WO2000018888A1 - Immunomodulation by genetic modification of dendritic cells and b-cells - Google Patents
Immunomodulation by genetic modification of dendritic cells and b-cells Download PDFInfo
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- WO2000018888A1 WO2000018888A1 PCT/US1999/022367 US9922367W WO0018888A1 WO 2000018888 A1 WO2000018888 A1 WO 2000018888A1 US 9922367 W US9922367 W US 9922367W WO 0018888 A1 WO0018888 A1 WO 0018888A1
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Definitions
- the present invention relates generally to immunology and adeno viral gene therapy. More specifically, the present invention relates to immunomodulation by genetic modification of dendritic cells and B-cells.
- dendritic cells play a pivotal role in the immune system [Bancheareau, J. and R. M. Steinman. 1998, Dendritic cells and the control of immunity. Nature. 392:245]. Foremost, dendritic cells are recognized to serve as a key mediator of T-cell based immunity. Stemming from their important function, dendritic cells have been proposed for utility in a number of clinical strategies, especially vaccinations. It has become clear that genetic modification of these cells can promote immunity against pathogenic entities, both infectious and tumorigenic [Reeves, M. R, et al. 1996. Retro viral transduction of human dendritic cells with a tumor-associated antigen gene. Cancer Res. 56:5672-7] .
- the projecting adenoviral fiber-knob protein mediates binding to the cell surface coxsackie-adenovirus receptor (CAR) followed by interaction with and internalization of the virion b y either of the av integrins avb3 or avb5 [Wickham, T. J., et al. 1993. Integrins ⁇ v ⁇ 3 and ⁇ v ⁇ 5 promote adenovirus internalization bu t not virus attachment. 73:309-19; Bergelson, J. M., et al. 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5, Science. 275: 1320-3] .
- CAR cell surface coxsackie-adenovirus receptor
- a bispecific antibody was generated through chemical conjugation of a neutralizing anti-fiber-knob monoclonal antibody to a monoclonal antibody with affinity for the dendritic cell receptor, CD40.
- the present invention demonstrates th at adenovirus complexed with this bispecific entity mediates dramatic enhancements in gene transfer to monocyte derived dendritic cells. More importantly, an upregulation of several dendritic cell maturational markers and enhanced allo-MLR performance after infection with CD40-targeted vector w as observed, indicating the vector itself possesses maturational properties .
- the present invention fulfills this long-standing need and desire in the art.
- a bispecific antibody was generated through chemical conjugation of antibodies with affinities for the adenovirus fiber- knob and a dendritic cell receptor, CD40.
- CD40 targeted adenovirus mediates dramatic enhancements in gene transfer to monocyte derived dendritic cells and that these enhancements can be attributed to a quantitative increase in the number of cells transduced. Additionally, the present invention shows that this enhancement is specific to th e epitope recognized by the G28.5 antibody through successful blockade with the parent monoclonal, G28.5, and failure of the conjugate to mediate gene transfer on CD40 negative lines.
- One object of the present invention is to provide an adenovirus vector capable of targeting and transducing immune system cells, such as dendritic cells and B-cells, wherein transduction of B-cells results in maturation of the B-cells.
- an immunomodulatory adenovirus comprising: a n adenoviral vector, and a bispecific antibody, comprising a n antibody, or fragment thereof, recognizing a fiber-knob protein of said adenovirus conjugated to an antibody, or fragment thereof, recognizing a CD40 antigen, wherein said adenovirus is targeted to and transduces immune system cells resulting in modulation of said cells.
- the bispecific antibody may be th e product of a gene fusion.
- a n immunomodulatory adenovirus comprising: a recombinant adenoviral vector, wherein the adenoviral gene encoding a fiber- knob protein has been replaced with a gene encoding an antibody, or fragment thereof, recognizing a CD40 antigen, or encoding th e natural ligand of CD40, the trimeric CD40 ligand.
- the adenovirus is targeted to and transduces immune system cells, the transduction results in modulation of the cells.
- the adenoviral vector may express a therapeutic gene, selected from the group consisting of a gene encoding a tumor antigen, a gene encoding an antigen for an infectious agent, a gene encoding a cytotoxic agent and a gene encoding an immunomodulatory agent; the antibody recognizing the CD40 antigen is G28.5; th e immune system cells are selected from the group consisting of dendritic cells and B-cells, as well as non-immune cells selected from the group consisting of vascular endothelium cells, epithelium cells, cells exhibiting chronic inflammation and cells and vessels of Karposi's sarcoma tumors; and maturation of th e immune cells is indicative of modulation of the immune cells.
- a therapeutic gene selected from the group consisting of a gene encoding a tumor antigen, a gene encoding an antigen for an infectious agent, a gene encoding a cytotoxic agent and a gene encoding an immunomodulatory agent
- a method of immunomodulation in an individual in need of such treatment comprising the step of: administering to the individual an immunomodulatory adenovirus, wherein the adenovirus modulates an immune response in the individual.
- This modulation is due to expression of a therapeutic gene by said adenovirus, and/or maturation of immune cells.
- the immune system cells are selected from the group consisting of dendritic cells and B-cells, a s well as non-immune cells selected from the group consisting of vascular endothelium cells, epithelium cells, cells exhibiting chronic inflammation and cells and vessels of Karposi's sarcoma tumors.
- the method will be useful in treating a n individual having a disease such as cancer, infectious diseases, allo transplant rejection, xeno transplant rejection and autoimmunity diseases. Additionally, administration of the immunomodulatory adenovirus is selected from the group consisting of systemic, intradermal and ex vivo.
- a disease such as cancer, infectious diseases, allo transplant rejection, xeno transplant rejection and autoimmunity diseases.
- administration of the immunomodulatory adenovirus is selected from the group consisting of systemic, intradermal and ex vivo.
- CD40 mediates enhanced magnitude of gene transfer that is specific for CD40.
- Monocyte derived dendritic cells Figure 1 A
- the glioma cell line D65 Figure IB
- AdCMVLuc either alone or complexed with Fab-anti-CD40. After 24 hour incubation, cells were assessed for expression of luciferase.
- FIG 2 shows that targeting of adenoviral to CD40 reduces the viral MOI necessary to attain a given level of gene expression.
- Nirus either in the presence or absence of Fab-anti- CD40 conjugate, was incubated briefly and subsequently serially diluted to correspond to Multiplicity of Infections (MOFs) of 1000, 100, 10, and 1.
- Monocyte derived dendritic cells were infected and cells were assayed at 24 hours for luciferase expression.
- Figure 3 shows CD40 targeted, ⁇ l integrin targeted and liposome complexed adenoviral mediate comparable gene transfer to monocyte derived dendritic cells.
- Monocyte derived dendritic cells were infected with adenoviral encoding Green Fluorescent Protein (GFP) preincubated with one of the following: PBS, Fab-anti-CD40, Fab-anti- ⁇ l integrin conjugate, Fab- anti-EGFR conjugate or Liposomes. After 24 hour incubation at 37°C, the conditions were assessed using flow cytometry for expression of GFP and are displayed as percent GFP positive cells based on analysis of 10,000 cells.
- GFP Green Fluorescent Protein
- Figure 4 shows that CD40-targeting mediates expression of dendritic cells maturational markers.
- Samples shown indicate expression of CD83, HLA-DR, HLA-DQ, CD86, and CD54 b y flow cytometry.
- Figure 5 shows that IL-12 production is enhanced after treatment with the anti-CD40 Ab or Fab-anti-CD40 targeting conjugate.
- Monocyte derived dendritic cells were treated with the indicated retargeted adenoviral or in the absence of adenoviral with unconjugated anti-CD40 Ab or the Fab-anti-CD40 conjugate.
- the supernatants were assessed by ELISA for production of IL-12, a marker of dendritic cells maturation. Of note, values below 8 ng are beyond the linear range of detection by this assay.
- FIG. 6 shows that targeting to CD40 mediates enhancement in the capacity to generate an allo-Mixed Lymphocyte Reaction.
- Monocyte derived dendritic cells were treated with the indicated conditions and mixed with non- adherent lymphocyte responder cells MLR at the indicated Responder/Stimulator ratios (R:S). Cells were subsequently 3 H labeled and assessed for cell associated cpm after 3 days.
- Figure 7 shows that primary B-cells are deficient in CAR ( Figure 7A ) and th e ⁇ v integrin, ⁇ v ⁇ 5 ( Figure 7B).
- Cells were FACS analyzed using the anti-CAR mAb RmcB and the anti- ⁇ v ⁇ 5 specific mAb P1F6. (analysis of ⁇ v ⁇ 3 was similar to ⁇ v ⁇ 5).
- Figure 8 shows that adenoviral targeted by Fab-anti- CD40 or Fab-anti- ⁇ l integrins mediates enhanced magnitude of gene transfer to primary normal B-cells.
- Purified primary B-cells were infected with AdCMVLuc either alone or complexed th e following as indicated Fab, Fab-anti-CD40, or Fab-anti- ⁇ l integrins. After 24 hour incubation, cells were assessed for expression of luciferase.
- FIG 9 shows that in nature, activation of dendritic cells is mediated by CD40-Ligand expressed on T-helper cells that enables maturation of dendritic cells such that they can properly stimulate cytotoxic T-lymphocytes (CTL' s).
- Figure 10 shows that CD40-targeted adenovirus may substitute for CD4+ T-helper function through activation of CD40 leading to maturation of dendritic cells. For this reason, CD40- targeted adenoviral may enable stimulation of a CTL response even in the absence of functioning T-helper cells.
- Adenovirus has been used in the context of dendritic cell transduction, but its efficiency of gene delivery has proven suboptimal.
- the present invention successfully demonstrates enhanced gene transfer to monocyte derived dendritic cells by retargeting the adenovirus to CD40, a marker widely expressed on dendritic cells.
- CD40- targeted virus demonstrated both dramatic and quantitative improvements in gene transfer compared to untargeted virus .
- the present invention is directed towards adenoviral vectors targeted for the CD40 cell-surface antigen of dendritic cells and B-cells.
- the present invention is further directed towards methods of dendritic cell and B-cell transduction using a targeted adenoviral vector.
- the present invention is also directed towards the method of dendritic cell and B-cell maturation following transduction with the targeted adenoviral vector of the present invention.
- a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of th e molecule, and does not limit it to any particular tertiary forms . Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
- linear DNA molecules e.g., restriction fragments
- viruses e.g., plasmids, and chromosomes.
- a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
- a "replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
- An "origin of replication” refers to those DNA sequences that participate in DNA synthesis.
- An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
- a coding sequence is "operably linked” and “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
- expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host.
- the expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells.
- the transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.
- a DNA "coding sequence” is a double- stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
- a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
- a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
- a "cDNA” is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript.
- An “exon” is an expressed sequence transcribed from the gene locus, whereas an “intron” is a non-expressed sequence that is from the gene locus.
- DNA regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
- a "cis-element” is a nucleotide sequence, also termed a “consensus sequence” or “motif, that interacts with other proteins which can upregulate or downregulate expression of a specicif gene locus.
- a “signal sequence” can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
- a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
- the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription a t levels detectable above background.
- Within the promoter sequence will be found a transcription initiation site, as well a s protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT” boxes.
- Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the - 1 0 and -35 consensus sequences.
- oligonucleotide is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of th e oligonucleotide.
- primer refers to a n oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
- the primer may be either single-stranded or double-stranded and must b e sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
- the exact length of the primer will depend upon many factors, including temperature, source of primer and use the method.
- the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
- Primers are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands . Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that th e primer sequence has sufficient complementarity with th e sequence or hybridize therewith and thereby form the template for the synthesis of the extension product.
- the terms “restriction endonucleases” and “restriction enzymes” refer to enzymes which cut double- stranded DNA at or near a specific nucleotide sequence.
- “Recombinant DNA technology” refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include “gene splicing", “molecular cloning” and “genetic engineering”. The product of these manipulations results in a “recombinant” or “recombinant molecule”.
- a cell has been "transformed”, “transfected” or “transduced” with exogenous or heterologous DNA when such DNA has been introduced inside the cell.
- the transforming DNA may or may not be integrated (covalently linked) into the genome of th e cell.
- the transforming DNA may be maintained on an episomal element such as a vector or plasmid.
- a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited b y daughter cells through chromosome replication.
- a "clone” is a population of cells derived from a single cell or ancestor by mitosis.
- a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
- An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed "transgenic".
- the term "host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells.
- a recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art.
- One preferred embodiment is the use of a vectors for purposes of prokaryotic transformation.
- Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis.
- Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells, and more preferentially, plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.
- Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
- heterologous' region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
- the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
- the coding sequence is a construct where th e coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
- fragment as applied to a antibody, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence.
- Antibody fragments can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant antibodies, by recombinant DNA techniques using an expression vector that encodes a defined fragment of an antibody, or b y chemical synthesis. The ability of a candidate fragment to exhibit binding to an antigen can be assessed by methods described herein.
- a standard Northern blot assay can be used to ascertain the relative amounts of mRNA in a cell or tissue obtained from plant or other transgenic tissue, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art.
- a standard Southern blot assay may be used to confirm the presence and th e copy number of a gene in transgenic systems, in accordance with conventional Southern hybridization techniques known to those of ordinary skill in the art.
- Both the Northern blot and Southern blot use a hybridization probe, e.g. radiolabelled cDNA, or a fragment of that DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 1 00 consecutive nucleotides in length).
- the DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art.
- the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to untraviolet light, and others.
- a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
- a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also b e labeled with a radioactive element or with an enzyme.
- the radioactive label can be detected by any of the currently available counting procedures.
- the preferred isotope may be selected from 3H, 14C, 3 p, 35 S , 36Q, 51 Cr , 57 C ⁇ , 58 C ⁇ , 59 Fe , 90 Y) 1251, 1311, and 186 Re.
- Enzyme labels are likewise useful, and can b e detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
- the enzyme is conjugated to the selected particle by reaction with bridging molecules such a s carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ - D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
- U.S. Patent Nos. 3 ,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
- immunomodulatory shall refer to the capacity to promote or suppress immunity towards cancer, infectious agents, autoimmune antigens, or allo/xeno transplants.
- maturation refers to expression of specific surface markers, production of defined soluble factors, or enhanced performance in a Mixed Lymphocyte Reaction all of which are known to be characteristic of a cell which has become more efficient in the capacity to elicit a response from effector cells, such as T-cells.
- CD40 antigen shall refer to a member of the TNF receptor (TNFR) family. It serves as th e receptor for CD40 Ligand (gp39). This molecule is known to b e expressed on B-lymphocytes, monocytes, dendritic cells, endothelium, epithelial cells, and fibroblasts. Of note, this molecule is known to be especially prevalent in areas of activated endothelium (such as chronic inflammation) and on the vessels of Kaposi's sarcoma.
- compositions may be prepared using the novel adenoviral vector of the present invention.
- the pharmaceutical composition comprises the novel adenoviral vector of the present invention and a pharmaceutically acceptable carrier.
- a person having ordinary skill in this art would readily be able to determine, without undue experimentation, the appropriate dosages and routes of administration of this adenoviral vector of the present invention.
- the adenoviral vector of the present invention is administered to the patient or an animal in therapeutically effective amounts, i.e., amounts that eliminate or reduce the tumor burden due to a n immunomodulatory effect. It will normally be administered parenterally, preferably intravenously, but other routes of administration will be used as appropriate.
- the dose and dosage regimen will depend upon the nature of the disease and its population, the characteristics of the particular vector, e.g., its therapeutic index, the patient, the patient's history and other factors.
- the amount of adenoviral vector of the present invention administered will typically be in the range of about 0.001 to about 500 mg/kg of patient weight.
- the schedule will be continued to optimize effectiveness while balanced against negative effects of treatment. See Remington's Pharmaceutical Science, 17th Ed. (1990) Mark Publishing Co., Easton, Penn.; and Goodman and Gilman's: Th e Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press.
- the adenoviral vector will most typically be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
- a pharmaceutically acceptable parenteral vehicle are preferably non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used a s carriers.
- the vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
- the adenoviral vector will typically be formulated in such vehicles at concentrations of about 0.001 mg/ml to 500 mg/ml.
- the present invention is directed to a gene delivery system for the genetic manipulation of immune system cells, comprising: (a) an adenovirus; and (b) a component recognizing CD40 antigen.
- the component recognizing the CD40 antigen is selected from the group consisting of a trimeric CD40 ligand conjugated to a fiber-knob protein of the adenovirus and a first antibody, or fragment thereof, directed to a fiber-knob protein of said adenovirus, wherein said first antibody is attached to a second antibody, or fragment thereof, directed to CD40 antigen.
- a representative antibody directed to CD40 antigen is G28.5.
- the first antibody and second antibody may be genetically fused together.
- This gene delivery system can be used to transduce and immunomodulate immune system cells.
- this system may also comprise a therapeutic gene.
- Representative therapeutic gene include a gene encoding a tumor antigen, a gene encoding an antigen for an infectious agent, a gene encoding an autoimmune antigen, an immunomodulatory gene and a gene encoding a cytotoxic agent.
- Representative immune sy stem cells which can be transduced and immunomodulated using this system include of dendritic cells and B-cells. In one aspect, the B- cells are matured following contact with said system.
- the present invention is also directed to a method of genetically manipulating immune system cells in an individual in need of such treatment, comprising the step of administering the gene delivery system described above to the individual.
- This method may be useful where the individual has a disease selected from the group consisting of cancer, an infectious disease, allotransplant rejection, xenotransplant rejection and autoimmune diseases.
- Representative immune system cells which can b e transduced and immunomodulated using this system include of dendritic cells and B-cells. In one aspect, the B-cells are matured following contact with said system.
- the present invention is also directed to a method of genetically manipulating immune system cells in an individual in need of such treatment, comprising the step of administering th e gene delivery system comprising a therapeutic gene to said individual.
- the present invention is also directed to a recombinant adenoviral vector for the genetic manipulation of immune system cells, wherein the adenoviral gene encoding a fiber-knob protein has been replaced with a gene encoding a protein recognizing a CD40 antigen.
- the gene recognizing said CD40 antigen is selected from the group consisting of a gene encoding a trimeric CD40 ligand and a gene encoding an antibody, or fragment thereof, directed to said CD40 antigen.
- a prefered antibody directed to CD40 antigen is G28.5.
- This recombinant adenoviral vector can b e used to transduce and immunomodulate immune system cells.
- the recombinant adenoviral vector may further comprise a therapeutic gene such as a gene encoding a tumor antigen, a gene encoding an antigen for an infectious agent, a gene encoding a n autoimmune antigen, an immunomodulatory gene and a gene encoding a cytotoxic agent.
- This recombinant adenoviral vector can be used in a method of genetic manipulating immune system cells in an individual in need of such treatment. Such individuals may have a disease such as cancer, an infectious disease, allo transplant rejection, xeno transplant rejection and autoimmune diseases.
- PBMC Peripheral Blood Mononuclear Cells
- Fresh or cryopreserved PBMC were suspended at a concentration of 3 to 5 million cells per ml in Iscove's modified Dulbecco's medium containing 50 U/mL penicillin-streptomycin, 1.6 mM L-Glutamine, 0.01 mM ⁇ -mercaptoethanol (complete medium), and 10% FCS and were allowed to adhere to the bottom of plastic culture flasks (NUNC, Intermed, Denmark). After 2 hours at 37°C, non- adherent cells were removed by rinsing with PBS.
- the adherent cells were cultured for a further 6 days in complete medium with 10% FCS supplemented with 1000 U/ml rIL-4 (CLB, Amsterdam, The Netherlands) and 100 ng/mL GM-CSF. Loosely adherent cells with typical dendritic cell morphology were harvested (adherent cells were detached by incubation with 0.5 mM EDTA in PBS) and us ed for FACS analysis or adenovirus mediated gene transfer.
- monocyte derived dendritic cells were added as stimulator cells to roundbottom 96-well culture plates (Nunclon Delta, Intermed, Denmark) at graded doses. Non- adherent lymphocyte fractions were used as a source for responder cells. Per well 1 X 10 5 lymphocytes were added to the allogeneic or autologous monocyte derived dendritic cells at the indicated Responder/Stimulator ratios (R:S). The cells were cultured for 3 days in complete medium with 10% Human Pooled Serum (CLB, Amsterdam, The Netherlands).
- [ 3 H]- thymidine was added (0.4 mCi per well) (Amersham, Aylesbury, UK), after which the cells were harvested onto fiberglass filters and [ 3 H]-thymidine incorporation was determined using a flatbed liquid scintillation counter (Wallac, Turku, Finland).
- Cell staining was performed using monoclonal antibodies (MoAbs) directly conjugated to Fluorescein Isothiocyanate (FITC) or to Phycoerthrin (PE).
- the antibodies used were HB 15 (CD83), BL6 (CDla), BU15 (CDl lc), MAB89 (CD40), (Immunotech, Marseille, France), SK7 (CD3), 4G7 (CD19), B73.1 (CD16), MoP9 (CD14), NCAM 16.2 (CD56), L243 (HLA-DR), 2A3 (CD25) (Becton Dickinson, San Jose, CA), 2331 (CD86), G46-2.6 (HLA A, B, C), HA58 (CD54), and TU169 (HLA-DQ) (Pharmingen, San Diego, CA).
- the samples were analyzed on a FACStar using Cellquest FACS analysis software (Becton Dickinson).
- AdCMVLuc a first generation E1-, E3-deleted vector expressing firefly luciferase from the CMV immediate early promoter, was obtained from Robert Gerard (University of Leuven, Leuven, Belgium). Viruses were propagated and plaque titered on the permissive line 293 and purified by double centrifugation on CsCl gradients. All virus aliquots were stored at -80°C until use. Murine monoclonal antibody RmcB to human coxsackie/adenovirus receptor (from Dr. Robert Finberg, Dana Farber Cancer Institute) has been described previously.
- Murine monoclonal antibody LM609 to avb3 and P1F6 to avb5 integrin were purchased from Chemicon (Temecula, CA) and Gibco BRL (Gaithersburg, MD) respectively.
- the neutralizing murine monoclonal antibody 1D6.14 specific for the carboxy-terminal, receptor binding domain of adenoviral serotype 5 has been described.
- the hybridomas G28.5, producing anti-CD40 monoclonal antibodies (ATCC#:9110-HB) and TS2/16.2.1 (ATCC#: 243-HB; "TS2") producing monoclonal antibodies against the ⁇ l integrin were purchased from ATCC. Both hybridomas were used to generate ascites in SCID mice.
- Antibodies were purified on an FPLC chromatography system using HiTrap Protein A column (Pharmacia) and the MAPS binding buffer system (Bio-Rad).
- Conjugate was purified on a HR 1 0/ 30 Superose 12 column using FPLC (Pharmacia, Piscataway, NJ) in Borate buffer pH 8.5, wherein the fractions were pooled that corresponding to a 1: 1 ratio of anti-receptor antibody to Fab, a t an approximate molecular weight of 200 kDa.
- Nonadherent monocyte derived dendritic cells w ere collected and mixed with the 0.5 mM EDTA released adherent cell fraction followed by washing in complete RPMI containing 2.5% FCS. Twenty-four thousand cells in a volume of 50 ⁇ l w ere distributed to individual microcentrifuge tubes in triplicate for each test condition. The use of microcentrifuge tubes enabled simplified infection and washing of cells, which represented both adherent and nonadherent fractions. Conjugate and virus w ere incubated for 30 minutes at room temperature in a minimal volume of under 10 ⁇ l per each test condition's worth of virus. Following incubation the mixture was diluted such that 100 ⁇ L was used to infect each microcentrifuge tube of cells.
- the amount of virus in this volume corresponded to a multiplicity of infection of 100.
- Microcentrifuge tubes containing the infection mixture were placed at 37°C for 1 hour. Subsequently, to remove unbound virus, cells were washed in the tubes with PBS, centrifuged, and the supernatant aspirated. Pelleted cells were resuspended in 1 mL of RPMI 10% FCS and moved to individual wells of a polylysine coated 24-well plate for overnight incubation.
- Use of polylysine coated wells enabled simpler processing in subsequent luciferase assays by anchoring of both adherent and suspension fractions to the well surface.
- the conjugate was titrated on a predetermined number of viral particles at a n MOI of 100, wherein gene transfer was measured in terms of luciferase expression as relative light units, RLU, in monocyte derived dendritic cells.
- Monocyte derived dendritic cells w ere infected with AdCMVLuc preincubated with increasing concentrations of Fab-G28.5.
- virus ratio proved to reduce the magnitude of retargeted gene transfer, presumably stemming from competition for CD40 binding by excess Fab-G28.5 conjugate.
- Fab-G28.5 was complexed with AdCMVLuc at a concentration corresponding to 1000 MOI. Subsequently, this mixture was serially diluted to MOI's of 500, 100, 50, 10, and 1. Simultaneously, samples of the same MOI's of adenovirus without retargeting conjugate were prepared for comparison with targeted samples. Monocyte derived dendritic cells were then infected and analyzed for luciferase as was done in the luciferase gene transfer experiments.
- Monocyte derived dendritic cells were generated b y treatment of monocytes isolated from peripheral blood with IL-4 and GM-CSF. The identity of these cells was validated in two ways. Purity was demonstrated through flow cytometry for lack of expression of CD14, CD3 and CD19. Further, the cells exhibited a dendritic cell phenotype with some veiled cells and a mixture of adherent and nonadherent fractions associated in multicellular clusters. These monocyte derived dendritic cells were negative for expression of dendritic cells maturational markers, such as CD83, and were thus immature. EXAMPLE 11
- the conjugate was titrated on a predetermined number of viral particles at an MOI of 100, wherein gene transfer was measured in terms of luciferase expression in monocyte derived dendritic cells.
- Monocyte derived dendritic cells were infected with AdCMVLuc preincubated with increasing concentrations of Fab-G28.5.
- CD40-targeted gene transfer reached a maximum with a Fab-G28.5 conjugate-virus ratio of 30 ng Fab-G28.5 per 2.4X 10 6 pfu (1.75 X 1 0 8 particles/mL as determined by OD 260 ).
- CD40 targeted adenoviral demonstrated a two logic enhancement in gene transfer to monocyte derived dendritic cells, as determined by expression of the Luciferase reporter gene. This optimal dose was analyzed in several ways for its specificity to CD40.
- Enhancements in gene transfer are due to quantitatively increased numbers of cells transduced While luciferase gene transfer had illustrated an overall increase in gene expression due to CD40-targeted adenovirus, the nature of this assay could indicate whether a n increased number of cells had actually been transduced.
- adenovirus containing a quantitative marker, Green Fluorescent Protein, GFP was used. The number of cells transduced was monitored through use of flow cytometry. It w as determined that compared to cells infected with untargeted adenovirus, CD40-targeted adenovirus quantitatively transduced more cells. Comparable levels of gene transfer were observed with two other methods, bl integrin targeted adenovirus and liposome complexed adenovirus. Once again, this enhanced gene transfer was absent when an irrelevant conjugate to EGFR w as used.
- Fab-G28.5 enhances adenovirus mediated gene transfer in different donors and such retargeting can reduce the viral dose required to achieve a given level of transgene expression
- a more rigorous index of dendritic cell maturation is the mixed lymphocyte reaction.
- MDDC treated using several vectors or conjugates were combined with responder cells from a n allogeneic donor and tested for the capacity to elicit responder cell proliferation. While adenoviral alone did not mediate enhancement in MLR, any treatments in the presence or absence of adenoviral were able to dramatically promote MDDC reactivity in the allo-MLR ( Figure 6). Moreover, while the effect of unconjugated mAb was significantly less than that seen with Fab- anti-CD40 conjugate in the presence of adenoviral, the effect of conjugate alone was comparable to that seen with the conjugate with virus.
- Peripheral dendritic cells's active in the process of antigen capture are referred to as “immature dendritic cells.”
- these cells do not express the appropriate panel of costimulatory molecules and cytokines necessary to activate effector cells such as cytotoxic T-lymphocytes (CTL's).
- CTL's cytotoxic T-lymphocytes
- immature dendritic cells must be differentiated to a n immunologically potent "mature" status by CD40 activation [Bennett, S. R. M., et al. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signaling. Nature. 393:478-480; Ridge, J. P., e t al. 1998.
- a conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature. 393 :474-7 ; Schoenberger, S. P., et al. 1998. Nature. 393:478-80; Ridge, J. P., e t al. 1998. T-cell help for cytotoxic T-lymphocytes is mediated b y cd40-cd40L interactions. 393:480-3] . For this reason, the effects the CD40-targeted adenoviral vector have on the maturational status of dendritic cells was examined.
- the present invention shows that retargeting adenoviral gene delivery to CD40 mediates dramatic increases in the magnitude of gene transfer and maturational effects that are specific for CD40. Consequently, despite th e comparable enhancements of conjugate targeted adenoviral and liposome complexed adenovirus ex vivo, the more cell specific targeting and maturational potential of CD40-targeted adenoviral would, in theory, lend itself more reliably to in vivo approaches.
- FACS analysis revealed a reduction in surface CD40 expression at 24 hours . Since the conjugate has been detected on the cell surface at 4 8 hours after treatment, it is possible that the retained conjugate might have obscured subsequent detection of CD40.
- the present invention shows that Fab-anti-CD40 conjugate mediates more dramatic MLR reactivity in MDDC's th an seen with unconjugated anti-CD40 mAb.
- Previous reports implicate CD40 crosslinking as a means to activate the CD40 pathway and herein are proposed two means by which th e present system has altered the crosslinking kinetics of this antibody.
- the inherent trimericity of the fiber-knob lends itself to binding of up to three conjugate molecules per each of twelve capsid vertices.
- Second is the semi-random nature of th e chemical crosslinking procedure which can result in heterodimers with ratios besides a simple 1 : 1 Fab to anti-CD40 mAb.
- adenovirus mediates minor effects on dendritic cells phenotype, but these effects are seen only when a sufficient number of particles enter each cell, such as by the high efficiency antibody-targeted or liposome- complexed adenoviral based gene transfer vectors. It is interesting to speculate as to whether the enhanced expression of costimulatory molecules seen with ⁇ l integrin-targeted or liposome-complexed adenoviral is a consequence of the capsid itself entering the cell, expression of the transgene, or b y background adenoviral gene expression.
- the dual role of CD40 in this scenario as both a surrogate adenoviral receptor and a powerful trigger of dendritic cell maturation will be useful as a retargeting strategy to this central cell type of the immune system.
- CD40-retargeted adenoviral vector by delivery of an antigen-encoding gene, a larger pool of dendritic cells's can be generated with the potential to prime effector cells against the antigen of interest, especially important in the case of cryptic antigens that might otherwise b e unaccessible to the immune system.
- CD40 Stemming from the important role of CD40 in T-helper activation of dendritic cells, such a system might also have applications in bypassing the need for CD4+ T-cell help in activation of CTL.
- bispecific-antibody based targeting of adenovirus for clinical purposes has been previously suggested, the limitations of this antibody based strategy for intensive clinical applications has been recognized. For this reason, a genetic fusion strategy between the trimeric adenovirus fiber and the natural ligand of CD40, trimeric CD40L, is useful.
- lymphocytes are a difficult cell type into which genes can b e delivered.
- hematopoetic cells have been documented for their failure to mediate binding and/ or internalization of adenoviral viral particles [Silver, L. and C.W. Anderson. 1988. Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection. J. of Virology. 62 : 341 - 5; Mentel, R., et al. 1997. Adenovirus-receptor interaction with human lymphocytes. J. of Med. Virology. 51:252-7; Wattel, E., et al. 1996.
- conjugates Fab-anti- CD40 and Fab-anti ⁇ l integrins directed against the B-cell markers CD40 and the ⁇ l integrins, respectively, were used. Both of these conjugates were expected to reconstitute binding to replace the absence of CAR and to provide an alternative method for virion internalization into the cells. By virtue of the previously described internalizing function of these receptors, these conjugates were also anticipated to reconstitute the internalizing function of the av integrins. By use of either of these retargeting strategies, gene transfer to primary B-cells has been enhanced b y a least 10-fold over untargeted adenoviral (Figure 8). These results are particularly interesting because targeting of adenoviral to CD40 or the ⁇ l integrins seems to have simultaneously overcome deficiency of both the primary binding receptor as well as the secondary, internalizing receptor.
Abstract
Description
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AT99954665T ATE310806T1 (en) | 1998-09-29 | 1999-09-28 | IMMUNE MODULATION USING GENETIC MODIFICATION OF DENDRITIC CELLS AND B CELLS |
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CA (1) | CA2344655A1 (en) |
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WO (1) | WO2000018888A1 (en) |
Cited By (2)
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US7354888B2 (en) | 2004-11-10 | 2008-04-08 | Danisco A/S | Antibacterial composition and methods thereof comprising a ternary builder mixture |
WO2009141335A1 (en) * | 2008-05-22 | 2009-11-26 | Proyecto De Biomedicina Cima, S.L. | An adapter molecule for the delivery of adenovirus vectors |
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US6136311A (en) | 1996-05-06 | 2000-10-24 | Cornell Research Foundation, Inc. | Treatment and diagnosis of cancer |
US20050095231A1 (en) * | 1998-02-17 | 2005-05-05 | Curiel David T. | Modified adenovirus containing a fiber replacement protein |
US6841540B1 (en) * | 1998-09-29 | 2005-01-11 | The Uab Research Foundation | Immunomodulation by genetic modification of dendritic cells and B cells |
AU5178800A (en) * | 1999-06-01 | 2000-12-18 | Cornell Research Foundation Inc. | Activation of dendritic cells to enhance immunity |
US20030059461A1 (en) * | 2000-06-06 | 2003-03-27 | Sibtech, Inc. | Molecular delivery vehicle for delivery of selected compounds to targets |
US7355019B2 (en) * | 2000-06-06 | 2008-04-08 | Sibtech, Inc. | Cysteine-containing peptide tag for site-specific conjugation of proteins |
AU2003252118A1 (en) * | 2002-07-22 | 2004-02-09 | Emd Lexigen Research Center | Targeted adenoviral vector displaying immunoglobulin-binding domain and uses thereof |
US7045348B2 (en) * | 2002-07-22 | 2006-05-16 | Vectorlogics, Inc. | Adenoviral vector incorporating zipper peptide-modified fiber protein and uses thereof |
US9701754B1 (en) | 2002-10-23 | 2017-07-11 | City Of Hope | Covalent disulfide-linked diabodies and uses thereof |
AU2003295502A1 (en) * | 2002-11-12 | 2004-06-03 | Yucheng Chang | Adenoviral vector vaccine |
WO2005024032A2 (en) * | 2003-09-03 | 2005-03-17 | Vectorlogics, Inc. | Targeting adenoviral vectors to dendritic cells |
JP2009531324A (en) | 2006-03-20 | 2009-09-03 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Engineered anti-prostatic stem cell antigen (PSCA) antibody for cancer targeting |
US20090074711A1 (en) * | 2006-09-07 | 2009-03-19 | University Of Southhampton | Human therapies using chimeric agonistic anti-human cd40 antibody |
EP2607477B1 (en) | 2007-05-03 | 2020-09-23 | The Brigham and Women's Hospital, Inc. | Multipotent stem cells and uses thereof |
WO2009032949A2 (en) | 2007-09-04 | 2009-03-12 | The Regents Of The University Of California | High affinity anti-prostate stem cell antigen (psca) antibodies for cancer targeting and detection |
WO2010096486A1 (en) | 2009-02-17 | 2010-08-26 | Cornell Research Foundation, Inc. | Methods and kits for diagnosis of cancer and prediction of therapeutic value |
RU2673908C2 (en) | 2009-12-02 | 2018-12-03 | Имэджинэб, Инк. | J591 minibodies and cys-diabodies for targeted delivery of human prostate specific membrane antigen (psma) and methods for their use |
AU2016304764C1 (en) | 2015-08-07 | 2023-06-01 | Imaginab, Inc. | Antigen binding constructs to target molecules |
EP3565578A1 (en) | 2016-12-21 | 2019-11-13 | Memgen LLC | Armed replication-competent oncolytic adenoviruses |
US11266745B2 (en) | 2017-02-08 | 2022-03-08 | Imaginab, Inc. | Extension sequences for diabodies |
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US5543328A (en) * | 1993-08-13 | 1996-08-06 | Genetic Therapy, Inc. | Adenoviruses having modified fiber proteins |
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JP4242447B2 (en) * | 1993-01-22 | 2009-03-25 | イミュネックス・コーポレーション | Detection and treatment of CD40 ligand gene mutations |
ATE267607T1 (en) * | 1993-12-23 | 2004-06-15 | Immunex Corp | USE OF SOLUBLE OLIGOMERIC CD40 LIGANDS OR MONOCLONAL ANTIBODIES FOR THE PRODUCTION OF A MEDICINAL PRODUCT FOR THE PREVENTION OR TREATMENT OF NEOPLASTIC DISEASES |
US5846782A (en) * | 1995-11-28 | 1998-12-08 | Genvec, Inc. | Targeting adenovirus with use of constrained peptide motifs |
US5872154A (en) * | 1995-02-24 | 1999-02-16 | The Trustees Of The University Of Pennsylvania | Method of reducing an immune response to a recombinant adenovirus |
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1999
- 1999-09-28 JP JP2000572336A patent/JP2002538767A/en not_active Withdrawn
- 1999-09-28 AT AT99954665T patent/ATE310806T1/en not_active IP Right Cessation
- 1999-09-28 EP EP99954665A patent/EP1117767B1/en not_active Expired - Lifetime
- 1999-09-28 WO PCT/US1999/022367 patent/WO2000018888A1/en active IP Right Grant
- 1999-09-28 AU AU10962/00A patent/AU759762B2/en not_active Ceased
- 1999-09-28 CA CA002344655A patent/CA2344655A1/en not_active Abandoned
- 1999-09-28 US US09/407,511 patent/US6284742B1/en not_active Expired - Lifetime
- 1999-09-28 DE DE69928556T patent/DE69928556T2/en not_active Expired - Lifetime
Patent Citations (1)
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US5543328A (en) * | 1993-08-13 | 1996-08-06 | Genetic Therapy, Inc. | Adenoviruses having modified fiber proteins |
Non-Patent Citations (1)
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TILLMAN B. ET AL.: "Dendritic cells are transduced with high efficiency by a CD40-targeted adenovirus that also induces dendritic cell maturation", J. LEUK. BIOL., vol. 2, 1998, pages 45, ABSTRACT B68, XP002925732 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7354888B2 (en) | 2004-11-10 | 2008-04-08 | Danisco A/S | Antibacterial composition and methods thereof comprising a ternary builder mixture |
WO2009141335A1 (en) * | 2008-05-22 | 2009-11-26 | Proyecto De Biomedicina Cima, S.L. | An adapter molecule for the delivery of adenovirus vectors |
Also Published As
Publication number | Publication date |
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EP1117767B1 (en) | 2005-11-23 |
JP2002538767A (en) | 2002-11-19 |
AU759762B2 (en) | 2003-05-01 |
CA2344655A1 (en) | 2000-04-06 |
EP1117767A1 (en) | 2001-07-25 |
DE69928556D1 (en) | 2005-12-29 |
DE69928556T2 (en) | 2006-08-10 |
US6284742B1 (en) | 2001-09-04 |
ATE310806T1 (en) | 2005-12-15 |
AU1096200A (en) | 2000-04-17 |
EP1117767A4 (en) | 2002-07-24 |
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