIVPYIGPALNI (SEQ ID NO: 5). The TT peptide 632DR fragment was used to replace the region of the CH2 domain beginning with Valine 276 and ending with Valine 292. No additional arginines were added to the epitope in the replacement clone. The replacement clone was constructed using PCR overlap extension as described above for the insertion clone but using the following primers. The ElOreplacementRev primer (5' GCC GAT GTA GGG CAC GAT GGT GCT CAC GTC GCT GAT CAC GCA TGT GAC CTC AGG GGT CCG GGA 3') (SEQ ID NO: 68) was used in place of the ElOinsertionRev primer in the creation of Fragment A, and the ElOreplacementFor primer (5' GTG AGC ACC ATC GTG CCC TAC ATC GGC CCC GCC CTG AAC ATC GAC GGC GTG GAG GTG CAT AAT GCC AAG 3') (SEQ ID NO: 69) was used in place of the ElOinsertionFor primer in the creation of Fragment B. The ElOreplacementRev primer anneals to the DNA region encoding amino acids 267 to 275 within the CH2 domain and has a tail encoding part of the TT 632DR peptide fragment. The
ElOinsertionFor primer anneals to the region of the CH2 domain encoding amino acids 295 to 305 and has a tail encoding part of the TT 632DR peptide fragment and which hybridizes to the tail of the ElOreplacementRev primer tail. Ligation and cloning into the vector was conducted as described above for the insertion clone. The sequences of the final cloned products were confirmed by DNA sequencing.
In a method similar to that described above for construction of the 632 DR peptide insertion (clone name E10chGl-HC632tt), the 947DR peptide (FNNFTVSFWLRVPKVSASHLE, SEQ ID NO: 62) was inserted into the CH2 region of ElO-IgG between glycines 249 and 250 to create the clone ElOchGl- HC947tt. Three flanking arginine residues were added to each end of the inserted peptide. Construction of the clone was performed using overlap PCR as above with the exception that fragment A was amplified using the reverse primer 947E10 REV (5' TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA GCG GCG GCG GCC CAG CAG TTC TGG TGC TGA 3') (SEQ ID NO: 115) and fragment B was amplified using the forward primer 947FOR (5' GTG TCC TTC
TGG CTG CGC GTG CCA AAA GTG TCC GCT TCC CAT CTG GAG CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 116). As above, fragments A and B were then gel purified and combined into an overlap extension PCR reaction. The overlap extension PCR product was gel isolated and
1023500CM g2 digested with Age I and Eco RI. The digested fragment was again gel isolated and cloned by ligation back into the plasmid vector piece of the ElO-IgG parental clone as above. Clones were sequenced to insure proper construction. Double insertion clones in ElO Gl An additional peptide was inserted into each of the above clones to create new clones having an insert containing two tandem peptides flanked and separated by arginine residues. To construct new clone E10chGl-HC632tt/947tt, the 947DR peptide was inserted into clone E10chGl-HC632tt downstream of the 632DR peptide after the second of the three flanking arginine residues. Additional arginine residues were placed after the inserted 947 peptide to create an insert with two upstream arginines, followed by the 632DR peptide, followed by two more arginines, then the 947DR peptide and lastly three more arginines (e.g., RR-632peptide-RR-947peptide- RRR). Construction was performed using overlap PCR as above with the exception that fragment A was amplified using the reverse primer 632ING 1 REV (5' CAC TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA TCT TCT GAT GTT CAG GGC GGG GCC 3') (SEQ ID NO: 117) and fragment B was amplified using the forward primer 632ING1 FOR (5' TCC TTC TGG CTG CGC GTG CCA AAA GTG TCC GCT TCC CAT CTG GAG AGA AGA AGA GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 118). To construct new clone El OchGl -HC947tt/632tt, the 632DR peptide was inserted into clone El OchGl - HC947tt downstream of the 947DR peptide after the second of the three flanking arginine residues. Additional arginine residues were placed after the inserted 632DR peptide to create an insert with three upstream arginines, followed by the 947DR peptide, followed by two more arginines, the 632DR peptide and lastly three more arginines (e.g., RR-947peptide-RR-632peptide-RRR). Construction was performed using overlap PCR as above with the exception that fragment A was amplified using the reverse primer 947IN REV (5' GAT GTA TGG CAC GAT GGT GGA CAC GTC GGA GAT TTT GTC GAT GCG GCG CTC CAG ATG GGA AGC GGA 3') (SEQ ID NO: 119) and fragment B was amplified using the forward primer 947IN FOR (5' GTG TCC ACC ATC GTG CCA TAC ATC GGC CCA GCT CTG AAC ATC CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 120). Construction of clones in the L-SIGN specific antibody Cl
Four additional peptide containing clones were constructed by inserting 632DR and 947DR peptides singly or in tandem into the L-SIGN specific antibody
10235000J 53 C7 (e.g., inserts of 632DR, 947DR, 632/947, and 947/632). The C7 antibody contains a G2G4 constant region. Both single peptide insertions and double peptide tandem insertions were constructed using overlap PCR methods similar to those described above. The peptide 632DR was inserted into antibody C7 to create the clone C7chG2G4-HC632tt. Fragment A was amplified using the forward primer
(E10Age5For : 5' TTC CCC GAA CCG GTG ACG GTG TCG T 3') (SEQ ID NO: 64), which anneals to C7, in combination with the backward primer PVA 632 REV (5' GAT GTA TGG CAC GAT GGT GGA CAC GTC GGA GAT TTT GTC GAT GCG GCG GCG TGC CAC AGG TGG TGC TGA GGA AGA GAT GGA GGT GGA 3') (SEQ ID NO: 121). Fragment B was generated using the forward primer 632 FOR (5' GTG TCC ACC ATC GTG CCA TAC ATC GGC CCA GCT CTG AAC ATC CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 122) and the reverse primer EcoRIG2G4REV (5' CTG ATT ATG ATC AAT GAA TTC TCA TCA TTT 3') (SEQ ID NO: 123). Fragments A and B were then gel purified and combined into an overlap extension PCR reaction. As above, the overlap extension PCR product was gel isolated and digested with Age I and Eco RI. The digested fragment was again gel isolated and cloned by ligation into the plasmid vector piece of the C7 parental clone, which had been digested with Age I and Eco RI and gel isolated. Clones obtained were sequenced to confirm proper construction. The peptide 947DR was inserted into antibody C7 to create the clone C7chG2G4- HC947tt. The method used was similar to that just described except that fragment A was amplified using the backward primer PVA 947 REV (5' TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA GCG GCG GCG TGC CAC AGG TGG TGC TGA GGA AGA GAT GGA GGT GGA 3') (SEQ ID NO: 124) and fragment B was generated using the forward primer 947 FOR (5' GTG TCC TTC TGG CTG CGC GTG CCA AAA GTG TCC GCT TCC CAT CTG GAG CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 116). As above, the overlap extension PCR product was gel isolated and digested with Age I and Eco RI. The digested fragment was again gel isolated cloned by ligation into the plasmid vector piece of the C7 parental clone. Clones were sequenced to insure proper construction. Clones containing double peptide tandem insertions were constructed using the above clones (C7chG2G4-HC632tt and C7chG2G4-HC947tt) and inserting the second peptide downstream using overlap PCR as detailed in the above examples. Briefly, to construct clone C7chG2G4-HC632tt/947tt, the 947DR peptide was
1023500O-1 64 inserted into clone C7chG2G4-HC632tt just downstream of the 632DR peptide. The resulting clone had two arginine residues between the peptides and three arginine residues flanking either end of the insertion (e.g., RRR-632peptide-RR-947peptide- RRR). The clone was constructed using overlap PCR as detailed above with the exception that for the construction of clone C7chG2G4-HC632tt/947tt, fragment A was amplified using the reverse primer 632G2G4IN REV (5' CAC TTT TGG CAC GCG CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA GCG GCG GAT GTT CAG AGC TGG GCC 3') (SEQ ID NO: 125) and fragment B was amplified using the forward primer 632G2G4IN FOR (5' TCC TTC TGG CTG CGC GTG CCA AAA GTG TCC GCT TCC CAT CTG GAG CGC CGC CGC GGA CCG TCA GTC TTC CTC TTC CCC CCA 3') (SEQ ID NO: 126). Clone C7chG2G4- HC947tt/632tt was similarly constructed by inserting the 632DR peptide into clone C7chG2G4-HC947tt just downstream of the 947DR peptide. Three arginine residues flanked either side of the insertion while two arginine residues were placed between the peptides (e.g., RRR-947peptide-RR-632peptide-RRR). Overlap PCR was used, as detailed above, with the exception that for the construction of clone C7chG2G4- HC947tt/632tt, fragment A was amplified using the reverse primer 947IN REV (described above) and fragment B was amplified using the forward primer 947IN FOR (described above). Overlap extension PCR products were then cloned into the vector C7 and sequenced as described above.
Two additional clones containing single peptide insertions were also created in the C7 antibody using overlap PCR as described for the C7 clones above. However, for these clones the amino acids proline, valine and alanine, (PVA) which are just upstream of the insertion site in C7 were converted to aspartate, leucine, leucine and glycine (ELLG) as is observed in the ElOGl clones. The peptide 632DR was inserted to create the clone C7chG2G4-HC-ELLG632tt. Fragment A was amplified using the forward primer E10Age5For in combination with the backward primer ELLG 632 REV (5' GAT GTA TGG CAC GAT GGT GGA CAC GTC GGA GAT TTT GTC GAT GCG GCG GCG GCC CAG CAG TTC TGG TGC TGA GGA AGA GAT GGA GGT GGA 3 ') (SEQ ID NO: 127). Fragment B was generated using the forward primer 632 and the reverse primer EcoRIG2G4REV as above. The peptide 947DR was inserted into the C7 antibody to create the clone C7chG2G4-HC- ELLG947tt using methods similar to those described above except that fragment A was amplified using the backward primer ELLG 947 REV (5' TTT TGG CAC GCG
10235000_1 65 CAG CCA GAA GGA CAC GGT GAA GTT GTT GAA GCG GCG GCG GCC CAG CAG TTC TGG TGC TGA GGA AGA GAT GGA GGT GGA 3') (SEQ ID NO: 128) and fragment B was generated using the forward primer 947 FOR. Overlap extension PCR products were then cloned into the vector C7 as described above. AU clones were sequenced to confirm construction. Construction of ElO G2G4 clones
To construct ElO G2G4 clones, the heavy chain constant region of clone ElOchGl was converted from a Gl to a G2G4 constant region. This was accomplished by partially digesting the ElOchGl clone with the restriction enzyme Apa I followed by complete digestion with EcoRI. An Apa I site is located near the start of the heavy chain constant region, whereas the EcoRI site is located past the terminal stop codon. This digestion effectively removes all but the first four amino acids of the heavy chain constant region. It should be noted that these first four amino acids are the same in both the Gl and G2G4 constant regions. This fragment was then replaced with the corresponding Apa I to EcoRI fragment from the G2G4 clone
C7chG2G4. Clones containing peptide insertions were then made by replacing an Age I to EcoRI fragment, removed from E10chG2G4, with an Age I to EcoRI fragment from each of the four different C7 clones containing single or double peptide insertions described above (e.g., C7chG2G4-HC632tt, C7chG2G4-HC947tt, C7chG2G4-HC632tt/947tt, and C7chG2G4-HC947tt/632tt). Thus, four new peptide insertion clones were created in E10chG2G4: E10chG2G4-HC632tt, E10chG2G4- HC947tt, E10chG2G4-HC632tt/947tt, and E10chG2G4-HC947tt/632tt. Production of peptide embedded antibodies
For production of the antibody, plasmids were transiently transfected into 293 EBNA cells using Effectine (Qiagen). Briefly, 1.2 x 107 293 cells were seeded in 15OmM tissue culture dishes in DMEM + 10% FBS. The following day, each dish was transfected with 16 ug of the IgG expression plasmid along with 4 ug of pAdVAntage and 800 ng of pE-GFP-1, using 1 ml of EC Buffer, 160 ul of Enhancer and 200 ul of Effectine according to the manufacturer's instructions. At twenty-four hours post transfection, the media was changed to serum free media conditions (IS PRO media from Irvine Scientific). After an additional 24 hours, 2.5 mis of 20 X TC Sugar Rush reagent (0.5M HEPES, 20% glucose) was added. Cells were incubated for an additional 4 days and the media supernatant was harvested and purified by protein A affinity chromatography.
10235000_l gg Expression and binding of the peptide embedded antibodies to the receptor
Peptide grafted antibodies generated by both insertion and replacement strategies were evaluated for expression and binding to DC-SIGN/L-SIGN receptors on cells. As shown in Figures 6A-B, the replacement clone showed relatively poor expression and commensurately less binding while the insertion clone showed expression and receptor binding equivalent to the native antibody. On the basis of these results, the insertion clone was selected for further study and a large batch (1.5 mg from 230 ml transient cultures) of the insertion clone (henceforth designated as E10-632DR) was made for antigen delivery studies. Figure 7A shows the affinity purified insertion clone, which is devoid of the partially formed antibody seen in the crude preps (compare with Figure 6A). The affinity purified peptide-inserted antibody migrates at a molecular weight greater than that of native antibody commensurate with the ~2kDa size difference. Furthermore, the relative affinities of the purified peptide-inserted antibody to both the DC-SIGN and L-SIGN receptors on the cell are very similar to that of the native antibody (see Figure 7B).
EXAMPLE 4: Activation ofT-cell responses by antibody mediated delivery of peptide antigen to DCs
Peptide inserted antibody, E10-632DR (insertion clone), was used for delivering peptide antigen to immature dendritic cells (iDCs) for the activation of T- cells isolated from TT vaccinated donors. The iDCs were differentiated from blood monocytes obtained from vaccinated donors by incubating with cytokines (IL-4 and GM-CSF) for six to nine days. The iDCs were treated with the native (ElO) and peptide inserted antibodies for one hour, washed and mixed with autologous T-cells and left for five days to proliferate. As shown in Figure 8, targeting with E10-632DR, elicited a significant level (p<0.005 vs. native antibody, ElO) of proliferative responses of T-cells obtained from donor 13. The proliferative response obtained with the peptide inserted antibody was similar to that obtained with the free 632DR peptide. However, no proliferative responses were induced in the other three donors (e.g., donors 5, 14 and 15) by targeting with E10-632DR peptide-inserted antibody commensurate with the lack of responses to the free peptide in these donors.
Furthermore, when iDC were treated with different doses of the antibody, T-cell responses directly correlated with dose of the antibody, with significant level of proliferation elicited even at picomolar concentrations of the targeting antibody (P<0.00005 vs. native antibody, see Figure 9A). Free peptide at a concentration (130
1Q235OOO 1 67 nM) equivalent to the concentration of the targeting antibody produces an equivalent T-cell response in these in vitro assays. However, targeting directly to the antigen presenting cells using peptide-inserted antibodies will have a clear advantage over free peptide in the in vivo setting. Furthermore, the proliferative responses induced by E10-632DR antibody could be blocked with excess native antibody, ElO (P = 0.00008 vs. E10-632DR) but not a control antibody (see Figure 9A). In addition, both the native antibody and peptide embedded antibody showed equivalent binding to immature dendritic cells (DCs) used in the T-cell assay (see Figure 9B). EXAMPLE 5: Antibody mediated epitope delivery produces a sustained T-cell response
We examined whether delivery of antigens linked to antibodies would require internalization and proteasomal cleavage of the antibody to release the linked antigen for presentation to T-cells. This type of processing may be advantageous, as prolonged presentation of antigen to T-cells could result in a long lasting immune response. To test this speculation, iDCs from donor 13 were incubated with ElO-
632DR or free 632DR peptide for 1 hour, washed and mixed with purified autologous T-cells either immediately, or two and four days after antigen pulsing. T-cell proliferative responses were enumerated after five days. As shown in Figure 10, only peptide-embedded antibody produces significant T-cell responses even four days after antigen pulsing. These results demonstrate that delivering antigen via antibodies can result in a sustained immune response not achievable with free peptide. EXAMPLE 6: Antibody fusion having disrupted Fc Receptor binding
We used E10-632DR peptide-inserted antibody to test whether inserting a peptide into the junction between the hinge and CH2 domain prevents binding to the Fc receptors. The relative binding of the native antibody (ElO) and peptide inserted antibody (E10-632DR) to Fc receptor bearing monocytic cell line U937 was tested by a competition flow cytometry analysis (Figure 11). As shown in Figure 11, the native antibody (ElO) competes away the binding of a biotinylated human IgGl in a dose dependent manner similar to the unbiotinylated to IgGl. In constrast, the peptide inserted antibody (E 10-632DR) does not compete for the binding at any dose tested similar to IgG2G4, an antibody genetically mutated not to bind the Fc receptor. Disruption of Fc receptor binding may be beneficial in a variety of applications because it prevents non-specific binding of the antibody fusion construct by Fc
10235000_l gg receptors thus allowing administration of a lower dose of the antibody fusion to obtain the desired therapeutic effect.
EXAMPLE 7: Design of peptide inserted antibody using sequence similarity replacement in the CH3 domain We designed an antibody fusion having peptide antigen TT 830-844 inserted into the proteolytic hot spot SVMHEALH (SEQ ID NO: 9) of the C-terminal hydrophobic patch VFSCSVMHEALH (SEQ ID NO: 70) in the CH3 domain. This hydrophobic patch is the most distant site from the antigen reactive portion of the antibody. Therefore, structural perturbances in this area may be less likely to affect the antigen binding properties of the antibody. The design for this antibody construct is schematically outlined in Figure 12.
EXAMPLE 8: Design of peptide inserted antibody by insertion into the CHl domain
We designed an antibody peptide fusion having a peptide epitope with flanking arginine (R) and/or lysine (K) residues (e.g., R and/or K residues at the amino- and carboxy termini of the peptide). Peptides with added proteasomal cleavage sites may theoretically be inserted anywhere in the antibody constant region for efficient cleavage by the proteasome. However, insertion of the peptide epitope into the large hydrophobic patch PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSN (SEQ ID NO: 71) of the CHl domain may be very favorable, since the hydrophobic effects of the inserted peptide epitope are best accommodated in this region of the antibody. The design for this antibody construct is outlined in Figure 13 and is exemplified using class II epitope, TT 830 with arginines (Format 1) (SEQ ID NO: 7) or lysines (Format 2) (SEQ ID NO: 8) or a combination of arginine and lysine residues as flanking residues. EXAMPLE 9: Design of peptide antibody fusion by sequence similarity replacement
We designed an antibody fusion by aligning the sequence of the peptide antigen with that of the antibody to identify a region with the closest sequence match. The mismatched residues may then be mutated in the antibody sequence to match the sequence of the peptide epitope. Such an epitope may then be provided with flanking proteolytic sites (e.g., RR, KK, RK, or KR) for efficient processing by the cellular proteasomes. This design may minimize effects on the native structure and folding of the antibody and therefore may be easier to produce and evaluate in terms of biological function. This strategy is illustrated by aligning the sequences of the
10235000J 69 tetanus toxoid peptide antigens listed in Table 1 and 2 with the G2G4 Ig constant region sequence. Alignment of the sequence from tetanus toxoid, site 230, for example, suggests a fit to the sequence SVMHEALHN (SEQ ID NO: 9) at the end of the CH3 region of the G2G4 sequence (see Figure 1). The alignment of these two regions is shown in Figure 14A. The lines indicate identity between amino acids, and the colons indicate conservative amino acid changes. The similarity of these two regions may permit the replacement of a portion of the G2G4 region with the TT 230 epitope, thereby producing an antibody fusion comprising the TT 230 peptide.
Figure 14B shows the sequence of the G2G4 constant region from a region within the CH3 region to the carboxy terminus of the protein and the symbol "S" underneath the sequence denotes regions that are predicted to be potential 2OS proteasome cleavage sites. The inserted peptide (underlined) may be processed by endogenous sites at the start and end of the peptide. However, in order to ensure that the peptide would be processed appropriately, it may be necessary to introduce strong proteasome cleavage signals, such as RR or KK residues, flanking one or both ends of the peptide. Examples of such insertions and the expected cleavage patterns resulting from them are shown in Figure 14C. For ease of viewing the inserted cleavage signals are written in lower case letters. It should be noted that either RR or KK could be used at either position or in combination (e.g., RR-peptide, KK-peptide, RK-peptide, KR- peptide, peptide-RR, peptide-KK, peptide-RK, peptide-KR, RR-peptide-RR, KK- peptide-KK, RR-peptide-KK, KK-peptide-RR, RK-peptide-RK, KR-peptide-KR, RK- peptide-KR, KR-peptide-RK, RR-peptide-RK, RR-peptide-KR, KK-peptide-RK, KK- peptide-KR, RK-peptide-RR, RK-peptide-KK, KR-peptide-RR., or KR-peptide-KK). Alternatively, the two lysines, KK, could replace either the histidine (H) and/or the tyrosine (Y) residues immediately following the TT 230 peptide. To maintain the size of the antibody after sequence similarity replacement with the TT 230 peptide, the histidine, tyrosine, threonine and glutamine residues following the location of introduction of the TT230 peptide could be replaced or deleted in order to accommodate the four residues introduced in the antibody fusion shown in the lower portion of Figure 14C (e.g., the two arginine and two lysine residues flanking the TT 230 peptide). In this example, the cysteine adjacent to the amino end of the introduced peptide is important to antibody structure and would not be removed or replaced with cleavage signal peptides.
10235000J 70 As shown in Figure 14D, the sequence from tetanus toxoid peptide 702 can be aligned with the G2G4 sequence at the carboxy terminus. As this peptide aligns with and effectively replaces the carboxy terminus, a cleavage signal after the terminal lysine would not be necessary. Proteasomal cleavage prediction suggests that the terminal peptide should be available as a cleavage product (Figure 14E). Should the cleavable region "QK" that includes the first lysine of the desired peptide epitope be insufficient for proper processing, the glutamine (Q) could be replaced with another lysine to generate the cleavage signal without altering the length of the carboxy terminus. The examples illustrated in Figures 14A-E utilize class I epitopes, however, the approach may also be possible with class II epitopes. For example, the tetanus toxoid class II epitope that begins at amino acid 632 can be aligned with the G2G4 sequence as shown in Figure 14F. In this case the period symbol indicates amino acids that are in a similar group, e.g. hydrophilic neutral, while the colon again represents conservative amino acid changes. The cysteine residue located within the aligned region would not be altered as it may be important in antibody structure. Analysis of the class II epitope shown has indicated that the first 7 amino acids can be removed and still permit recognition of the peptide as a class II epitope (Reece, J. C. et al., J Immunol 151: 6175-6184 (1993); Diethelm-Okita, B. M. et al., J Infect Dis 181: 1001-1009 (2000)). The altered region and the sites where it would be subject to proteasome degradation are shown in Figure 14G.
The class I epitopes have more constraints in terms of the size of the epitope, such that efficient processing sites must surround the introduced peptide. In contrast, class II epitopes are more flexible in terms of the size of the epitope. The antibody- class II epitope fusion described above may be processed efficiently by the naturally occurring surrounding proteasome sites without the necessity of introducing additional lysine or arginine residues. However, addition of lysine and/or arginine residues may be added if the processing observed is insufficient.
The same strategy of replacement following alignment can be performed with other IgG heavy chain constant regions such as, for example, the Gl heavy chain.
Since the Gl and G2G4 heavy chains have fairly similar amino acid sequences, some of the regions identified in the G2G4 region for sequence similarity replacement would be similar or identical to the location for placement in the Gl heavy chain. For
10235000J η\ example, the TT 230 (A2) epitope could be placed in the exact same region of the Gl heavy chain as shown in Figure 14A for the G2G4 heavy chain. EXAMPLE 10: Design of antibody peptide fusion having peptide fused to the C- terminus of the antibody We designed an antibody peptide fusion having the peptide antigen fused to the carboxy-terminus of the antibody by a flexible spacer composed of two tandem repeats of four glycines and one serine. This design allows for independent folding of the antibody and the attached peptide. This strategy has been successfully used in the past by (Peschen, D. et al., Nat Biotechnol 22: 732-738 (2004)) to produce an antifungal peptide (AFP) fused to scFv in both bacteria and plants. However, to facilitate efficient cleavage by cellular proteasomes, we have modified this design to incorporate proteasomal cleavage sites (e.g., RR or KK) between the gly-ser spacer and the peptide antigen as shown in Figure 15. EXAMPLE 11: Design of peptide antibody fusions having multiple disease specific epitopes
We developed a strategy to deliver multiple disease specific epitopes via antibodies by grafting the relevant epitopes into a disease specific protein, e.g., a tumor antigen (or autoantigen) and then attaching the epitope grafted carrier protein to a targeting antibody. Design of such antibody peptide fusions first involves identification of the endogenous epitopes in the carrier protein. The carrier protein is also analyzed to identify the proteasomal cleavage sites for grafting of other disease specific epitopes into the carrier protein. As an example, various disease antigens responsible for the autoimmune form of diabetes and human cancers were analyzed for the presence of 2OS proteasomal cleavage hotspots and endogenous epitopes that have been shown to be the most active in human patients. A total of four human autoantigens (namely, glutamic-acid decarboxylase 65 (GAD65), heat shock protein 60 (HSP60), insulinoma associated protein 2 (IA-2) and proinsulin (PI)) responsible for causing either Type 1 diabetes mellitus (TlDM) or insulin dependent diabetes mellitus (IDDM) and four human tumor antigens (namely, gplOO and Tyrosinase associated with Melanoma, Late Membrane Protein 2 (LMP-2) associated with Lymphoma and Carcinoembryonic Antigen associated with various types of adenocarcinomas) were analyzed by computer-aided algorithms to locate hydrophobic patches and proteasomal cleavage hot spots. Additionally, endogenous epitopes that are capable of stimulating a human immune response were surveyed in the literature.
1Q235000_1 72 The results of this analysis for autoimmune antigens are summarized in Table 3 and Table 4 (below) and illustrated in detail by Figures 16 through 19. The results of this analysis for tumor antigens are summarized in Table 5 and Table 6 (below) and illustrated in detail by Figures 20 through 23.
Table 3. Ranking of hydrophobic sequences of autoantigens containing most proteasomal cleavage sites (hotspots) for epitope grafting.
Figure imgf000074_0001
Cleavage hot spot refers to a hydrophobic patch having a higher cleavage score than the entire protein score (shown in bold).
2 Cleavage score is determined as number of individual cleavage sites (S)/number of amino acid residues.
Table 4. T-cell epitopes of autoantigens found to be most active in TlDM or IDDM patients.
Figure imgf000074_0002
10235000 1 73
Figure imgf000075_0001
Table 5. Ranking of proteasomal cleavage hotspots in tumor antigens for epitope grafting.
Figure imgf000075_0002
1 Cleavage hot spot refers to a hydrophobic patch having a higher cleavage score than the entire protein score (shown in bold).
2 Cleavage score is determined as number of individual cleavage sites (S)/number of amino acid residues.
Table 6. Tumor antigen derived epitopes currently in human clinical trials.
Figure imgf000075_0003
10235000 1 74
Figure imgf000076_0001
EXAMPLE 12: Selection of a carrier protein for grafting of multiple disease specific epitopes
Multiple factors are considered when selecting a disease protein as a carrier for grafting epitopes. First, the disease being targeted is considered such as, for example, diabetes or cancer, or a particular type of cancer, such as, skin cancer or blood cancer. After determining the disease indication, the selection of an autoantigen as a carrier protein is based on the presence of a "maximum number of 2OS proteasomal hotspots." This method will allow for the incorporation of the greatest number of epitopes from other autoantigens responsible for the disease, e.g., diabetes in this example. For example, IA-2 has the greatest number of proteasomal cleavage hotspots when compared to the other potential carrier proteins that were analyzed. This is illustrated in Figures 16-19 which show the proteasomal cleavage hot spots for the diabetes carrier proteins indicated by underlined regions. Figure 18 shows the sequence for IA-2 which has six proteasomal cleavage hot spots as compared to four, four, and two, hot spots for GAD65 (Figure 16), HSP60 (Figure 17), and PI (Figure 19), respectively. Therefore, each of the clinically relevant diabetic epitopes listed in Table 4 may be incorporated into the IA-2 carrier protein at a proteasomal cleavage hotspot. Furthermore, selection of an optimal location for grafting of epitopes into a proteasomal cleavage hot spot of a carrier protein may be based on sequence similarity between the epitope and a region of the carrier protein having a proteasomal cleavage hotspot. Additionally, when selecting a carrier protein, consideration will also be given to the size of the carrier protein. A smaller carrier protein will facilitate production and subsequent conjugation to an antibody. Accordingly, the smallest disease protein that contains enough proteasomal hot spots to accommodate all of the disease epitopes desired to be delivered may be selected.
10235000 1 75 An exemplary scheme for selection of a disease specific carrier protein for grafting of multiple epitopes is illustrated in Figure 24.
Carrier proteins comprising multiple disease specific epitopes may be associated with an antibody by expressing the carrier protein as a fusion with the antibody molecule, e.g., embedded into the constant region or attached to the C-terminus of the heavy or light chain constant domain. When attaching the carrier protein to the C- terminus of the antibody, the fusion may additionally comprise a peptide linker between the antibody and the carrier protein, such as a cleavable peptide linker as described below. Alternatively, the carrier protein may be conjugated to the antibody, for example, using glutaraldehyde (see e.g., Reichlin M. Methods Enzymol. 70(A): 159-65 (1980); Yao TJ et al., Clin Cancer Res. 5: 77-81 (1999); Mittelman A et al, Clin Cancer Res. 1: 705-13 (1995)).
In other embodiments, the selected antigenic peptides from disease proteins can be directly grafted onto the targeting antibody using the strategies described in the examples above.
EXAMPLE 13: Targeted destruction of cancer cells using peptide embedded antibodies
An anti-cancer antibody having a toxin polypeptide embedded in the Fc region may be designed for use in selectively targeting and destroying cancer cells. An exemplary pro-apoptotic polypeptide that may be used for targeted cytotoxicity of cancer cells is a 14-amino-acid amphipathic peptide KLAKLAKKLAKLAK (SEQ ID NO: 55) that has been successfully used for selectively killing malignant hematopoietic cells and cells lining tumor blood vessels (Ellerby, H. M. et al., Nat Med 5: 1032-1038 (1999); Marks, AJ. et al., Cancer Res 65: 2373-2377 (2005)). This polypeptide, or another cytotoxic polypeptide, may be introduced into the constant region of a cancer specific antibody using the methods described above for introduction of an antigenic polypeptide (e.g., insertion or sequence similarity replacement into a region flanked by naturally occurring proteasomal cleavage sites and/or by introduction of flanking proteasomal cleavage sites). The resulting peptide toxin embedded antibodies may be screened for target cell killing using a MTT or MTS dye reduction assay (Marks, A. J. et al., Cancer Res 65: 2373-2377 (2005); Perchellet, E. M. et al., J Med Chem 48: 5955-5965 (2005)), a soft agar assay (Dakappagari, N. K. et al., J Biol Chem 280: 54-63 (2005)), 3H-Thymidine incorporation assay (Dakappagari, N. K. et al., Cancer Res 60: 3782-3789 (2000)),
10235000 1 76 apoptosis assay (Sommer, U. et al., J Biol Chem 280: 23853-23860 (2005)), or any other method for evaluation of cell proliferation or cell death known to those skilled in the art.
EXAMPLE 14: Screening method for identifying tumor internalizing antibodies Peptide toxins mediate their toxic effects upon internalization into the cells.
This property permits a rapid, high-throughput screen for identifying internalizing antibodies from a library of antibodies. For example, a peptide toxin may be embedded in or attached to the C-terminus of a library of antibodies, such as, for example, a library of tumor cell reactive antibodies. The antibody fusions are then mixed with cells and screened for cell proliferation or cell death using an assay having an easy read out, such as, for example, an assay based on dye reduction methods (e.g., MTT). EXAMPLE 15: Screening method for identifying novel peptide toxins
A library of peptides may be screened for toxic activity by fusing the library of peptides to an internalizing antibody, such as, for example, a known tumor cell internalizing antibody (e.g., growth receptor antibody, 4D5). The peptide antibody fusions are then mixed with cells and screened for cell proliferation or cell death as described above. Peptides having toxic activity may thus be identified. EXAMPLE 16: Design ofcleavable linkers for peptide embedded toxins into the DC-SIGN/L-SIGN antibody (clone ElO)
We analyzed several linkers for embedding or attaching the peptide toxin KLAKLAKKLAKLAK (SEQ ID NO: 55) to a full IgG or Fab fragment of antibody clone ElO which has specificity for the DC-SIGN/L-SIGN receptor. The ElO antibody clone was chosen as a model system for a variety of reasons. First, the ElO antibody clone has been found to internalize well both on L-SIGN and DC-SIGN transfected cells. Second, Fabs, chimeric IgGs and peptide embedded IgGs of ElO were found to produce equally well. Third, stable cell lines expressing the SIGN molecules for cell killing assays are readily available. Finally, it is possible to test the therapeutic applicability of this antibody for killing dendritic cells implicated in disease pathogenesis. Computer-based prediction was used to assess cleavable linkers for embedding peptide toxin into the hinge region of full IgGs and for attachment to the C-terminus of the light and heavy chains of the Fab. Based on this analysis, appropriate primers for incorporation of the peptide toxin into the Fabs (bacterial
10235000J 77 expression) and full IgGs (mammalian cell expression) were designed for grafting of peptide toxin onto the antibodies as described above in Example 3.
Table 7. Analysis of linkers for cleavage by the 2OS proteasome for attachment of peptide toxin to the C-terminus of Fab clone ElO. The linker is in bold, predicted 2OS proteasomal cleavage sites are indicated with an S, the antibody sequence is shown in lower case, and the proapoptotic peptide domain (toxin) is KLAKLAKKLAKLAK (SEQ ID NO: 55).
Figure imgf000079_0001
An exemplary cleavable linker for attachment of the peptide toxin to the Fab heavy chain of clone ElO is illustrated in Figure 25A. Figure 25B shows a table indicating the 2OS proteasome cleavage scores for the sequence shown in Figure 25A. The site with highest cleavage score is highlighted.
An exemplary cleavable linker for attachment of the peptide toxin to the C- terminus of Fab light chain of clone ElO is illustrated in Figure 26A. Figure 26B shows a table indicating the 2OS proteasome cleavage scores for the sequence shown in Figure 26A. The site with highest cleavage score is highlighted.
An exemplary cleavable linker for inserting the peptide toxin into the hinge region of chimeric IgGl (clone ElO) is illustrated in Figure 27A. Figure 27A shows a table indicating the 2OS proteasome cleavage scores for the sequence shown in Figure 27A. The site with highest cleavage score is highlighted. EXAMPLE 17: Design of peptide antibody fusions utilizing peptide linkers to increase proper expression
10235000 1 78 Display of a 24 amino acid peptide at the carboxy terminus of the light chain alone can produce antibody that assembles correctly (Figure 29b). Insertion of two peptides at the end of the light chain, in effect making a longer insertion, in this case of 47 amino acids, also produces antibody that is correctly assembled (Figure 29c). Display of peptides of 27 amino acids in the region adjacent to the hinge of the heavy chain produces primarily correctly assembled antibody (Figure 29d). Increasing the length of the heavy chain peptide insertion to 48 amino acids by adding a second peptide can result in an increase in the amount of a protein of about 75 kilodaltons (Figure 29f, h, arrow). This 75 kilodalton protein appears to be a half antibody (Hab); Figure 30 shows that both anti-kappa and anti-gamma chain antibodies bind to it, and its size is correct for half an antibody. This probably results from some interference on the part of the larger inserted peptides on the formation of the disulfide bridge between the two heavy chains. When a peptide is added to the carboxy terminus of the light chain in combination with the insertion of a peptide of 25 amino acids in the site adjacent to the hinge of the heavy chain, the ability to form the disulfide bridge between the two heavy chains also appears to be compromised, and Hab is also obtained (Figure 29i, j). The proportion of Hab can vary; this may reflect the length of the peptides inserted or their composition. This effect appears to be independent of whether Gl or G2G4 heavy chains are used. In order to improve the formation of the disulfide bridge in antibodies when peptides are inserted in both the heavy and light chains, a linker peptide is added to the end of the light chain and in front of the arginine stretch preceding the inserted peptide, so that steric hindrance between inserted peptide regions in the light and heavy chains is alleviated. These linkers are composed of amino acids that promote flexibility of the protein backbone, such as glycine, and additional small amino acids with limited reactivity, such as alanine. Two examples of such linkers would be the amino acid sequence GGGAAG (SEQ ID NO: 129), a six amino acid linker, or the sequence GGGAAAGAAG (SEQ ID NO: 130), a ten amino acid linker. Other sequences and lengths of linkers may be envisioned by those skilled in the art. Two examples of the use of such linkers in the presentation of the 947tt peptide are shown in Table 8. Such linkers may also improve the ability of cells to express peptides at the C terminus of the light chain in general.
Table 8. Amino acid linker sequences. The first sequence following the ellipses represents the carboxy terminus of the ElO light chain. Linker sequences are
10235000J 79 underlined. The three arginines preceding the engrafted peptide are indicated in bold type. In both cases, the sequence following the arginine group is the 947DR tetanus toxoid epitope.
Figure imgf000081_0001
EXAMPLE 18: Engrafting peptides with surrounding proteasomal cleavage sites at the C-terminal end of the ElO light chain
The 632DR and 947DR peptides were chosen for insertion, either singly or in tandem, at the C-terminal end of the ElO light chain. In order to facilitate proteasomal cleavage, three arginines (RRR) were designed into the engrafted constructs between the C-terminal end of the light chain and either 632DR or 947DR. When the peptides were incorporated in tandem, either as 632DR-947DR or as 947DR-632DR, three arginines were also incorporated between the two peptides. Table 9 shows the final sequences from the end of the light chain through the engrafted peptides for all the light chains generated.
Table 9. Engrafted peptides at the end of the L-SIGN antibody ElO light chain. The three arginines are indicated in bold type. The first sequence following the ellipses represents the end of the ElO light chain. The following sequences (between or after arginine groups) are as indicated, either 632DR or 947DR.
Figure imgf000081_0002
10235000 1 80 Table 10. DNA primers used for the construction of light chain-engrafted peptides.
Figure imgf000082_0001
The above light chain constructs were generated by overlap PCR using the primers listed in Table 10. Primers were synthesized by Operon. The ElO light chain construct with the 632DR peptide engrafted was designated E10chGl-LC632tt. For this construct, a PCR fragment of 586 bp was first generated by pairing primers 060427 UP FOR and 060427 UP REV and using the ElO plasmid DNA as a template. The fragment was gel purified and used in a PCR reaction with fragments 632DR for and 632DR rev (which overlap each other), along with additional 060427 UP FOR and 632 s rev, to generate a 670 bp fragment. After gel purification, this fragment and ElO were digested with Acc65l andNotl and subjected to gel electrophoresis. A 612
10235Q00 1 81 bp fragment was isolated from the PCR fragment digest and an 11542 bp fragment was isolated from the plasmid digest and ligated together to generate ElOchGl- LC632tt.
The ElO light chain construct with the 947DR peptide engrafted, ElOchGl- LC947tt, was generated in a similar manner. The purified 586 bp fragment generated above was used in a PCR reaction with fragments 947DR for and 947DR rev (which overlap each other), along with additional 060427 UP FOR and 947 s rev to generate a 673 bp fragment. After gel purification, this fragment was subjected to gel electrophoresis and a 615 bp fragment was isolated and ligated to the 11542 bp Acc65VNotl ElO fragment (above) to generate E10chGl-LC947tt.
The tandem epitope insertions were generated using the single epitope insertion plasmids as PCR templates. Construct E10chGl-LC947tt/632tt was generated using E10chGl-LC632tt as template, pairing primers 060427 UP FOR with 060616 947 IR to generate a 636 bp fragment, and primer 060616 947 IF with 632DR s rev to generate a 130 bp fragment, which were gel purified. These purified fragments were then subjected to PCR with added 060427 UP FOR and 632DR s rev to generate a 742 bp fragment, which was digested with Acc65I and Notl along with E10chGl-LC632tt. Fragments of 684 and 11542 bp were gel purified from the PCR fragment digest and plasmid digest, respectively, and were ligated together to generate E 1 OchG 1 -LC947tt/632tt.
Construct E10chGl-LC632tt/947tt was generated using E10chGl-LC947tt as template, pairing primers 060427 UP FOR with 060724 632 IR to generate a 637 bp fragment, and primer 060724 632 IF with 947DR s rev to generate a 128 bp fragment, which were gel purified. These purified fragments were then subjected to PCR with added 060427 UP FOR and 947DR s rev to generate a 742 bp fragment, which was digested with Acc65I and Notl to generate a 684 bp fragment. This was gel purified and ligated to the 11542 bp E10chGl-LC632tt plasmid fragment (above) to generate E10chGl-LC632tt/947tt. The 11542 bp plasmid fragment is the empty plasmid fragment which does not contain the 632-sρecific sequences. All constructs were confirmed to be correct by sequencing. Gel and Western analysis of supernatants from transient transfection of CHO cells with the constructs indicated above showed that the altered light chains were expressed and incorporated into antibody.
10235000J 82 The light chain variants developed above are shown in the first four rows of Table 11. These light chains were transferred to a number of other constructs bearing alternate heavy chains with or without inserted peptides. The heavy chain constructs are described above. The light chain variants were transferred into different heavy chain contexts by digestion of the constructs above with-ϊδαl and Notl, isolation of the light chain containing fragment, and replacement of the corresponding light chain containing XballNotl region in the heavy chain construct of interest. In a few cases, the replacement was done with Acc65llNot\ fragments. In this way, the remaining constructs of Table 11 were generated.
Table 11. Constructs including TT peptides in light chains, with and without TT peptides in heavy chains.
Figure imgf000084_0001
EXAMPLE 19: Construction of light chains containing linker sequences
Light chain constructs containing linker sequences were generated using the PCR primers indicated in Table 12. Primer 060925 SL 947 (short linker) was used with primer 947 s rev (see above) and ElO chGl LC947 as a template in order to generate a 128 bp PCR fragment. This was used in overlap PCR with the 586 bp PCR fragment previously generated with primers 060427 UP FOR and 060427 UP REV described above to generate a 691 bp fragment. Digestion of this fragment with Acc65I and Notl created a 632 bp fragment. For the longer linker, primer 060925 LL 947 (long linker) was used with primer 947 s rev and ElO chGl LC947 as a template in order to generate a 140 bp PCR fragment. This was used in overlap PCR with the 586 bp PCR fragment previously generated with primers 060427 UP FOR and 060427 UP REV described above to generate a 703 bp fragment. Digestion of this fragment
10235000 1 83 with^4cc65I and Notl created a 644 bp fragment. The 632 and the 644 bp fragments were ligated separately into ElO chGl HC632tt from which the light chain region had been removed by AccβSllNotl digestion to generate ElO chGl HC632tt LCsl947tt and ElO chGl HC632tt LC11947tt, respectively. Table 12. DNA primers sequences.
Figure imgf000085_0001
EXAMPLE 20: Construction of light chains containing linker sequences
Materials
Custom oligonucleotide primers were purchased from Sigma-Genosys. All PCR steps were performed using the Expand High Fidelity PCR System (Roche Applied Science). PinAI restriction enzyme was obtained from Roche Applied Science. All other restriction enzymes and T4 DNA Ligase were obtained from New England Biolabs. Conversion ofscFv-2G12 into a rabbit-human chimeric IgGl
The rabbit anti-human CD 19 single-chain antibody scFv-2G12 was converted to a rabbit-human chimeric whole IgGl by overlap extension PCR using the following primers:
Figure imgf000085_0002
The scFv-2G12 kappa light chain V region was amplified from vector PAX243-scFv-2G12 using primers R2G12VK-F1 and R2G12VK-hCK-R. The human kappa constant region was amplified from a human kappa light
10235000 1 84 chain library using primers R2G12VK-hCK-F and hCK-Rl . Overlap extension PCR was performed on the two products using primers R2G12VK- Fl and hCG-R to make the rabbit-human chimeric kappa light chain. The PCR product was digested with Xbal/Sacl and cloned into the human Fab expression vector PAX243hGK. The resulting construct was designated PAX243-2G12VK-hCK.
The scFv-2G12 heavy chain V region was amplified from vector PAX243-scFv-2G12 using primers R2G12VH-F1 and R2G12VH-hCG-R. The human IgGl heavy chain CHl region was amplified from a rabbit-human chimeric IgGl Fab expression vector using primers R2G12VH-hCG-F and hCG-Rl . Overlap extension PCR was performed on the two products using primers R2G12VH-F1 and hCG-Rl to make the rabbit-human chimeric Fab heavy chain. The PCR product was digested with XhoI/PinAI and cloned into PAX243-2G12VK-hCK. The resulting chimeric Fab construct was designated PAX243-2G12/cFab.
To convert 2G12/cFab into a whole IgGl, a human CMV immediate early promoter cassette was cloned into the Notl/Xhol sites of PAX243- cFab/2G12 (in front of the heavy chain). The resulting vector was digested with Xbal/PinAI and the insert containing the chimeric Fab and CMV promoter was cloned into the human IgGl expression vector 3 B. IBB. The resulting construct was designated 3B.lBB-2G12/cIgGl. Insertion of sequences encoding the peptide toxins KLAK and p21 into 2G12/cIgGl
Sequences encoding two copies of the KLAK peptide toxin flanked by proteasomal cleavage sites (amino acid sequence RRRR
KLAKLAKKLAKLAK RRR KLAKLAKKLAKLAK (SEQ ID NO: 159)) were added to the end of the 2G12/cIgGl light chain by overlap extension PCR using the following primers:
Figure imgf000086_0001
10235000 1 85
Figure imgf000087_0001
The 2G12 light chain was amplified from plasmid 3B.1BB- 2G12/cIgGl using primers R2G12VK-F1 and hCK[KLAK]x2-R2. The resulting product was then subjected to a second round of PCR with primers R2G12VK-F1 and hCK[KLAK]x2-R3. The final PCR product was digested with Xbal/Notl and cloned into the Xbal/Notl sites of vector 3B. IBB- 2G12/cIgGl . The resulting construct was designated 3B.1BB-2G12/LC- KLAKx2/cIgGl.
Sequences encoding three copies of the p21 peptide toxin flanked by proteasomal cleavage sites (amino acid sequence RRRR PVKRRLFG RRRR PVKRRLFG RRRR PVKRRLFG RRRR (SEQ ID NO: 162)) were inserted between Gly249 and Gly250 of the 2G12/cIgGl heavy chain (amino acid sequence PAPELLG249G250PSVFLFPPK (SEQ ID NO: 163)) by overlap extension PCR using the following primers:
Figure imgf000087_0002
The 5' segment of the human IgGl heavy chain ending with codon Gly249 was amplified from plasmid 3B.lBB-2G12/cIgGl using primers E10Age5For and hCH2[p21]x3-R2. The resulting product was then subjected to a second round of PCR with primers E10Age5For and hCH2[p21]x3-R3. The 3 'segment of the human IgGl heavy chain beginning with codon Gly250
10235000 1 86 was amplified from plasmid 3B. lBB-2G12/dgGl using primers hCH2[p21]x3-F2 and 3B. IBBSaB-R. The resulting product was then subjected to a second round of PCR with primers hCH2[p21]x3-F3 and 3B. IBBSaB-R. A third round of overlap extension PCR was then performed on the two second round PCR products using primers E10Age5For and 3B. IBBSaB-R. The final product was digested with PinAI/Sall and cloned into the PinAI /Sail sites of vector 3B. lBB-2G12/dgGl . The resulting construct was designated 3B.lBB-2G12/HC-p21x3/cIgGl.
The 2G12 light chain containing the KLAK peptide sequences was combined with the 2Gl 2 heavy chain containing the p21 peptide sequences by digesting plasmid 3B.lBB-2G12/LC-KLAKx2/cIgGl with Xbal/NotI and cloning the light chain insert into the Xbal/NotI sites of plasmid 3B.1BB- 2G12/HC-p21x3/cIgGl . The resulting construct was designated 3B. IBB- 2G12/LC-KLAKx2/HC-p21x3/cIgGl . Antibody expression and purification
The 2G12/cIgGl and 2G12/LC-KLAKx2/HC-p21x3/cIgGl antibodies were expressed in 293 -EBNA cells by transient transfection using Effectene reagent (Qiagen) and purified by protein A affinity chromatograph.
EXAMPLE 21: Evaluation of immune responses to a large panel of peptide embedded antibodies targeting antigen-presenting cells in an autologous T-cell assay system
To test the versatility and utility of the peptide embedding approach for antibody targeted vaccines, a large number of antibodies were designed to evaluate the following parameters (1) number (and length) of epitopes that can be embedded without substantial loss of expression, (2) the position of the epitopes e.g., structural hinge versus C-terminal end of the light chain, (3) effect of epitope orientation on processing, and (4) effect of Fc region e.g., IgGl vs. IgG2G4 hybrid.
A panel of peptide embedded antibodies was successfully produced. We evaluated whether these antibodies successfully deliver the embedded peptides to the antigen processing and presentation machinery of antigen presenting cells, resulting in stimulatory T cell responses to these peptides. To evaluate T cell responses, sixteen human donors were first screened for their T cell response to three universal tetanus toxoid HLA-DR binding peptides (described earlier). One hundred thousand to
10235000 1 87 200,000 peripheral blood lymphocytes (PBL) were incubated with 500 nM peptide for five days. Proliferation responses were determined by 3H-thymidine incorporation in the last 18 hrs. Statistically significant immune responses (P<0.01) were induced in 25% (4 of 15) of the donors by peptides 632DR and 947DR (see Table 13). Based on this result, these two peptides were selected for embedding into DC-SIGN/L-SIGN reactive antibody clone ElO (described earlier). A large panel of peptide-embedded antibodies was then evaluated for the elicitation of immune responses in an autologous DC-T cell assay system. Immature DCs (>90% DC-SIGN positive) from three donors were incubated with antibodies at 10 μg/mL (Figure 3 IA and Figure 32A) or 0.1 μg/mL (Figure 3 IB and 32B), free peptide (1 μg/mL) and TT protein (100 ng/mL) for 2 hrs at 370C, unbound antibody removed and co-cultured with 100,000 purified autologous T cells (>95% CD3 positive) and incubated for five days. Proliferation was assessed by 3H Thymidine incorporation in the last 24 hrs of the assay. The assay was performed in four replicate wells for each treatment. All peptide-embedded antibodies irrespective of the orientation, position, copy number or nature of Fc region produced a 2 fold to 10 fold increase in proliferative responses compared with the native ungrafted antibodies, which reached statistical significance in most cases (PO.01, see Table 14 and Table 15). In addition, significant immune responses were observed even at subnanomolar concentration of the antibodies (compare Figure 3 IA with Figure 3 IB). Furthermore, the proliferative responses produced by peptide embedded antibodies were equal to or higher than those produced by a 10-fold excess of free peptides. These results demonstrate that it is possible to successfully elicit robust proliferative response by incorporating peptides into the selected regions in the heavy chain and light chains respectively. This approach can therefore be invaluable for design of novel vaccines for a variety of indications.
Table 13. Human PBL responses to universal HLA-DR binding tetanus toxoid peptides. + = Statistically significant response, PO.01; [++] very strong response. Mean/SD of six well replicates.
Figure imgf000089_0001
10235000 1
Figure imgf000090_0001
Table 14. Statistical analysis of T-cell proliferation responses shown in Figure 31. Significant differences were analyzed by comparing T-cell responses induced by peptide grafted antibodies with ungrafted native antibody using two-tailed unpaired Student's t test. Significance was accepted when/? < 0.01.
Figure imgf000090_0002
Table 15. Statistical analysis of T-cell proliferation responses shown in Figure 32. Significant differences were analyzed by comparing T-cell responses induced by peptide-grafted antibodies with ungrafted native antibody using two-tailed unpaired Student's t test. Significance was accepted when/? < 0.01.
Figure imgf000090_0003
10235000 1 89
Figure imgf000091_0001
EXAMPLE 22: Assessment of cell growth inhibition by peptide toxin embedded antibodies targeting B-cell leukemic cells
Selection and Evaluation of Growth Inhibitory Peptides As a first step towards evaluating the utility of tumor targeting antibodies for delivering growth inhibitory peptides to cancer cells, four growth-inhibitory peptides were selected based on their ability to interfere with the function of key cellular proteins, e.g., p53, CDK etc. (Chen, Y. N., et al. 1999. Proc Natl Acad Sci U S A 96, 4325-4329; Datta, K., et al. 2001. Cancer Res 61, 1768-1775; Kim, A. L., et al. 1999. J Biol Chem 274, 34924-34931; and Marks, A. J., et al. 2005. Cancer Res 65, 2373- 2377). Each of these four peptides was then chemically synthesized collinearly with additional sequences (e.g., HIV TAT peptide) capable of naturally traversing the mammalian cell membrane and facilitate transport of the attached growth inhibitory peptide into the cytoplasm (see Table 16 for the sequence design). All four growth inhibitory peptides were then evaluated on a panel of eleven tumor cell lines in a cell growth inhibition assay. Various tumor cell lines were incubated with a 2-fold titrated dose (range 100 μM to 1.5 μM) of growth inhibitory peptides for two days. Growth inhibition was assessed by MTS bioreduction in the last two hours of the assay. Two peptides, Ant-p21 and TAT-KLAK showed the strongest growth inhibition. While KLAK peptide inhibited the growth of both suspension and adherent cell lines, p21 peptide was ineffective on adherent cells e.g., PC3 (see Table 17). Overall, suspension cells were more susceptible to growth inhibition than adherent cell lines. Of all cell lines tested Raji and Namalwa were most susceptible to growth inhibition by p21 and
10235000 1 90 KLAK peptides, over 50% growth inhibition was observed with less than 20 μM peptide (see Table 17). The IC50 values for growth inhibition by all four peptides are provided in Table 17. Only statistically significant (p<0.01) growth inhibition values are shown in Table 17. P values were determined by comparing peptide treated cells with medium treated cells by unpaired student's T test. Based on these growth inhibition studies, p21 and KLAK would be most suitable for grafting into antibodies reactive with RAJI or Namalwa cells.
Internalization Studies with CDl 9 antibody, clone 2Gl 2 and Proteasomal Cleavage Analysis for Embedding Selected Growth Inhibitory Peptides An antibody, clone 2G12 reactive with CD19 receptor expressed on normal
B-cells and B-cell leukemic cells e.g., Raji or Namalwa was selected for exploring the utility of embedding peptides p21 and KLAK on the inhibition of RAJI cell growth. A prerequisite for efficient delivery of peptides into the cytoplasm of cells is its ability to undergo internalization by the targeted tumor cells. The internalization of 2Gl 2 was therefore determined on RAJI cells. RAJI cells were incubated with rabbit single chain antibody, clone 2G12 or a known internalizing CD19 mouse niAb, clone BC3 at 40C and 370C. The level of cell surface antibody remaining after 2 hrs was measured by flow cytometry. As illustrated in Figure 33, RAJI cells internalized over 60% of 2Gl 2 single chain antibody in two hours similar to the known internalizing CD 19 mAb, clone BC3. Based on the peptide growth inhibition and antibody internalization studies, cleavage analysis with 2OS proteasome was performed for embedding single copies of KLAK and p21 peptides into the linker region of single chain (scFv) antibody (see Table 18) and multiple copies of the peptide into the hinge region or the C-terminus of full length (IgGl) antibody, clone 2G12 (see Table 19). Binding and Cell Growth Inhibition Studies with Peptide Toxin Embedded Antibodies
Binding and growth inhibition of the peptide embedded antibodies was performed on RAJI tumor cells. One million Raji cells were incubated with 10 μg/mL of full length IgGl antibodies embedded with three copies of p21 peptide (in the hinge) and one copy of the KLAK peptide (at the C-terminal end of light chain) antibodies for 30 min at 4C in FACS buffer, washed with ImL buffer and incubated with goat anti-human PE (1/100 dilution) for 30 min at 40C, washed and binding level analyzed on a FACScalibur. The results of this study illustrated in Figure 34 indicated a similar level of binding for both the native and peptide toxin embedded full length
10235000_l 9 J antibodies. Likewise, one million Raji cells were incubated with peptide embedded single chain (HA tagged) antibodies for 45 min at 40C in FACS buffer, washed with ImL buffer and incubated with Rat anti-HA (1:10 dilution) for 30 min at 40C, washed with ImL buffer and incubated with anti-Rat PE (1 :50uL) for 30 min at 40C, washed and analyzed by flow cytometry. As depicted in Figure 35, peptide embedded single chain antibodies showed reduced binding to Raji cells compared with non-embedded single chain antibodies.
After the completion of binding studies, the growth inhibitory effects of peptide toxin embedded antibodies were tested on Raji tumor cells in a 3-day cell proliferation assay. Raji tumor cells (10,000 cells in 3-4 well replicates) were incubated with a 2-fold titrated dose (25 μM to 1 nM) of peptide toxin embedded full length antibodies (Figure 36A and 36B), single chain antibodies (Figure 36C and 36D) or free synthetic peptides, TAT-KLAK and Ant-p21 as positive controls (Figure 36E) for three days. Cell proliferation was assessed by 3H-thymidine incorporation in the last 18 hrs. While the peptide toxin embedded full length (IgGl) antibodies produced a highly significant (p<0.01 vs. non-embedded antibodies) reduction in tumor cell growth of approximately 90% at doses almost one thousand times lower than the free synthetic peptides (compare Figure 36B with Figure 36E), peptide toxin embedded single chain antibodies had little effect on tumor cell growth (see Figure 36C and 36D). Furthermore, quite contrary to our expectations, higher doses (over 500 nM) of peptide toxin embedded full length antibodies were not effective in inhibiting tumor cell growth (see Figure 36A). Overall, these experiments provide proof that targeting internalizing receptors with mAbs harboring growth inhibitory peptides is a powerful novel approach to inhibit growth of cancer cells. Such an approach is expected to be useful in the treatment of a variety of cancers.
Table 16. Sequences of selected chemically synthesized growth inhibitory peptides. Growth inhibiting peptide sequence is shown in bold and membrane translocating sequences are underlined.
Figure imgf000093_0001
10235000 1 92
Figure imgf000094_0001
Table 17. Growth inhibitory activities of selected synthetic peptides. If the growth inhibition was less than 50%, the corresponding values are indicated in parenthesis. NE = No Effect on cell growth.
Figure imgf000094_0002
Table 18. 2OS Constitutive Proteasome Cleavage Analysis for insertion of growth inhibitory peptides, KLAK and p21 into the CD 19 antibody, clone 2Gl 2. Peptide toxin sequence is inserted into the glycine linker region of single chain
10235000 1 93 antibody and hinge region of chimeric whole IgGl. Legend: bold = cleavable linkers; CAPS = epitope; small case = Ig sequence; underlined sequence is the final insert. 'S' predicted 2OS proteasomal cleavage site (>90% probability).
Figure imgf000095_0001
Table 19. 2OS Constitutive Proteasome Cleavage Analysis for insertion of multiple copies of growth inhibitory peptides, KLAK and p21 into chimeric CD 19 antibody, clone 2Gl 2-IgGl. Peptide toxin sequence is inserted into either the hinge region of chimeric whole IgGl or attached at the C-terminus of the light chain. Legend: bold = cleavable linkers; CAPS = epitope; small case = Ig sequence; underlined sequence is the final insert; 'S' predicted 2OS proteasomal cleavage site (>90% probability); superscript in the clone name indicates the number of toxin copies in the antibody.
Figure imgf000095_0002
EQUIVALENTS The present invention provides among other things antibody-peptide fusion proteins and methods for producing and using antibody-peptide fusions proteins. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will
10235000 1 94 become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
INCORPORATION BY REFERENCE
AU publications and patents mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) (www.tigr.org) and/or the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov).
Also incorporated by reference are the following: Arif, S. et al., J Clin Invest 113: 451-463 (2004); Atkinson, M. A. et al., J Clin Invest 94: 2125-2129 (1994); Bessalle, R. et al., FEBS Lett 274: 151-155 (1990); Billetta, R. et al., Proc Natl Acad Sci U S A 88: 4713-4717 (1991); Blondelle, S. E., and Houghten, R. A. Biochemistry 31: 12688-12694 (1992); Bonifaz, L. C. et al., J Exp Med 199: 815-824 (2004); Burton DR and Woof JM, Adv. Immun. 51: 1-18 (1992); Burton DR, Molecular Genetics of Immunoglobulin, Chapter 1 (Calabi, F. and Neuberger, M.S., eds; Elsevier Science Publishers B.V. (1987)); Burton DR, Fc Receptors and the Action of Antibodies, pages 31-54 (Metzger H. ed.; American Society for Microbiology, Washington, DC (1990)); Canfield SM and Morrison SL, J. Exp. Med. 173: 1483- 1491 (1991); Casten, L. A. and Pierce, S. K., J Immunol 140: 404-410 (1988); Chiong, B. et al., J Immunother 27: 368-379 (2004); de Kroon, A. I. et al., Biochim Biophys Acta 1325: 108-116 (1997); Clynes, R. A. et al., Nat Med 6: 443-446 (2000); Diethelm-Okita, B. M. et al., J Infect Dis 181: 1001-1009 (2000); Eidem, J. K. et al., J Immunol Methods 245: 119-131 (2000); Ellerby, H. M. et al., Nat Med 5: 1032-1038 (1999); Ellerby, H. M. et al., J Neurosci 17: 6165-6178 (1997); Emmerich, N. P. et al., J Biol Chem 275: 21140-21148 (2000); Hawiger, D. et al., J Exp Med 194: 769- 779 (2001); Hovius, R. et al., FEBS Lett 330: 71-76 (1993); Javadpour, M. M. et al., J Med Chem 39: 3107-3113 (1996); Knolle, P. A. et al., Gastroenterology 116: 1428-
10235000_I 95 1440 (1999); Lautscham, G. et al., J Virol 77: 2757-2761 (2003); Lee, P. et al., J Clin Oncol 19: 3836-3847 (2001); Limmer, A. et al., Nat Med 6: 1348-1354 (2000); Liu, K. J. et al., Clin Cancer Res 10: 2645-2651 (2004); Livingston, B. D. et al., Vaccine 19: 4652-4660 (2001); Lunde, E. et al., Nat Biotechnol 17: 670-675 (1999); Marks, AJ. et al., Cancer Res 65: 2373-2377 (2005); Mehlen, P. et al., Nature 395: 801-804 (1998); Mueller, J. P. et al., MoI Immunol 34: 441-452 (1997); Parker, K.C. et al., J Immunol 152: 163-175 (1994); Peschen, D. et al., Nat Biotechnol 22: 732-738 (2004); Pop, L. M. et al., Int Immunopharrnacol 5: 1279-1290 (2005); Rasmussen, I. B. et al., Proc Natl Acad Sci U S A 98: 10296-10301 (2001); Raz, I. et al., Lancet 358: 1749- 1753 (2001); Saxova, P. et al., Int Immunol 15: 781-787 (2003); Schjetne, K.W. et al., Eur J Immunol 33: 3101-3108 (2003); Slingluff, C. L. et al., Clin Cancer Res 7: 3012- 3024 (2001); Sundaram, R. et al., Vaccine 21 : 2767-2781 (2003); Toes, R. E. et al., J Exp Med 194: 1-12 (2001); Valmori, D. et al., J Immunol 152: 2921-2929 (1994); Xiong, S. et al., Nat Biotechnol 15: 882-886 (1997); Zaghouani, H. et al., Science 259: 224-227 (1993).
10235000_l 96

Claims

What is claimed is:
1. An antibody fusion comprising: an antibody, and at least one amino acid sequence incorporated into the constant region of the antibody, wherein said amino acid sequence is flanked by one or more proteasomal cleavage sites at or directly adjacent to each end of said amino acid sequence.
2. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated into a hydrophobic region of the constant region of the antibody.
3. The antibody fusion of claim 1 , wherein said hydrophobic region comprises amino acid residues 135-146, 149-198, 243-259, 271-292, 320-329, 388- 400, or 453-464 of the heavy chain constant domain based on the Kabat numbering system.
4. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated between amino acid residues 224-251 based on the Kabat numbering system.
5. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated into the CH2 domain.
6. The antibody fusion of claim 5, wherein said amino acid sequence is incorporated into a hydrophobic region of the CH2 domain.
7. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated into the constant region of the antibody at a region having structural flexibility.
8. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated into a region comprising (i) the hinge region, (ii) 50 amino acid residues flanking the N-terminus of the hinge region, and (iii) 50 amino acid residues flanking the C-terminus of the hinge region.
9. The antibody fusion of claim 8, wherein said amino acid sequence is incorporated into a region comprising (i) the hinge region, (ii) 10 amino acid residues flanking the N-terminus of the hinge region, and (iii) 10 amino acid residues flanking the C-terminus of the hinge region.
10. The antibody fusion of claim 9, wherein said amino acid sequence is incorporated into the hinge region.
10235000 1 97
11. The antibody fusion of claim 1 , wherein said amino acid sequence is incorporated into the lower hinge region.
12. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated between the C-terminus of the hinge region and the downstream heavy chain constant region sequence.
13. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated between amino acid residues 249 and 250 based on the Kabat numbering system.
14. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated between two structural domains of the constant region.
15. The antibody fusion of claim 14, wherein said amino acid sequence is incorporated between the CH2 and CH3 domains.
16. The antibody fusion of claim 1, wherein the antibody comprises an IgG, IgA, or IgD heavy chain, or a portion thereof.
17. The antibody fusion of claim 16, wherein the antibody comprises a human IgGl, IgG2, IgG3, or IgG4 constant region, or a portion thereof.
18. The antibody fusion of claim 1, wherein the antibody comprises a hybrid constant region, or a portion thereof.
19. The antibody fusion of claim 18, wherein the hybrid constant region is a G2G4 hybrid constant region.
20. The antibody fusion of claim 18, wherein the hybrid constant region comprises glycine residues at positions 249 and 250 based on the Kabat numbering system.
21. The antibody fusion of claim 1 , wherein the antibody is an antibody fragment.
22. The antibody fusion of claim 21, wherein the antibody fragment comprises at least a portion of a heavy chain constant region, a portion of a light chain constant region, or portions of both.
23. The antibody fusion of claim 21, wherein the antibody comprises an scFv fused to a heavy chain constant region or a portion thereof.
24. The antibody fusion of claim 22, wherein the antibody fragment is an Fab, F(ab')2, or minibody.
25. The antibody fusion of claim 21, wherein the antibody comprises less than the full length heavy chain constant region.
I 10235000J 93
26. The antibody fusion of claim 25, wherein the antibody does not contain a CH2 domain.
27. The antibody fusion of claim 25, wherein the antibody does not contain a light chain constant domain.
28. The antibody fusion of claim 1, wherein said amino acid sequence is flanked by proteasomal cleavage sites naturally occurring in the antibody.
29. The antibody fusion of claim 1, wherein said amino acid sequence is flanked by proteasomal cleavage sites normative to the antibody.
30. The antibody fusion of claim 1, wherein said amino acid sequence is flanked at one end by a proteasomal cleavage site naturally occurring in the antibody and at one end by a proteasomal cleavage site normative to the antibody.
31. The antibody fusion of claim 1, wherein said amino acid sequence is flanked by at least two proteasomal cleavage sites at or directly adjacent to each end of said amino acid sequence.
32. The antibody fusion of claim 1, wherein said amino acid sequence is flanked by at least three proteasomal cleavage sites at or directly adjacent to each end of said amino acid sequence.
33. The antibody fusion of claim 1, wherein said amino acid sequence is flanked by at least one lysine or arginine at either end of the amino acid sequence.
34. The antibody fusion of claim 1, wherein said amino acid sequence is flanked by Xn at either end of said amino acid sequence wherein X is lysine or arginine independently at each position and n is at least 2.
35. The antibody fusion of claim 1, wherein said amino acid sequence is flanked by Xn at either end of the amino acid sequence wherein X is lysine or arginine independently at each position and n is at least 3.
36. The antibody fusion of claim 1, wherein said amino acid sequence further comprises one or more amino acid residues between a proteasomal cleavage site and the antibody sequence.
37. The antibody fusion of claim 1, wherein said amino acid sequence comprises about 5 to about 50 amino acids.
38. The antibody fusion of claim 1, wherein said antibody fusion comprises at least two amino acid sequences incorporated into the constant region of the antibody at the same or different locations and wherein said amino acid sequences
10235000J 99 are flanked by proteasomal cleavage sites at or directly adjacent to each end of each amino acid sequence.
39. The antibody fusion of claim 1, wherein said amino acid sequence comprises a chemotactic peptide or a functional fragment thereof.
40. The antibody fusion of claim 1, wherein said amino acid sequence comprises a growth factor or a functional fragment thereof.
41. The antibody fusion of claim 1, wherein said amino acid sequence comprises a cancer antigen.
42. The antibody fusion of claim 1, wherein said amino acid sequence comprises a cytotoxic peptide or a growth inhibitory peptide.
43. The antibody fusion of claim 42, wherein said growth inhibitory peptide comprises the amino acid sequence PVKRRLFG (SEQ ID NO: 179).
44. The antibody fusion of claim 42, wherein said cytotoxic peptide is pro-apoptotic.
45. The antibody fusion of claim 42, wherein said cytotoxic peptide comprises the amino acid sequence KXAKLAKKLAKLAK (SEQ ID NO: 55).
46. The antibody fusion of claim 42, wherein said antibody binds to a tumor-associated antigen.
47. The antibody fusion of claim 46, wherein said antibody binds to at least one of CA125, CA19-9, CA15-3, D97, gplOO, CD20, CD21, TAG-72, EGF receptor, Epithelial cell adhesion molecule (Ep-CAM), Carcino embryonic antigen (CEA), Prostate specific antigen (PSA), PMSA, CDCPl, CD26, Hepsin, HGF (hepatocyte growth factor), Met, CAIX(G250), EphhB4 (Ephrin type-B receptor 4), EGFRl, EGFR2, PDGF, VEGFR, DPP6, syndecan 1, IGFBP2 (Human insulin-like growth factor binding protein 2), CD3, CD19, CD28, CTL4, VEGF, Her2/Neu receptor, tyrosinase, MAGE 1, MAGE 3, MART, BAGE, TRP-I, CA 50, CA 72-4, MUC 1, NSE (neuron specific enolase), α-fetoprotein (AFP), SSC (squamous cell carcinoma antigen), BRCA-I, BRCA-2 or hCG.
48. The antibody fusion of claim 42, wherein said antibody binds to an immune cell specific surface protein.
49. The antibody fusion of claim 48, wherein said antibody binds to at least one of DC-SIGN, CD32, CD3, or FcεRl.
10235000_1 IQQ
50. The antibody fusion of claim 1, wherein said antibody binds to a dendritic cell specific surface protein.
51. The antibody fusion of claim 50, wherein said antibody binds to at least one of CD83, CD205/DEC-205, CD197/CCR7, or CD209/DC-SIGN.
52. The antibody fusion of claim 1, wherein said antibody binds to an internalizing receptor.
53. The antibody fusion of claim 52, wherein said antibody binds to CDl 9.
54. The antibody fusion of claim 1, wherein one or more disease specific epitopes are incorporated into the constant region of the antibody at the same or different locations.
55. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated into the constant region of the antibody by insertion.
56. The antibody fusion of claim 1, wherein said amino acid sequence is incorporated into the constant region of the antibody by replacing a portion of the constant region amino acid sequence.
57. The antibody fusion of claim 56, wherein said portion of the constant region amino acid sequence has at least about 50% sequence similarity to the amino acid sequence being incorporated.
58. The antibody fusion of claim 1 , wherein incorporation of said amino acid sequence disrupts Fc receptor binding.
59. The antibody fusion of claim 1, wherein the antibody comprises a constant region, or a portion thereof, that does not interact with the Fc receptor.
60. The antibody fusion of claim 59, wherein the antibody comprises a G2/G4 hybrid constant region, or a portion thereof.
61. The antibody fusion of claim 1 , wherein the antibody is a humanized antibody or a fragment thereof.
62. The antibody fusion of claim 1, wherein the antibody is a fully human antibody or a fragment thereof.
63. The antibody fusion of claim 1, further comprising an amino acid sequence attached to the C-terminus of the heavy chain constant region.
64. The antibody fusion of claim 63, wherein said amino acid sequence comprises a cytotoxic peptide.
65. The antibody fusion of claim 1, further comprising an amino acid sequence attached to the C-terminus of the light chain constant region.
10235000_l J Ol
66. The antibody fusion of claim 65, wherein said amino acid sequence comprises a cytotoxic peptide.
67. An antibody fusion comprising an antibody, and at least one amino acid sequence attached to the C-terminus of a constant region of the antibody by a cleavable amino acid linker.
68. The antibody fusion of claim 67, wherein said at least one amino acid sequence is attached to the C-terminus of the light chain constant region.
69. The antibody fusion of claim 67, wherein said at least one amino acid sequence is attached to the C-terminus of the heavy chain constant region.
70. The antibody fusion of claim 67, wherein said cleavable amino acid linker comprises the amino acid sequence GGXXX (SEQ ID NO: 113) wherein X is lysine or arginine independently at each position.
71. The antibody fusion of claim 67, wherein said cleavable amino acid linker comprises the amino acid sequence GGGGSGGGSXX (SEQ ID NO: 114) wherein X is lysine or arginine independently at each position.
72. The antibody fusion of claim 67, wherein said amino acid sequence is a cytotoxic peptide.
73. The antibody fusion of claim 67, wherein said amino acid sequence comprises multiple disease specific epitopes.
74. The antibody fusion of claim 73, wherein said amino acid sequence comprises multiple diabetes specific epitopes.
75. The antibody fusion of claim 74, wherein said amino acid sequence comprises multiple epitopes from one or more of glutamic-acid decarboxylase 65 (GAD65), heat shock protein 60 (HSP60), insulinoma associated protein 2 (IA-2), or proinsulin (PI).
76. The antibody fusion of claim 73, wherein said amino acid sequence comprises multiple tumor antigens.
77. The antibody fusion of claim 76, wherein said amino acid sequence comprises multiple epitopes from one or more of gplOO, Tyrosinase, Late Membrane Protein 2 (LMP-2), or Carcinoembryonic Antigen.
78. The antibody fusion of claim 67, wherein said antibody is an antibody fragment.
79. The antibody fusion of claim 67, wherein said antibody is an Fab.
10235000J 102
80. The antibody fusion of claim 67, wherein said amino acid sequence extends serum half-life of the antibody fusion.
81. The antibody fusion of claim 80, wherein said amino acid sequence is albumin, transferrin, or a functional fragment thereof.
82. The antibody fusion of claim 73, wherein said amino acid sequence is a disease specific carrier protein having two or more proteasomal cleavage hotspots.
83. An antibody fusion comprising an antibody and at least one amino acid sequence incorporated into, or directly adjacent to, the hinge region of the antibody.
84. A method for identifying an internalizing antibody comprising: contacting a cell with an antibody fusion, wherein said antibody fusion comprises an antibody that binds to a surface protein on said cell and a cytotoxic peptide, and determining survival of the cell as compared to a control cell not contacted with the antibody fusion, wherein a decrease in the level of cell survival is indicative of an internalizing antibody.
85. The method of claim 84, wherein a population of cells is contacted with a library of antibody fusions comprising a plurality of antibodies fused to a cytotoxic peptide.
86. A method for identifying a cytotoxic peptide comprising: a) contacting a cell with an antibody fusion, wherein said antibody fusion comprises an internalizing antibody that binds to a surface protein on said cell and a peptide, and b) determining survival of the cell as compared to a control cell not contacted with the antibody fusion, wherein a decrease in the level of cell survival is indicative that the peptide is cytotoxic.
87. The method of claim 86, wherein a population of cells is contacted with a library of antibody fusions comprising an internalizing antibody fused to a plurality of peptides.
88. A method for constructing an antibody fusion having one or more amino acid sequences incorporated into the constant region of the antibody, comprising: a) identifying a region in an antibody constant region having one or more of the following characteristics: hydrophobicity, structural flexibility, at least 50%
10235000J 103 sequence similarity to an amino acid sequence to be incorporated into the constant region, or multiple proteasomal cleavage sites; and b) incorporating an amino acid sequence into the identified region, thereby constructing an antibody fusion.
89. The method of claim 88, wherein said amino acid sequence is incorporated into the identified region between proteasomal cleavage sites.
90. The method of claim 89, wherein said proteasomal cleavage sites are located directly adjacent to one or both ends of said incorporated amino acid sequence.
91. The method of claim 90, wherein said proteasomal cleavage sites are located directly adjacent to each end of said incorporated amino acid sequence.
92. The method of claim 89, wherein said proteasomal cleavage sites are naturally occurring in the antibody.
93. The method of claim 89, further comprising inserting proteasomal cleavage sites into said antibody fusion at or directly adjacent to one or both ends of said amino acid sequence.
94. The method of claim 93, wherein said proteasomal cleavage sites are Xn wherein X is lysine or arginine independently at each position and n is at least 1.
95. The method of claim 94, wherein n is at least 2.
96. The method of claim 88, wherein said characteristic is hydrophobicity.
97. The method of claim 96, wherein the region further comprises at least 50% sequence similarity to the amino acid sequence to be incorporated into the constant region.
98. The method of claim 96, wherein the region has structural flexibility.
99. The method of claim 98, wherein the region is a hinge region.
100. The method of claim 88, wherein said amino acid sequence is incorporated into the identified region by insertion.
101. The method of claim 88, wherein said amino acid sequence is incorporated into the identified region by replacing at least a portion of the identified region.
102. The method of claim 88, wherein incorporation of said amino acid sequence into the identified region disrupts Fc receptor binding.
103. The method of claim 88, wherein the antibody comprises an IgG, IgA, or IgD heavy chain, or a portion thereof.
10235000_l 104
104. The method of claim 88, wherein the antibody comprises a human IgGl, IgG2, IgG3, or IgG4 constant region, or a portion thereof.
105. The method of claim 88, wherein the antibody comprises a hybrid constant region, or a portion thereof.
106. The method of claim 88, wherein the antibody is an antibody fragment.
107. The method of claim 88, wherein the antibody fragment comprises at least a portion of a heavy chain constant region, a portion of a light chain constant region, or portions of both.
108. A method for modulating an immune response in a subject, comprising administering to a subject a therapeutically effective amount of an antibody fusion comprising an antibody having at least one amino acid sequence incorporated into the constant region between one or more proteasomal cleavages sites at or directly adjacent to each end of said amino acid sequence.
109. The method of claim 108, wherein said method stimulates the immune response in said subject.
110. The method of claim 109, wherein said subject is suffering from a pathogen infection.
111. The method of claim 110, wherein said amino acid sequence comprises one or more microbial peptide epitopes.
112. The method of claim 110, wherein said antibody fusion is administered in combination with an anti-infective agent.
113. The method of claim 110, wherein said subject is suffering from an influenza infection.
114. The method of claim 113, wherein said influenza is H5N1.
115. The method of claim 109, wherein said subject is suffering from cancer.
116. The method of claim 115, wherein said amino acid sequence comprises one or more tumor associated antigens.
117. The method of claim 115, wherein said antibody fusion is administered in combination with a chemotherapeutic agent.
118. The method of claim 109, wherein said antibody fusion is administered in combination with an immunostimulatory agent.
119. The method of claim 109, wherein said antibody is specific for a dendritic cell specific surface protein.
!O235ooo_i 105
120. The method of claim 119, wherein said antibody is specific for at least one of the following: CD83, CD205/DEC-205, CD197/CCR7, or CD209/DC-SIGN.
121. The method of claim 108, wherein said method reduces the immune response in said subject.
122. The method of claim 121, wherein said method tolerizes the subject to one or more peptide epitopes.
123. The method of claim 121, wherein said subject is suffering from an autoimmune disorder, allergies or inflammation or has received a transplant.
124. The method of claim 123, wherein said amino acid sequence comprises a peptide epitope associated with an autoimmune disorder.
125. The method of claim 121, wherein said antibody is specific for a LSEC specific surface protein.
126. The method of claim 125, wherein said antibody is specific for L- SIGN.
127. The method of claim 121, wherein said antibody fusion is administered in combination with one or more of the following: an anti-inflammatory agent, an immunosuppressive agent, or anti-infective agent.
128. The method of claim 108, wherein said antibody is an internalizing antibody.
129. The method of claim 108, wherein said antibody fusion is administered in a pharmaceutical composition.
130. The method of claim 108, wherein said subject is a human.
131. A method for treating a subject suffering from a disease or disorder associated with the presence or growth of an undesired cell population, comprising administering to a subject a therapeutically effective amount of an antibody fusion comprising an antibody specific for a protein expressed on the surface of a cell in the undesired cell population and having at least one cytotoxic peptide or growth inhibitory peptide (i) incorporated into the constant region between one or more proteasomal cleavage sites at or directly adjacent to each end of said amino acid sequence, (ii) attached to the C-terminus of a constant region, or (iii) both (i) and (ii).
132. The method of claim 131, wherein said undesired cell population comprises at least one of the following cell types: bacterial cells, fungal cells, cells infected with a virus, cells associated with cancer, cells associated with an autoimmune response, or cells associated with an inflammatory response.
10235000_l 106
133. The method of claim 131, wherein said antibody is an internalizing antibody.
134. The method of claim 131, wherein said antibody binds to an internalizing receptor.
135. The method of claim 131, wherein said cytotoxic peptide is at least one of the following: anthrax lethal factor, diphtheria toxin A fragment, nonbinding active fragments of diphtheria toxin, exotoxin A, ricin A chain, abrin A chain, modeccin A chain, α-sacrin, a protein from Aleurites fordii, a protein from Dianthin, PAP, PAPII, PAP-S, Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phomycin, neomycin, or a peptide comprising the amino acid sequence KLAKLAKKLAKLAK (SEQ ID NO: 55), or fragments thereof.
136. The method of claim 131, wherein said growth inhibitory peptide is a tumor suppressor protein, or a fragment thereof.
137. The method of claim 131, wherein said antibody fusion is administered in a pharmaceutical composition.
138. The method of claim 131, wherein said subject is a human.
139. A method for treating a subject suffering from a disease or disorder associated with the presence or growth of an undesired B cell population, comprising administering to a subject a therapeutically effective amount of an antibody fusion comprising an antibody specific for a protein expressed on the surface of a B cell and having at least one cytotoxic peptide or growth inhibitory peptide (i) incorporated into the constant region between one or more proteasomal cleavage sites at or directly adjacent to each end of said amino acid sequence, (ii) attached to the C-terminus of a constant region, or (iii) both (i) and (ii).
140. The method of claim 139, wherein the subject is suffering from leukemia.
141. The method of claim 139, wherein the antibody binds to CD 19.
142. The method of claim 141, wherein the antibody comprises at least one growth inhibitory peptide incorporated between residues G249G250 based on the Kabat numbering system.
143. The method of claim 142, wherein the growth inhibitory peptide comprises the amino acid sequence PVKRRLFG (SEQ ID NO: 179).
10235000_1 107
144. The method of claim 143, wherein said antibody further comprises a cytotoxic peptide attached to a C-terminus of the antibody.
145. The method of claim 144, wherein the cytotoxic peptide is attached to the C-terminus of the light chain.
146. The method of claim 145, wherein the cytotoxic peptide comprises the amino acid sequence KLAKLAKKLAKLAK (SEQ ID NO: 55).
10235000 1 108
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