WO2017158421A1

WO2017158421A1 - Anti-viral engineered immunoglobulins

Info

Publication number: WO2017158421A1
Application number: PCT/IB2017/000314
Authority: WO
Inventors: Maria BOTTERMANN; Inger Sandlie; Leo James; Stian FOSS; Jan Terje ANDERSEN
Original assignee: University Of Oslo; Medical Research Council
Priority date: 2016-03-14
Filing date: 2017-03-14
Publication date: 2017-09-21

Abstract

The present invention relates to compositions and methods for antibody-mediated immunity against viral targets. In particular, provided herein are engineered immunoglobulins with anti-viral activity.

Description

ANTI- VIRAL ENGINEERED IMMUNOGLOBULINS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Antibody-mediated immunity forms a crucial part of the anti-pathogen (e.g., antiviral) immune response, and its induction is a principal objective of vaccination. Recently, a novel mechanism termed antibody-dependent intracellular neutralization (ADIN) was described where it was shown that antibodies can mediate neutralization intracellularly by recruiting a cytosolic antibody binding receptor named tripartite motif-containing protein 21 (TRIM21). The engagement of antibody-virus complexes by TRIM21 promotes the degradation of both antibodies and virus by the proteasome. Cells depleted of TRIM21 are inefficient in neutralization whereas interferon-stimulated cells have robust TRIM21 expression and efficient neutralization. The importance of ADIN in virus neutralization has been confirmed by the genetic knockout of TRIM21 in mouse cells and site-directed mutagenesis to ablate TRIM21 binding, either of which is sufficient to substantially diminish the potency of cytosolic neutralizing monoclonal antibodies. Recognition of intracellular antibodies by TRIM21 also activates immune signaling that results in production of pro-inflammatory cytokines, modulation of natural killer stress ligands and induction of an antiviral state. Thus, the antibody-TRIM21 detection system provides potent, comprehensive activation of the innate immune system.

The neonatal Fc receptor (FcRn) is a key player in several immunological and non- immunological processes, as it mediates maternal -fetal transfer of IgG, regulates the serum persistence of IgG and albumin, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation and cross-presentation. In contrast to TRIM21 that is found in the cytosol, FcRn is a transmembrane receptor that resides predominantly within acidified endosomes and transports its ligands either via a recycling pathway or a transcytotic pathway across polarized cell layers. However, FcRn may also enhance processing of immune complexes followed by presentation of antigenic peptides to T cells. A hallmark of the FcRn-IgG interaction is that FcRn binds the IgG Fc in a strictly pH dependent fashion, binding at acidic pH and no binding or release at neutral pH, which is a prerequisite for FcRn-mediated transport in and out of cells. Engineering of the FcRn-IgG Fc interaction has given rise to antibodies with shorter or longer serum half-life or altered capacity to be transported across cell layers. Both TRIM21 and FcRn are broadly expressed and as such are found on distinct cellular locations in both hematopoietic and non- hematopoietic cells. Furthermore, TRIM21 and FcRn share partially overlapping binding sites of the IgG Fc.

Improved therapeutic antibodies are needed.

SUMMARY OF THE INVENTION

For example, in some embodiments, the present invention provides the use of a variant immunoglobulin comprising at least one mutation in the Fc region and/or hinge region to modulate inflammatory signaling in a subject.

In further embodiments, the present invention provides the use of a variant immunoglobulin comprising at least one mutation in the Fc region and/or hinge region to neutralize a viral infection in a subj ect.

Further embodiments provide a method of modulating inflammatory signaling in a subject, comprising: administering a variant immunoglobulin comprising at least one mutation in the Fc region and/or hinge region to the subject.

Additional embodiments provide a method of neutralizing a viral infection in a subject, comprising: administering a variant immunoglobulin comprising at least one mutation in the Fc region and/or hinge region to the subject.

In some embodiments, the immunoglobulin exhibits altered binding (e.g., increased or decreased binding affinity) to TRIM21 relative to an immunoglobulin without the mutation. In some embodiments, the immunoglobulin exhibits altered binding to FcRn and/or altered activation of NFkB relative to an immunoglobulin without the mutation. In some

embodiments, the immunoglobulin further comprises one or more mutation in the hinge region. In some embodiments, the immunoglobulin is an IgGl, IgG2, IgG3, or IgG4 subclass. In some embodiments, the immunoglobulin has mutations at one or more of positions selected from, for example, 131, 31 1 , 345, 385, 433, 434, 435, 436, or 428. In some embodiments, the mutation is selected from, for example, M428L/N434S,

M252Y/S254T/T256E, H433K/N434F, IgGl-Q31 1R/N434W/M428E, IgGl- Q31 1R/N434W, IgGl-Q311R, IgGl -N434W, IgGl-N434Y, IgGl -Q31 1H, IgGl-H433R, IgGl-E345R, IgGl-MST/G385E/M428E, IgGl-Y436W, IgG3(b)-

Q311R/N434F/H433R/R435H, IgGl-M428E, IgGl-Q311R, IgGl-Q311R/M428E, IgGl-

Q311H/M428E, IgGl-G385E/M428E, IgGl-M428F, IgGl-N434K, IgGl-Q311F, IgGl-

N434H, IgG3(b)-Q311R/G385E/M428E/R435H, IgGl-Q311R/G385E/M428E, IgGl- Q311R/N434Y/M428F, IgGl-Q311R/G385E/M428E/N434Y, IgGl-

Q311R/G385E/M428F/N434Y, IgG3-R435H, IgGl-N434W, IgGl-N434F, IgGl-N434Y,

IgG3-N434F/R435H, IgGl-S 131C-IgG3Hinge, IgG3-C131 S-IgGlHinge, IgGl-IgG3Fc,

IgG3-IgGlFc, IgG3-AHinge_exonl, IgG3_AHinge_exonl_2, IgG3_AHinge_exonl_2_3,

IgG3_AHinge_exonl_2_3_4, IgG3-AHinge_exon2, IgG3_AHinge_exon2_3,

IgG3_AHinge_exon2_3_4, or IgG3-C131S/R435H-IgGlHinge. In some embodiments, the constant region of the immunoglobulin is described by an amino acid sequence selected from, for example, of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51 and sequences that are at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, or 99%) identical to SEQ ID NOs: SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51.

In some embodiments, the immunoglobulin neutralizes a viral infection in the subject.

In some embodiments, the immunoglobulin is IgGl Q311R/N434W/M428E.

Yet other embodiments provide a composition, comprising: a therapeutic

immunoglobulin with anti-viral activity, wherein the immunoglobulin comprises at least one mutation in the Fc region and/or hinge region.

Still other embodiments provide a composition, comprising: an immunoglobulin comprising a at least one mutation in the Fc region and/or hinge region, wherein the immunoglobulin modulates inflammatory TRIM21 signaling and/or FcRn mediated signaling.

Some embodiments provide a composition, comprising: an immunoglobulin comprising a at least one mutation in the Fc region and/or hinge region of said

immunoglobulin, wherein the immunoglobulin modulates TRIM21 signaling, and wherein the mutation is selected from, for example, IgGl-Q311R/N434W/M428E, IgGl- Q311R/N434W, IgGl-MST/G385E/M428E, IgGl-Y436W, IgG3(b)- Q311R/N434F/H433R/R435H, IgGl-Q311R, IgGl-Q311R/M428E, IgGl-Q311H/M428E, IgGl-G385E/M428E, IgGl-Q311F, IgG3(b)-Q311R/G385E/M428E/R435H, IgGl- Q311R/G385E/M428E, IgGl-Q311R/N434Y/M428F, IgGl-Q311R/G385E/M428E/N434Y, IgGl-Q311R/G385E/M428F/N434Y, IgGl-N434F, IgG3-N434F/R435H, IgGl-S131C- IgG3Hinge, IgG3-C131S-IgGlHinge, IgGl-IgG3Fc, IgG3-IgGlFc, IgG3-AHinge_exonl, IgG3_AHinge_exonl_2, IgG3_AHinge_exonl_2_3, IgG3_AHinge_exonl_2_3_4, IgG3- AHinge_exon2, IgG3_AHinge_exon2_3, IgG3_AHinge_exon2_3_4, or IgG3-C131S/R435H- IgGlHinge.

Further embodiments provide vaccine compositions comprising the variant immunoglobulins or Fc fusions thereof described herein (e.g., fused to an immunogen) and uses thereof to generate an immune response in a subject.

Additional embodiments are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows neutralization of AdV5-GFP by human IgG subclasses. (A) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 subclasses in HeLa cells. (B)

Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 IgGl-WT and IgG3(b)- WT in HeLa cells treated with either T21 or control siRNA. (C) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 subclasses in HeLa cells treated with either expoximicin or DMSO. (D) WT HEK293T cells. (E) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C 12 IgGl -WT and IgG3(b)-WT in FcRn KO cells. (F)

Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 IgGl-WT and IgG3(b)- WT in T21 KO cells.

FIG. 2 shows NF-KB induction by IgG subclasses. (A) NF-κΒ induction in response to infection of HEK293T cells with AdV5-GFP in complex with h9C12 IgG subclasses. (B) NF-KB induction in response to infection of HEK293T cells treated with T21 or control siRNA with AdV5-GFP in complex with h9C12 IgGl-WT and IgG3(b)-WT.

FIG. 3 shows neutralization and NF-κΒ induction. (A) NF-κΒ induction in response to infection of HEK293T cells with AdV5-GFP in complex with h9C12 variant. (B) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variant in HEK293T cells.

FIG. 4 shows neutralization and NF-κΒ induction. (A) NF-κΒ induction in response to infection of WT, T21 KO, FcRn KO and T21/FcRn KO HEK293T cells with AdV5-GFP in complex with h9C12 variants. (B) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variant in HEK293T cells.

FIG. 5 shows neutralization and NF-κΒ induction. (A) NF-κΒ induction in response to infection of HEK293T cells with AdV5-GFP in complex with h9C12 variants. (B) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variant in HeLa cells. (C) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variants in WT or T21KO mouse embryonic fibroblasts (MEFs). FIG. 6 shows neutralization and NF-κΒ induction. (A) NF-κΒ induction in response to infection of HEK293T cells with AdV5-GFP in complex with h9C12 variants. (B) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variant in HeLa cells.

FIG. 7 shows neutralization and NF-κΒ induction. (A) NF-κΒ induction in response to infection of HEK293T cells with AdV5-GFP in complex with h9C12 variants. (B)

Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variant in HeLa cells.

FIG. 8 shows neutralization and NF-κΒ induction. (A) NF-κΒ induction in response to infection of HEK293T cells with AdV5-GFP in complex with h9C12 variants. (B) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variant in HeLa cells.

FIG. 9 shows alignment showing the amino acid sequence of IgGl and IgG3 variants.

FIG. 10 shows sequences from Figure 9.

FIG. 11 shows alignment showing the amino acid sequence of IgGl and IgG3 variants.

FIG. 12 shows exemplary immunoglobulin constant region sequences.

FIG. 13 shows neutralization and NF-κΒ induction. (A) NF-κΒ induction in response to infection of HEK293T cells with AdV5-GFP in complex with h9C12 variants. (B) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variant in HeLa cells.

Fig 14 shows that IgG3 Fc engineering improves the anti-AdV5 activity of IgG3. (A) Relative infection (I/IO) levels of AdV5-GFP in complex with h9C12 variants in WT, T21 KO and FcRn KO HEK293T cells. (B) NF-κΒ induction in WT, T21 KO and FcRn KO HEK293T cells infected with AdV5-GFP in complex with h9C12 variants.

DEFINITIONS

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. Also included are antibody fragments having an Fc region, and fusion proteins that comprise a region equivalent to the Fc region of an immunoglobulin.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab').sub.2, single-chain antibody molecules (e.g. scFv), diabodies, and multispecific antibodies formed from antibody fragments.

The term "Fc region" herein is used to define a C-terminal region of an

immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

"Effector functions" are used herein refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cytokine secretion; immune-complex-mediated antigen uptake by antigen presenting cells; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. The term "wildtype" when used in reference to a protein refers to proteins encoded by the genome of a cell, tissue, or organism, other than one manipulated to produce synthetic proteins.

The term "antigen binding domain" refers to the part of an antigen binding molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. For chimeric antibodies, for example, the non-antigen binding components may be derived from a wide variety of species, including primates such as chimpanzees and humans. Humanized antibodies are a particularly preferred form of chimeric antibodies.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG₂, IgG₃, IgG₄, IgAi, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, β, δ, ε, γ, and μ.

A "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al, Science 247: 1306-10 (1990)).

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) (or CDR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)- FR4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al, supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al, supra.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term "hypervariable region" or "HVR", as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. With the exception of CDRl in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other.

Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

The term "variant" and "mutant" when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is

phenylalanine, tyrosine, and tryptophan; unnatural amino acids like p-aminophenylalanine, a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on). For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of lysine with alanine at position 573 is designated as "K573A" and the substitution of lysine with proline at position 573 is designated as K573P. Multiple mutations are separated by addition marks ("+") or "/", e.g., "Gly205Arg + Ser41 lPhe" or "G205R/S41 IF", representing mutations at positions 205 and 411 substituting glycine (G) with arginine (R), and serine (S) with phenylalanine (F), respectively.

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity". For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as

implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters 11644.000-EP7 used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

The expression "amino acid position corresponding to" a position in a reference sequence and similar expression is intended to identify the amino acid residue that in the primary or spatial structure corresponds to the particular position in the reference sequence. The skilled person will appreciate that this can be done by aligning a given sequence with the reference sequence and identifying the amino acid residue that aligns with the particular position in the reference sequence. For example in order to find the amino acid residue in a given albumin sequence that corresponds to position 573 in HSA, the given albumin sequence is aligned with HSA and the amino acid that aligns with position 573 in HSA is identified as the amino acid in the given albumin sequence that corresponds to position 573 in HSA.

The expression Xnnn is intended to mean an amino acid residue X located in a position corresponding to position nnn in HSA and the expression XnnnY is intended to mean a substitution of any amino acid X located in a position corresponding to position nnn in HSA with the amino acid residue Y.

As used herein, the term "affinity" refers to a measure of the strength of binding between two members of a binding pair, for example, an albumin and FcRn. K_<j is the dissociation constant and has units of molarity. The affinity constant is the inverse of the dissociation constant. An affinity constant is sometimes used as a generic term to describe this chemical entity. It is a direct measure of the energy of binding. The natural logarithm of K is linearly related to the Gibbs free energy of binding through the equation AGo = -RT LN(K) where R= gas constant and temperature is in degrees Kelvin. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units (GE Healthcare).

As used herein, the term "under conditions such that said subject generates an immune response" refers to any qualitative or quantitative induction, generation, and/or stimulation of an immune response (e.g., innate or acquired).

A used herein, the term "immune response" refers to a response by the immune system of a subject. For example, immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion). Additional examples of immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells. An immune response may be to immunogens that the subject's immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign). Thus, it is to be understood that, as used herein, "immune response" refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade) cell- mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system) and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids). The term "immune response" is meant to encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response).

As used herein, the term "immunity" refers to protection from disease (e.g., preventing or attenuating (e.g., suppression) of a sign, symptom or condition of the disease) upon exposure to a microorganism (e.g., pathogen) capable of causing the disease. Immunity can be innate (e.g., non-adaptive (e.g., non-acquired) immune responses that exist in the absence of a previous exposure to an antigen) and/or acquired (e.g., immune responses that are mediated by B and T cells following a previous exposure to antigen (e.g., that exhibit increased specificity and reactivity to the antigen)).

As used herein, the term "immunogen" refers to an agent (e.g., a microorganism (e.g., bacterium, virus or fungus) and/or portion or component thereof (e.g., a protein antigen)) that is capable of eliciting an immune response in a subject. In some embodiments, immunogens elicit immunity against the immunogen (e.g. , microorganism (e.g., pathogen or a pathogen product)).

The term "test compound" refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample. Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g. , through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.

The term "sample" as used herein is used in its broadest sense. As used herein, the term "sample" is used in its broadest sense. In one sense it can refer to a tissue sample. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include, but are not limited to blood products, such as plasma, serum and the like. These examples are not to be construed as limiting the sample types applicable to the present invention. A sample suspected of containing a human chromosome or sequences associated with a human chromosome may comprise a cell, chromosomes isolated from a cell (e.g. , a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like. A sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.

As used herein, the term "affinity" refers to a measure of the strength of binding between two members of a binding pair, for example, an immunoglobulin and FcRn. ¾ is the dissociation constant and has units of molarity. The affinity constant is the inverse of the dissociation constant. An affinity constant is sometimes used as a generic term to describe this chemical entity. It is a direct measure of the energy of binding. The natural logarithm of K is linearly related to the Gibbs free energy of binding through the equation AGo = -RT LN(K) where R= gas constant and temperature is in degrees Kelvin. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units (GE Healthcare).

DETAILED DESCRIPTION OF THE INVENTION

The immunoglobulin molecule is composed of two identical heavy and two identical light polypeptide chains, held together by interchain disulfide bonds. Each individual light and heavy chain folds into regions of about 110 amino acids, assuming a conserved three- dimensional conformation. The light chain comprises one variable region (termed VL) and one constant region (CL), while the heavy chain comprises one variable region (VH) and three constant regions (CHI, CH2 and CH3). Pairs of regions associate to form discrete structures. In particular, the light and heavy chain variable regions, VL and VH, associate to form an "FV " area that contains the antigen-binding site.

The variable regions of both heavy and light chains show considerable variability in structure and amino acid composition from one antibody molecule to another, whereas the constant regions show little variability. Each antibody recognizes and binds an antigen through the binding site defined by the association of the heavy and light chain, variable regions into an FV area. The light-chain variable region VL and the heavy-chain variable region VH of a particular antibody molecule have specific amino acid sequences that allow the antigen-binding site to assume a conformation that binds to the antigen epitope recognized by that particular antibody.

Within the variable regions are found regions in which the amino acid sequence is extremely variable from one antibody to another. Three of these so-called "hypervariable" regions or "complementarity -determining regions" (CDR's) are found in each of the light and heavy chains. The three CDRs from a light chain and the three CDRs from a corresponding heavy chain form the antigen-binding site.

The amino acid sequences of many immunoglobulin heavy and light chains have been determined and reveal two important features of antibody molecules. First, each chain consists of a series of similar, although not identical, sequences, each about 110 amino acids long. Each of these repeats corresponds to a discrete, compactly folded region of protein structure known as a protein domain. The light chain is made up of two such immunoglobulin domains, whereas the heavy chain of the IgG antibody contains four.

The second important feature revealed by comparisons of amino acid sequences is that the amino-terminal sequences of both the heavy and light chains vary greatly between different antibodies. The variability in sequence is limited to approximately the first 110 amino acids, corresponding to the first domain, whereas the remaining domains are constant between immunoglobulin chains of the same isotype. The amino-terminal variable or V domains of the heavy and light chains (VH and VL, respectively) together make up the V region of the antibody and confer on it the ability to bind specific antigen, while the constant domains (C domains) of the heavy and light chains (CH and CL, respectively) make up the C region. The multiple heavy-chain C domains are numbered from the amino-terminal end to the carboxy terminus, for example CHT, CH2, and so on. The protein domains described above associate to form larger globular domains. Thus, when fully folded and assembled, an antibody molecule comprises three equal-sized globular portions joined by a flexible stretch of polypeptide chain known as the hinge region. Each arm of this Y-shaped structure is formed by the association of a light chain with the 5 amino-terminal half of a heavy chain, whereas the trunk of the Y is formed by the pairing of the carboxy-terminal halves of the two heavy chains. The association of the heavy and light chains is such that the VH and VL domains are paired, as are the CHT and CL domains. The CH3 domains pair with each other but the (¾2 domains do not interact; carbohydrate side chains attached to the (¾2 domains lie between the two heavy chains. The two antigenic⁾ binding sites are formed by the paired VH and VL domains at the ends of the two arms of the Y.

Proteolytic enzymes (proteases) that cleave polypeptide sequences have been used to dissect the structure of antibody molecules and to determine which parts of the molecule are responsible for its various functions. Limited digestion with the protease papain cleaves

15 antibody molecules into three fragments. Two fragments are identical and contain the

antigen-binding activity. These are termed the Fab fragments, for Fragment antigen binding. The Fab fragments correspond to the two identical arms of the antibody molecule, which contain the complete light chains paired with the VH and CHT domains of the heavy chains. The other fragment contains no antigen-binding activity but was originally observed to

20 crystallize readily, and for this reason was named the Fc fragment, for Fragment

crystallizable. This fragment corresponds to the paired CH and CH3 domains and is the part of the antibody molecule that interacts with effector molecules and cells. The functional differences between heavy-chain isotypes lie mainly in the Fc fragment. The hinge region that links the Fc and Fab portions of the antibody molecule is in reality a flexible tether,

25 allowing independent movement of the two Fab arms, rather than a rigid hinge.

The present invention relates to compositions and methods for antibody-mediated immunity against viral targets. In particular, provided herein are engineered immunoglobulins with antiviral activity.

The present invention provides mutant immunoglobulins comprising one or more 30 mutations, preferably in the Fc region or hinge region of the immunoglobulin. In preferred embodiments, the mutation alters the function of the immunoglobulin in a desired way. For example, in some embodiments, the present invention provides the use of a variant immunoglobulin comprising at least one mutation in the Fc region or hinge region to modulate TRIM21 signaling in a subject. In further embodiments, the present invention provides the use of a variant immunoglobulin comprising at least one mutation in the Fc region and/or hinge region to neutralize a viral infection in a subject.

embodiments, the immunoglobulin further comprises one or more mutation in the hinge region. In some embodiments, the immunoglobulin is an IgGl or IgG3 subclass. In some embodiments, described herein are constant chain (e.g., Fc or hinge region) variants. The immunoglobulins described herein are not limited to particular CDR or variable region sequences and can be generated or engineered to recognize any antigen or epitope desired. In some embodiments, the immunoglobulin has mutations at one or more of positions selected from, for example, 131, 311, 345, 385, 433, 434, 435, 436, or 428 as numbered by the Kabat system. Throughout this disclosure, reference is made to the numbering system from Kabat, E. A., et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991). In these compendiums, Kabat lists many amino acid sequences for antibodies for each subclass, and lists the most commonly occurring amino acid for each residue position in that subclass. Kabat uses a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. The Kabat numbering scheme is followed in this description. For purposes of this invention, to assign residue numbers to a candidate antibody amino acid sequence which is not included in the Kabat compendium, one follows the following steps. Generally, the candidate sequence is aligned with any immunoglobulin sequence or any consensus sequence in Kabat. Alignment may be done by hand, or by computer using commonly accepted computer programs; an example of such a program is the Align 2 program. Alignment may be facilitated by using some amino acid residues which are common to most Fab sequences. For example, the light and heavy chains each typically have two cysteines which have the same residue numbers; in VL domain the two cysteines are typically at residue numbers 23 and 88, and in the VH domain the two cysteine residues are typically numbered 22 and 92. Framework residues generally, but not always, have approximately the same number of residues, however the CDRs will vary in size. For example, in the case of a CDR from a candidate sequence which is longer than the CDR in the sequence in Kabat to which it is aligned, typically suffixes are added to the residue number to indicate the insertion of additional residues. For candidate sequences which, for example, align with a Kabat sequence for residues 34 and 36 but have no residue between them to align with residue 35, the number 35 is simply not assigned to a residue.

Thus, in some embodiments, the mutation is selected from, for example, IgGl- Q311R/N434W/M428E, IgGl-Q311R/N434W, IgGl-Q311R, IgGl-N434W, IgGl-N434Y, IgGl-Q311H, IgGl-H433R, IgGl-E345R,

IgGl-MST/G385E/M428E, IgGl-Y436W, IgG3(b)-Q311R/N434F/H433R/R435H, IgGl- M428E, IgGl-Q311R, IgGl-Q311R/M428E, IgGl-Q311H/M428E, IgGl-G385E/M428E, IgGl-M428F, IgGl-N434K, IgGl-Q311F, IgGl-N434H, IgG3(b)-

Q311R/G385E/M428E/R435H, IgGl-Q311R/G385E/M428E, IgGl-Q311R/N434Y/M428F IgGl-Q311R/G385E/M428E/N434Y, IgGl-Q311R/G385E/M428F/N434Y, IgG3-R435H IgGl-N434W, IgGl-N434F, IgGl-N434Y, IgG3-N434F/R435H, IgGl-S131C-IgG3Hinge, IgG3-C131S-IgGlHinge, IgGl-IgG3Fc, IgG3-IgGlFc, IgG3-AHinge_exonl,

IgG3_AHinge_exonl_2, IgG3_AHinge_exonl_2_3, IgG3_AHinge_exonl_2_3_4, IgG3- AHinge_exon2, IgG3_AHinge_exon2_3, IgG3_AHinge_exon2_3_4, or IgG3-C131S/R435H- IgGlHinge. In some embodiments, the constant region of the immunoglobulin is described by an amino acid sequence selected from, for example, of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51 and sequences that are at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NOs: SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51. In some embodiments, the immunoglobulin neutralizes a viral infection in the subject. In some embodiments, the immunoglobulin is IgGl Q311R/N434W/M428E.

The mutations described above may be introduced into any suitable immunoglobulin molecule. In some embodiments, the immunoglobulin is a monoclonal antibody and is preferably produced by recombinant techniques.

Monoclonal antibodies against target antigens (e.g., a cell surface protein, such as receptors) are produced by a variety of techniques including conventional monoclonal antibody methodologies such as the somatic cell hybridization techniques of Kohl er and Milstein, Nature, 256:495 (1975). Although in some embodiments, somatic cell

hybridization procedures are preferred, other techniques for producing monoclonal antibodies are contemplated as well (e.g., viral or oncogenic transformation of B lymphocytes).

The preferred animal system for preparing hybridomas is the murine system.

Hybridoma production in the mouse is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Human monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice carrying the complete human immune system rather than-the mouse system. Splenocytes from the transgenic mice are immunized with the antigen of interest, which are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein. (See e.g., Wood et al, WO 91/00906, Kucherlapati et al, WO 91/10741 ; Lonberg et al, WO 92/03918; Kay et al, WO 92/03917 [each of which is herein incorporated by reference in its entirety]; N. Lonberg et al, Nature, 368:856-859 [1994]; L.L. Green et al, Nature Genet, 7: 13-21 [1994]; S.L. Morrison et al, Proc. Nat. Acad. Sci. USA, 81 :6851-6855 [1994]; Bruggeman et al, Immunol, 7:33-40 [1993];

Tuaillon et al, Proc. Nat. Acad. Sci. USA, 90:3720-3724 [1993]; and Bruggernan et al. Eur. J. Immunol, 21 : 1323-1326 [1991]).

Monoclonal antibodies can also be generated by other methods known to those skilled in the art of recombinant DNA technology. An alternative method, referred to as the "combinatorial antibody display" method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies. (See e.g., Sastry et al, Proc. Nat. Acad. Sci. USA, 86:5728 [1989]; Huse et al., Science, 246: 1275 [1989]; and Orlandi et al, Proc. Nat. Acad. Sci. USA, 86:3833 [1989]). After immunizing an animal with an immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known for obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and the PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies. (See e.g., Larrick et al, Biotechniques, 11 : 152-156 [1991]). A similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (See e.g., Larrick et al., Methods: Companion to Methods in Enzymology, 2: 106- 110 [1991]).

Chimeric mouse-human monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted. (See e.g., Robinson et al, PCT/US86/02269;

European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; WO 86/01533; US 4,816,567; European Patent Application 125,023 [each of which is herein incorporated by reference in its entirety]; Better et al, Science, 240: 1041-1043 [1988]; Liu et al., Proc. Nat. Acad. Sci. USA, 84:3439-3443 [1987]; Liu et al, J. Immunol, 139:3521-3526 [1987]; Sun et al, Proc. Nat. Acad. Sci. USA, 84:214- 218 [1987]; Nishimura et al, Cane. Res., 47:999-1005 [1987]; Wood et al, Nature, 314:446- 449 [1985]; and Shaw et al, J. Natl. Cancer Inst, 80: 1553-1559 [1988]).

The chimeric antibody can be further humanized by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General reviews of humanized chimeric antibodies are provided by S.L. Morrison, Science, 229: 1202-1207 (1985) and by Oi et al, Bio. Techniques, 4:214 (1986). Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from 7E3, an anti-GPIIbllla antibody producing hybridoma. The recombinant DNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Suitable humanized antibodies can alternatively be produced by CDR substitution (e.g., US 5,225,539 (incorporated herein by reference in its entirety); Jones et al, Nature, 321 :552-525 [1986]; Verhoeyan et al., Science, 239: 1534 [1988]; and Beidler et al., J.

Immunol, 141 :4053 [1988]). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor.

An antibody can be humanized by any method that is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. The human CDRs may be replaced with non-human CDRs; using oligonucleotide site-directed mutagenesis.

Also within the scope of the invention are chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added. In particular, preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, in a humanized antibody having mouse CDRs, amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances. In some embodiments, the monoclonal antibody is a murine antibody or a fragment thereof. In other preferred embodiments, the monoclonal antibody is a bovine antibody or a fragment thereof. For example, the murine antibody can be produced by a hybridoma that includes a B cell obtained from a transgenic mouse having a genome comprising a heavy chain transgene and a light chain transgene fused to an immortalized cell. The antibodies can be of various isotypes, including, but not limited to: IgG (e.g., IgGl, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4); IgM; IgAl; IgA2; IgAsec; IgD; and IgE. In some preferred embodiments, the antibody is an IgG isotype. In other preferred embodiments, the antibody is an IgM isotype. The antibodies can be full-length (e.g., an IgGl, IgG2, IgG3, or IgG4 antibody) or can include only an antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment).

In preferred embodiments, the immunoglobulin is a recombinant antibody (e.g., a chimeric or a humanized antibody), a subunit, or an antigen binding fragment thereof (e.g., has a variable region, or at least a complementarity determining region (CDR)).

In some embodiments, the immunoglobulin is monovalent (e.g., includes one pair of heavy and light chains, or antigen binding portions thereof). In other embodiments, the

immunoglobulin is a divalent (e.g., includes two pairs of heavy and light chains, or antigen binding portions thereof).

In some embodiments, the present invention provides vaccine compositions comprising a variant immunoglobulin or Fc fusion thereof and an immunogen. The present invention is not limited by the particular formulation of a vaccine composition. Indeed, a vaccine composition of the present invention may comprise one or more different agents in addition to the fusion protein. These agents or cofactors include, but are not limited to, adjuvants, surfactants, additives, buffers, solubilizers, chelators, oils, salts, therapeutic agents, drugs, bioactive agents, antibacterials, and antimicrobial agents (e.g., antibiotics, antivirals, etc.). In some embodiments, a vaccine composition comprising a fusion protein comprises an agent and/or co-factor that enhance the ability of the immunogen to induce an immune response (e.g., an adjuvant). In some preferred embodiments, the presence of one or more co-factors or agents reduces the amount of immunogen required for induction of an immune response (e.g., a protective immune respone (e.g., protective immunization)). In some embodiments, the presence of one or more co-factors or agents can be used to skew the immune response towards a cellular (e.g., T cell mediated) or humoral (e.g., antibody mediated) immune response. The present invention is not limited by the type of co-factor or agent used in a therapeutic agent of the present invention. Adjuvants are described in general in Vaccine Design—the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995. The present invention is not limited by the type of adjuvant utilized (e.g., for use in a composition (e.g., pharmaceutical composition). For example, in some embodiments, suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate. In some embodiments, an adjuvant may be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.

In general, an immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. Immune responses may be broadly categorized into two categories: humoral and cell mediated immune responses (e.g., traditionally characterized by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed Thl-type responses (cell- mediated response), and Th2-type immune responses (humoral response).

Stimulation of an immune response can result from a direct or indirect response of a cell or component of the immune system to an intervention (e.g., exposure to an immunogen). Immune responses can be measured in many ways including activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells etc.); up-regulated or down-regulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly (including increased spleen cellularity); hyperplasia and mixed cellular infiltrates in various organs. Other responses, cells, and components of the immune system that can be assessed with respect to immune stimulation are known in the art.

Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, compositions and methods of the present invention induce expression and secretion of cytokines (e.g., by macrophages, dendritic cells and CD4+ T cells).

Modulation of expression of a particular cytokine can occur locally or systemically. It is known that cytokine profiles can determine T cell regulatory and effector functions in immune responses. In some embodiments, Thl-type cytokines can be induced, and thus, the immunostimulatory compositions of the present invention can promote a Thl type antigen- specific immune response including cytotoxic T-cells (e.g., thereby avoiding unwanted Th2 type immune responses (e.g., generation of Th2 type cytokines (e.g., IL-13) involved in enhancing the severity of disease (e.g., IL-13 induction of mucus formation))). Cytokines play a role in directing the T cell response. Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including B and other T cells. Most mature CD4+T helper cells express one of two cytokine profiles: Thl or Th2. Thl -type CD4+ T cells secrete IL-2, IL-3, IFN-γ, GM-CSF and high levels of TNF-a. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL- 9, IL-10, IL-13, GM-CSF and low levels of TNF-a. Thl type cytokines promote both cell- mediated immunity, and humoral immunity that is characterized by immunoglobulin class switching to IgG2a in mice and IgGl in humans. Thl responses may also be associated with delayed-type hypersensitivity and autoimmune disease. Th2 type cytokines induce primarily humoral immunity and induce class switching to IgGl and IgE. The antibody isotypes associated with Thl responses generally have neutralizing and opsonizing capabilities whereas those associated with Th2 responses are associated more with allergic responses.

Several factors have been shown to influence skewing of an immune response towards either a Thl or Th2 type response. The best characterized regulators are cytokines. IL-12 and IFN-γ are positive Thl and negative Th2 regulators. IL-12 promotes IFN- γ production, and IFN-γ provides positive feedback for IL-12. IL-4 and IL-10 appear important for the establishment of the Th2 cytokine profile and to down-regulate Thl cytokine production.

Thus, in preferred embodiments, the present invention provides a method of stimulating a Thl -type immune response in a subject comprising administering to a subject a composition comprising an immunogen. However, in other embodiments, the present invention provides a method of stimulating a Th2-type immune response in a subject (e.g., if balancing of a T cell mediated response is desired) comprising administering to a subject a composition comprising an immunogen. In further preferred embodiments, adjuvants can be used (e.g., can be co-administered with a composition of the present invention) to skew an immune response toward either a Thl or Th2 type immune response. For example, adjuvants that induce Th2 or weak Thl responses include, but are not limited to, alum, saponins, and SB-As4. Adjuvants that induce Thl responses include but are not limited to MPL, MDP, ISCOMS, IL-12, IFN- γ, and SB-AS2.

Several other types of Thl -type immunogens can be used (e.g., as an adjuvant) in compositions and methods of the present invention. These include, but are not limited to, the following. In some embodiments, monophosphoryl lipid A (e.g., in particular 3-de-O- acylated monophosphoryl lipid A (3D-MPL)), is used. 3D-MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. In some embodiments, diphosphoryl lipid A, and 3-O-deacylated variants thereof are used. Each of these immunogens can be purified and prepared by methods described in GB 2122204B, hereby incorporated by reference in its entirety. Other purified and synthetic

lipopolysaccharides have been described (See, e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al, 1986, Int. Arch. Allergy. Immunol, 79(4):392-6; Hilgers et al, 1987, Immunology, 60(1): 141-6; and EP 0 549 074, each of which is hereby incorporated by reference in its entirety). In some embodiments, 3D-MPL is used in the form of a particulate formulation (e.g., having a small particle size less than 0.2 μιτι in diameter, described in EP 0 689 454, hereby incorporated by reference in its entirety).

In some embodiments, saponins are used as an immunogen (e.g.,Thl-type adjuvant) in a composition of the present invention. Saponins are well known adjuvants (See, e.g., Lacaille-Dubois and Wagner (1996) Phytomedicine vol 2 pp 363-386). Examples of saponins include Quil A (derived from the bark of the South American tree Quillaja

Saponaria Molina), and fractions thereof (See, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0 362 279, each of which is hereby incorporated by reference in its entirety). Also contemplated to be useful in the present invention are the haemolytic saponins QS7, QS17, and QS21 (HPLC purified fractions of Quil A; See, e.g., Kensil et al. (1991). J. Immunology 146,431-437, U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0 362 279, each of which is hereby incorporated by reference in its entirety). Also contemplated to be useful are combinations of QS21 and polysorbate or cyclodextrin (See, e.g., WO 99/10008, hereby incorporated by reference in its entirety.

In some embodiments, an immunogenic oligonucleotide containing unmethylated CpG dinucleotides ("CpG") is used as an adjuvant. CpG is an abbreviation for cytosine- guanosine dinucleotide motifs present in DNA. CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis et al, J.Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.Immunol, 1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660, each of which is hereby incorporated by reference in its entirety). For example, in some embodiments, the immunostimulatory sequence is Purine-Purine-C-G-pyrimidine-pyrirnidine; wherein the CG motif is not methylated.

Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, the presence of one or more CpG oligonucleotides activate various immune subsets including natural killer cells (which produce IFN-γ) and macrophages. In some embodiments, CpG oligonucleotides are formulated into a composition of the present invention for inducing an immune response. In some embodiments, a free solution of CpG is co-administered together with an antigen (e.g., present within a solution (See, e.g., WO

96/02555; hereby incorporated by reference). In some embodiments, a CpG oligonucleotide is covalently conjugated to an antigen (See, e.g., WO 98/16247, hereby incorporated by reference), or formulated with a carrier such as aluminium hydroxide (See, e.g., Brazolot- Millan et al, Proc.Natl.AcadSci., USA, 1998, 95(26), 15553-8).

In some embodiments, adjuvants such as Complete Freunds Adjuvant and Incomplete

Freunds Adjuvant, cytokines (e.g., interleukins (e.g., IL-2, IFN-γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.), detoxified mutants of a bacterial ADP- ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. Coli heat- labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (See, e.g., WO93/13202 and W092/19265, each of which is hereby incorporated by reference), and other immunogenic substances (e.g., that enhance the effectiveness of a composition of the present invention) are used with a composition comprising an immunogen of the present invention.

Additional examples of adjuvants that find use in the present invention include poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi

ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants may be added to a composition comprising an immunogen, or, the adjuvant may be formulated with carriers, for example liposomes, or metallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) prior to combining with or co-administration with a composition.

In some embodiments, a composition comprising an immunogen comprises a single adjuvant. In other embodiments, a composition comprises two or more adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241 ; and WO 94/00153, each of which is hereby incorporated by reference in its entirety).

In some embodiments, a composition comprising an immunogen comprises one or more mucoadhesives (See, e.g., U.S. Pat. App. No. 20050281843, hereby incorporated by reference in its entirety). The present invention is not limited by the type of mucoadhesive utilized. Indeed, a variety of mucoadhesives are contemplated to be useful in the present invention including, but not limited to, cross-linked derivatives of poly(acrylic acid) (e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (e.g., alginate and chitosan), hydroxypropyl methylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, use of a mucoadhesive (e.g., in a composition comprising an immunogen) enhances induction of an immune response in a subject (e.g., administered a composition of the present invention) due to an increase in duration and/or amount of exposure to an immunogen that a subject experiences when a mucoadhesive is used compared to the duration and/or amount of exposure to an immunogen in the absence of using the mucoadhesive.

In some embodiments, the immunoglobulin and/or vaccine compositions are used in conjunction with appropriate salts and buffers to render delivery of the compositions to a subject. Buffers also are employed when the compositions are introduced into a patient. Aqueous compositions comprise an effective amount of composition dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.

The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

In some embodiments of the present invention, the active compositions include classic pharmaceutical preparations. Administration of these compositions according to the present invention is via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.

The compositions may also be administered parenterally or intraperitoneally.

Solutions of the active compounds as free base or pharmacologically acceptable salts are prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution is suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the present invention, the active particles or agents are formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses may be administered.

Additional formulations that are suitable for other modes of administration include vaginal suppositories and pessaries. A rectal pessary or suppository may also be used.

Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or the urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably \%- 2%. Vaginal suppositories or pessaries are usually globular or oviform and weighing about 5 g each. Vaginal medications are available in a variety of physical forms, e.g., creams, gels or liquids, which depart from the classical concept of suppositories.

"Treating" within the context of the instant invention, means an alleviation, in whole or in part, of symptoms associated with a disorder or disease, or slowing, inhibiting or halting of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder in a subject at risk for developing the disease or disorder. Thus, e.g., treating a viral infection may include inhibiting or preventing replication of the virus in a subject or decreasing symptoms of viral infection in the subject. As used herein, a

"therapeutically effective amount" of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with a disorder or disease, or slows, inhibits or halts further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disease or disorder in a subject at risk for developing the disease or disorder.

A subject is any animal that can benefit from the administration of a compound as described herein. In some embodiments, the subject is a mammal, for example, a human, a primate, a dog, a cat, a horse, a cow, a pig, a rodent, such as for example a rat or mouse. Typically, the subject is a human.

A therapeutically effective amount of a compound as described herein used in the present invention may vary depending upon the route of administration and dosage form. Effective amounts of invention compounds typically fall in the range of about 0.001 up to 100 mg/kg/day, and more typically in the range of about 0.05 up to 10 mg/kg/day. Typically, the compound or compounds used in the instant invention are selected to provide a formulation that exhibits a high therapeutic index. The therapeutic index is the dose ratio between toxic and therapeutic effects which can be expressed as the ratio between LD50 and ED50. The LD50 is the dose lethal to 50% of the population and the ED50 is the dose therapeutically effective in 50% of the population. The LD50 and ED50 are determined by standard pharmaceutical procedures in animal cell cultures or experimental animals.

The instant invention also provides for pharmaceutical compositions and medicaments which may be prepared by combining one or more compounds described herein, pharmaceutically acceptable salts thereof, stereoisomers thereof, tautomers thereof, or solvates thereof, with pharmaceutically acceptable carriers, excipients, binders, diluents or the like to inhibit or treat primary and/or metastatic prostate cancers. Such compositions can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The instant compositions can be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, or via implanted reservoir. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular injections. The following dosage forms are given by way of example and should not be construed as limiting the instant invention.

For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the instant invention, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in

administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or antioxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. Pharmaceutical formulations and medicaments may be prepared as liquid suspensions or solutions using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or parenteral administration. As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, com oil and olive oil. Suspension preparations may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but not limited to, poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations.

Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. Typically, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.

For rectal administration, the pharmaceutical formulations and medicaments may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum. Rectal suppositories are prepared by mixing one or more compounds of the instant invention, or pharmaceutically acceptable salts or tautomers of the compound, with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is present in a solid phase at normal storing temperatures, and present in a liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum. Oils may also be employed in the preparation of formulations of the soft gelatin type and suppositories. Water, saline, aqueous dextrose and related sugar solutions, and glycerols may be employed in the preparation of suspension formulations which may also contain suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives.

Compounds of the invention may be administered to the lungs by inhalation through the nose or mouth. Suitable pharmaceutical formulations for inhalation include solutions, sprays, dry powders, or aerosols containing any appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. Formulations for inhalation administration contain as excipients, for example, lactose, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Aqueous and nonaqueous aerosols are typically used for delivery of inventive compounds by inhalation.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the compound together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (TWEENs, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. A nonaqueous suspension (e.g., in a fluorocarbon propellant) can also be used to deliver compounds of the invention.

Aerosols containing compounds for use according to the present invention are conveniently delivered using an inhaler, atomizer, pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, pressurized dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, nitrogen, air, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Delivery of aerosols of the present invention using sonic nebulizers is advantageous because nebulizers minimize exposure of the agent to shear, which can result in degradation of the compound. For nasal administration, the pharmaceutical formulations and medicaments may be a spray, nasal drops or aerosol containing an appropriate solvent(s) and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. For

administration in the form of nasal drops, the compounds maybe formulated in oily solutions or as a gel. For administration of nasal aerosol, any suitable propellant may be used including compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

Dosage forms for the topical (including buccal and sublingual) or transdermal administration of compounds of the invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier or excipient, and with any preservatives, or buffers, which may be required. Powders and sprays can be prepared, for example, with excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The ointments, pastes, creams and gels may also contain 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.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the invention to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the inventive compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the instant invention. Such excipients and carriers are described, for example, in "Remingtons Pharmaceutical Sciences" Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.

The formulations of the invention may be designed to be short-acting, fast-releasing, long-acting, and sustained-releasing as described below. Thus, the pharmaceutical formulations may also be formulated for controlled release or for slow release.

The instant compositions may also comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged storage and/or delivery effect. Therefore, the pharmaceutical formulations and medicaments may be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants such as stents. Such implants may employ known inert materials such as silicones and biodegradable polymers.

Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the instant invention.

In some embodiments of the present invention, methods and compositions are provided for the prevention and/or treatment of viral disease.

In some embodiments, the immunoglobulins described herein are administering in combination with an anti-viral agent. In some embodiments, treatment with the

immunoglobulin described herein precedes or follows the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and immunoglobulin are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the multiple therapies would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that cells are contacted with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2 to 7) to several weeks (1 to 8) lapse between the respective administrations.

In some embodiments, more than one administration of the immunotherapeutic composition of the present invention or the other agent is utilized. Various combinations may be employed, where the immunogloublin is "A" and the other agent is "B", as exemplified below:

A/B/A, B/A/B, B/B/A, A/A/B, B/A/A, A/B/B, B/B/B/A, B/B/A/B,

A/A/B/B, A/B/A/B, A/B/B/A, B/B/A/A, B/A/B/A, B/A/A/B, B/B/B/A,

A/A/A/B, B/A/A/A, A/B/A/A, A/A/B/A, A/B/B/B, B/A/B/B, B/B/A/B.

Other combinations are contemplated.

In some embodiments of the invention, one or more compounds of the invention and an additional active agent are administered to a subject, more typically a human, in a sequence and within a time interval such that the compound can act together with the other agent to provide an enhanced benefit relative to the benefits obtained if they were administered otherwise. For example, the additional active agents can be co-administered by co-formulation, administered at the same time or administered sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. In some embodiments, the compound and the additional active agents exert their effects at times which overlap. Each additional active agent can be administered separately, in any appropriate form and by any suitable route. In other embodiments, the compound is administered before, concurrently or after administration of the additional active agents. In various examples, the compound and the additional active agents are administered less than about 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In other examples, the compound and the additional active agents are administered concurrently. In yet other examples, the compound and the additional active agents are administered concurrently by co-formulation.

In other examples, the compound and the additional active agents are administered at about 2 to 4 days apart, at about 4 to 6 days apart, at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeks apart.

In certain examples, the inventive compound and optionally the additional active agents are cyclically administered to a subject. Cycling therapy involves the administration of a first agent for a period of time, followed by the administration of a second agent and/or third agent for a period of time and repeating this sequential administration. Cycling therapy can provide a variety of benefits, e.g., reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one or more of the therapies, and/or improve the efficacy of the treatment.

In other examples, one or more compound of some embodiments of the present invention and optionally the additional active agent are administered in a cycle of less than about 3 weeks, about once every two weeks, about once every 10 days or about once every week. One cycle can comprise the administration of an inventive compound and optionally the second active agent by infusion over about 90 minutes every cycle, about 1 hour every cycle, about 45 minutes every cycle, about 30 minutes every cycle or about 15 minutes every cycle. Each cycle can comprise at least 1 week of rest, at least 2 weeks of rest, at least 3 weeks of rest. The number of cycles administered is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles. Courses of treatment can be administered concurrently to a subject, i.e., individual doses of the additional active agents are administered separately yet within a time interval such that the inventive compound can work together with the additional active agents. For example, one component can be administered once per week in combination with the other components that can be administered once every two weeks or once every three weeks. In other words, the dosing regimens are carried out concurrently even if the therapeutics are not administered simultaneously or during the same day.

The additional active agents can act additively or, more typically, synergistically with the inventive compound(s). In one example, one or more inventive compound is

administered concurrently with one or more second active agents in the same pharmaceutical composition. In another example, one or more inventive compound is administered concurrently with one or more second active agents in separate pharmaceutical compositions. In still another example, one or more inventive compound is administered prior to or subsequent to administration of a second active agent. The invention contemplates administration of an inventive compound and a second active agent by the same or different routes of administration, e.g., oral and parenteral. In certain embodiments, when the inventive compound is administered concurrently with a second active agent that potentially produces adverse side effects including, but not limited to, toxicity, the second active agent can advantageously be administered at a dose that falls below the threshold that the adverse side effect is elicited.

The immunoglobulin compositions described herein find use in the treatment and/or prevention of a variety of viral disease. Examples include, but are not limited to, DNA viruses, RNA viruses, etc. Viruses from the families Adenoviridae (e.g., Adenovirus), Herpesviridae (e.g., Herpes simplex, type 1, Herpes simplex, type 2, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus, type 8), Papillomaviridae (e.g., Human papillomavirus), Polyomaviridae (e.g., BK virus, JC virus), Poxviridae (e.g., Smallpox), Hepadnaviridae (e.g., Hepatitis B virus), Parvoviridae (e.g., Parvovirus),

Astroviridae (e.g., Human astrovirus), Caliciviridae (e.g., Norwalk virus), Picornaviridae (e.g., coxsackievirus, hepatitis A virus, poliovirus, rhinovirus), Coronaviridae (e.g., Severe acute respiratory syndrome virus), Flaviviridae (e.g., Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus) Togaviridae (e.g., Rubella virus), Hepeviridae (e.g., Hepatitis E virus), Retroviridae (e.g., Human immunodeficiency virus (HIV)),

Orthomyxoviridae (e.g., Influenza virus), Arenaviridae (e.g., Lassa virus), Bunyaviridae (e.g., Crimean-Congo hemorrhagic fever virus, Hantaan virus), Filoviridae (e.g., Ebola virus, Marburg virus), Paramyxoviridae (e.g., Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus), Rhabdoviridae (e.g., Rabies virus), Hepatitis D, and Reovirida (e.g., Rotavirus, Orbivirus, Coltivirus, Banna virus) are specifically contemplated.

EXPERIMENTAL

Example 1

Fc-engineered IgGl and IgG3 variants with altered anti-viral effector functions

Antibody-mediated immunity forms a important part of the antiviral immune response, and its induction is a principal objective of vaccination. IgGl and IgG3 variants with anti-viral activity were engineered.

These IgG Fc variants have previously been described by others and shown to extend the half-life (M428L/N434S, M252Y/S254T/T256E and H433K/N434F) or to increase the clearance of endogenous IgG (M252Y/S254T/T256E/H433K/N434F).

IgGl-H433K/N434F: [1]

IgGl-M428L/N434S: [2]

IgGl-M252Y/S254T/T256E/H433K/N434F: [3]

IgGl-M252Y/S254T/T256E: [4, 5]

Materials and Methods

Cell culture. The cell lines HEK293E, HEK293T and HeLa were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin at 37^°C in a humid 5% CO2, 95% air incubator. Where appropriate, cells were selected in 1 mg/ml G418 (Life Technologies). Mouse embryonic fibroblasts (MEFs) were derived from wild- type C57BL/6 (WT) or TRIM21 knockout (K21) embryos as described (McEwan, W.A., et al., Regulation of virus neutralization and the persistent fraction by TRIM21. J Virol, 2012. 86(16): p. 8482-91). Production of soluble recombinant human FcRn. Monomeric His-tagged human FcRn was produced using a Baculovirus expression vector system (Kim, J.K., et al, Mapping the site on human IgG for binding of the MHC class 1-r elated receptor, FcRn. Eur J

Immunol, 1999. 29(9): p. 2819-25). A viral stock encoding His-tagged human FcRn was a kind gift from Dr. Sally Ward (University of Texas, Southwestern Medical Center, Dallas, TX). Briefly, human FcRn was purified using a HisTrap HP column supplied with Ni²⁺ ions (GE Healthcare). The column was preequilibrated with 1 ^χ PBS with 0.05% sodium azide, and the pH of the supernatant was adjusted with 1 ^χ PBS, 0.05% sodium azide (pH 10.9) to pH 7.2 before being applied to the HisTrap HP column with a flow rate of 5 ml/min. The column was washed using 200 ml of 1 ^χ PBS followed by 50 ml of 25 mm imidazole, 1 ^χ PBS (pH 7.3), and human FcRn was eluted with 250 mm imidazole, 1 ^χ PBS (pH 7.4). The collected protein was buffer-exchanged to l x PBS using Amicron Ultra- 10 filter units (Millipore) followed by isolation of the monomeric fraction. A HiLoad 26/600 Superdex 200 prep grade column (GE Healthcare) was used to isolate the monomeric fraction before the protein was concentrated using Amicon Ultra columns (Millipore) and stored at 4°C.

Production of recombinant TRIM21. Recombinant TRIM21 PRYSPRY was overexpressed in BL21 E.coli cells under standard conditions and purified by Ni-NTA resin (Qiagen) and Superdex 75 gel filtration (GE Healthcare) as described (James, L.C., et al, Structural basis for PRY SPRY -mediated tripartite motif (TRIM) protein function. Proc Natl Acad Sci U S A, 2007. 104(15): p. 6200-5).

Construction and production of Fc-engineered IgGl variants. Vectors encoding humanized 9C12 IgGl variants were based on the pLNOH2/pLNOk expression system (Norderhaug, L., et al, Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells. J Immunol Methods, 1997. 204(1): p. 77-87).

Specifically, heavy (H) and light (L) chain variable (V) genes derived from the hybridoma cell line TC31-9C12.C9 (Developmental Studies Hybridoma Bank, University of Iowa) Varghese, R., et al, Postentry neutralization of adenovirus type 5 by an antihexon antibody. J Virol, 2004. 78(22): p. 12320-32) were synthesized as a cloning cassette flanked by the restriction sites recognized by the endonucleases Bsml/BsiWI. The gene fragments were then subcloned into pLNOH2-^Nn>hIgGl -WT-oriP and ^Nn>pLNOk-oriP, resulting in pLNOH2-

^HexonhIgGl-WT-oriP and ^HexonpLNOk-oriP encoding a chimeric human H chain and L chain, respectively. The H chain encoding vector pLNOH2-^HexonhIgGl-WT-oriP was further used for the generation of h9C12 variants by exchanging CH2 and CH3 gene fragments with fragments containing the desired mutations. CH2 fragments were exchanged using the unique restriction sites recognized by the endonucleases Agel and Sfil, while CH3 fragments were exchanged using Sfil and BamHI (All from New England Biolabs). Following co-transfection of both H chain and L chain vectors into HEK293E cells using Lipofectamine 2000 (Life Technologies), h9C12 IgGl variants were purified from collected supernatant using a CHT specific CaptureSelect column (Life Technologies). Monomeric fractions were isolated by SEC chromatography using a Superdex 200 column (GE Healthcare). Protein integrity was verified by non-reducing SDS-PAGE (Life Technologies).

Virus Production. Replication deficient AdV5 carrying GFP reporter gene (AdV5- GFP) was grown in trans-complementation cell line 293F for 72 hours, before three rounds of freeze-thaw to release virus particles and filtration at 0.45 μιτι. Virus stocks was purified by two rounds of ultracentrifugation banding on a cesium chloride gradient, dialyzed into PBS/10% glycerol, and frozen at -80^°C until required.

SPR. A Biacore 3000 instrument (GE Healthcare) was used for all kinetics measurements. 9C12 IgG variants were immobilized by amine coupling on CM5 sensor chips according to the manufacturer instructions. The coupling was performed by injecting 1-2.5 μg/ml of the IgGl variants dissolved in 10 mM sodium acetate, pH 4.5 (GE Healthcare). HBS-P buffer (0.01 M HEPES, 0.15 NaCl, 0.005% surfactant P20, pH 7.4) was used as running and dilution buffer. Subsequently, concentration series of recombinant human TRIM21 PRYSPRY or human FcRn were injected. Kinetics analysis was performed using the BIAevaluation Software and the binding data were fitted to a simple first order (1 : 1) Langmuir bio molecular interaction model. Steady state affinity constants were obtained by immobilizing IgG variants at -2500 RU and determined by an equilibrium (Req) binding model. Binding was measured at both pH 6.0 and pH 7.4 for human FcRn.

Neutralization assay. The assay was performed essentially as previously described (McEwan, W.A., et al, Regulation of virus neutralization and the persistent fraction by TRIM21. J Virol, 2012. 86(16): p. 8482-91; Mallery, D.L., et al., Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proc Natl Acad Sci U S A, 2010. 107(46): p. 19985-90). Briefly, HeLa cells were plated at a density of 6 x 10⁴ cells per well in 24-well plates 1 day prior to infection. 2.5 x 10⁴ infectious units (IU) AdV5- GFP was mixed 1 : 1 with antibody to give the final stated antibody concentration, and incubated for 30 min at RT. This was then added to the cells and allowed to incubate for 24 h at 37^°C. Cells were then collected by trypsination and fixed in 4% PFA solution. AdV5-GFP reporter gene expression was determined by FACS analysis. Where stated, cells were transfected with 150 pmol control or TRIM21 siRNA following the manufacturer instructions (Life Technologies). Relative Infection (I/IO) were calculated as described (McEwan, W.A., et al, Regulation of virus neutralization and the persistent fraction by TRIM21. J Virol, 2012 86(16): p. 8482-91).

NF-kB reporter assay. The assay was performed essentially as previously described (McEwan, W.A., et al, Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nat Immunol, 2013. 14(4): p. 327-36). Briefly, HEK293T cells stably transfected with pGL4.32 NF-kB luciferase (Promega) were plated at a density of 1 x 10⁴ cells per well in 96-well plates 1 day prior to infection. AdV5-GFP was mixed 1 : 1 with antibody to give the final antibody concentration, and incubated for 30 min at RT. AdV5- GFP was used at a final concentration of 7.4 x 10⁵ IU per well. This was then added to cells and allowed to incubate for 7 hours at 37^° C before addition of 100 μΐ Steady lite Plus luciferase reagent (Perkin Elmer) and analysis on a BMG Pherastar FS plate reader. Where stated, cells were transfected with 150 pmol control or TRIM21 siRNA following the manufacturer instructions (Life Technologies).

Results

Results are shown in the Tables below and Figures 1-14. Fig 14 shows that IgG3 Fc engineering improves the anti-AdV5 activity of IgG3. (A) Relative infection (I/I0) levels of AdV5-GFP in complex with h9C12 variants in WT, T21 KO and FcRn KO HEK293T cells. (B) NF-DB induction in WT, T21 KO and FcRn KO HEK293T cells infected with AdV5- GFP in complex with h9C12 variants. Presented as fold change compared to AdV5-GFP only. Error bars represents mean±S.D. of triplicates from a representative experiment.

IgGl-IgG3Fc 6.5 NT 2.0

IgG3(b)-IgGlFc No change NT No change

IgG3 (b)-AHinge exonl As IgG3(b)-WT NT 1.8

IgG3 (b)-AHinge exonl 2 As IgG3(b)-WT NT 1.5

IgG3 (b)-AHinge exon 1 2 3 As IgG3(b)-WT NT No change

IgG3 (b)-AHinge exon 2 As IgG3(b)-WT NT No change

IgG3 (b)-AHinge exon 2 3 As IgG3(b)-WT NT No change

IgG3 (b)-AHinge exon 2 3 4 As IgG3(b)-WT NT No change

IgGl-H433K/N434F No change - 13.5

IgGl-M428L/N434S No change No change -

IgGl- -5.0 4.5

M252Y/S254T/T256E/H433K

/N434S

IgGl-M252Y/S254T/T256E No change No change -

NT: not tested

Table 2 | SPR-derived kinetics for binding of h9C12 variants to human TRIM21

PRYSPRY

Human PRYSPRY

IgGl- 32.0±0.2 8.1±0.3 253.1

M252Y/S254T/T256E

aThe kinetic rate constants were obtained using the first-order (1: 1) Langmuir biomolecular interaction model. The kinetic values represent the average of triplicates.

NA, not acquired because of fast binding kinetics.

"Steady State Affinity

"Steady State affinity All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

1. The use of a variant immunoglobulin comprising at least one mutation in the Fc region or hinge region to modulate antiviral response in a subject.

2. The use of a variant immunoglobulin comprising at least one mutation in the Fc region or hinge region to neutralize a viral infection in a subject.

3. The use of claim 1 or 2, wherein said immunoglobulin exhibits altered binding to TRIM21 relative to an immunoglobulin without said mutation.

4. The use of claim 3, wherein said altered binding is increased or decreased binding affinity.

5. The use of any one of claims 1 to 4, wherein said immunoglobulin exhibits altered binding to FcRN and/or altered activation of NFkB relative to an immunoglobulin without said mutation.

6. The use of any one of claims 1 to 5, wherein said immunoglobulin further comprises one or more mutation in the hinge region of said immunoglobulin.

7. The use of any one of claims 1 to 6, wherein said immunoglobulin is an IgGl, IgG2, IgG3, or IgG4 subclass.

8. The use of any one of claims 1 to 7, wherein said immunoglobulin has mutations at one or more of positions selected from the group consisting of 131, 311, 345, 385, 433, 434, 435, 436, and 428. 9. The use of any one of claims 1 to 8, wherein said mutation is selected from the group consisting of M428L/N434S, M252Y/S254T/T256E, H433K/N434F,

IgGl-Q311R/N434W/M428E, IgGl-Q311R/N434W, IgGl-Q311R,

IgGl-N434W, IgGl-N434Y, IgGl-Q311H, IgGl-H433R, IgGl-E345R,

IgGl-MST/G385E/M428E, IgGl-Y436W, IgG3(b)-Q311R/N434F/H433R/R435H, IgGl- M428E, IgGl-Q311R/M428E, IgGl-Q311H/M428E, IgGl-G385E/M428E, IgGl-M428F, IgGl-N434K, IgGl-Q311F, IgGl-N434H, IgG3(b)-Q311R/G385E/M428E/R435H, IgGl- Q311R/G385E/M428E, IgGl -Q311R/N434Y/M428F

IgGl-Q311R/G385E/M428E/N434Y, IgGl-Q311R/G385E/M428F/N434Y, IgG3-R435H, IgGl-N434F, IgG3-N434F/R435H, IgGl-S 131C-IgG3Hinge,

IgG3-C131S-IgGlHinge, IgGl-IgG3Fc, IgG3-IgGlFc, IgG3-AHinge_exonl,

IgG3_AHinge_exonl_2, IgG3_AHinge_exonl_2_3, IgG3_AHinge_exonl_2_3_4, IgG3- AHinge_exon2, IgG3_AHinge_exon2_3, IgG3_AHinge_exon2_3_4, and IgG3- C 131 S/R435H-IgGlHinge. 10. The use of claim 9, wherein the constant region of said immunoglobulin is described by an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51 and sequences that are at least 90% identical to SEQ ID NOs: 5, 6, 7, 8,

9,

10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51.

11. The use of any one of claims 1 to 10, wherein said immunoglobulin neutralizes a viral infection in said subject.

12. The use of any one of claims 1 to 11, wherein said immunoglobulin is IgGl

Q311R/N434W/M428E.

13. A method of modulating antiviral response in a subject, comprising:

administering a variant immunoglobulin comprising at least one mutation in the Fc region or hinge region to said subject.

14. A method of neutralizing a viral infection in a subject, comprising: administering a variant immunoglobulin comprising at least one mutation in the Fc region or hinge region to said subject.

15. The method of claim 13 or 14, wherein said immunoglobulin exhibits altered binding to TRIM21 relative to an immunoglobulin without said mutation.

The method of claim 15, wherein said altered binding is increased or decreased

17. The method of any one of claims 13 to 16, wherein said immunoglobulin exhibits altered binding to FcRN and/or altered activation of NFkB relative to an immunoglobulin without said mutation.

18. The method of any one of claims 13 to 17, wherein said immunoglobulin further comprises one or more mutation in the hinge region of said immunoglobulin.

19. The method of any one of claims 13 to 18, wherein said immunoglobulin is an IgGl, IgG2, IgG3, or IgG4 subclass.

20. The method of any one of claims 13 to 19, wherein said immunoglobulin has mutations at one or more of positions selected from the group consisting of 131, 311, 345, 385, 433, 434, 435, 436, and 428. 21. The method of any one of claims 13 to 20, wherein said mutation is selected from the group consisting of M428L/N434S, M252Y/S254T/T256E, H433K/N434F,

IgGl-Q311R/N434W/M428E, IgGl-Q311R/N434W, IgGl-Q311R,

IgGl-N434W, IgGl-N434Y, IgGl-Q311H, IgGl-H433R, IgGl-E345R,

IgGl-Q311R/G385E/M428E/N434Y, IgGl-Q311R/G385E/M428F/N434Y, IgG3-R435H, IgGl-N434F, IgG3-N434F/R435H, IgGl-S131C-IgG3Hinge,

IgG3-C131S-IgGlHinge, IgGl-IgG3Fc, IgG3-IgGlFc, IgG3-AHinge_exonl,

IgG3_AHinge_exonl_2, IgG3_AHinge_exonl_2_3, IgG3_AHinge_exonl_2_3_4, IgG3- AHinge_exon2, IgG3_AHinge_exon2_3, IgG3_AHinge_exon2_3_4, and IgG3- C 131 S/R435H-IgGlHinge. 22. The method of claim 21, wherein the constant region of said immunoglobulin is described by an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51 and sequences that are at least 90% identical to SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20,

21,

22, 23, and 26-51.

23. The method of any one of claims 13 to 22, wherein said immunoglobulin neutralizes a viral infection in said subject.

24. The method of any one of claims 13 to 23, wherein said immunoglobulin is IgGl Q31 1R/N434W/M428E.

25. A composition, comprising:

a therapeutic immunoglobulin with anti-viral activity, wherein said immunoglobulin comprises at least one mutation in the Fc region or hinge region of said immunoglobulin.

26. A composition, comprising:

an immunoglobulin comprising a at least one mutation in the Fc region or hinge region of said immunoglobulin, wherein said immunoglobulin modulates antiviral response is a subject.

27. The composition of claim 25 or 26, wherein said immunoglobulin exhibits altered binding to TRIM21 relative to an immunoglobulin without said mutation.

28. The composition of claim 27, wherein said altered binding is increased or decreased binding affinity.

29. The composition of any one of claims 25 to 28, wherein said immunoglobulin exhibits altered binding to FcRN and/or altered activation of NFkB relative to an immunoglobulin without said mutation.

30. The composition of any one of claims 25 to 29, wherein said immunoglobulin further comprises one or more mutation in the hinge region of said immunoglobulin.

31. The composition of any one of claims 25 to 30, wherein said immunoglobulin is an IgGl, IgG2, IgG3, or IgG4 subclass.

32. The composition of any one of claims 25 to 31 , wherein said immunoglobulin has mutations at one or more of positions selected from the group consisting of 131, 31 1, 345,

385, 433, 434, 435, 436, and 428.

33. The composition of any one of claims 25 to 32, wherein said mutation is selected from the group consisting of IgGl-Q311R/N434W/M428E, IgGl-Q311R/N434W, IgGl- Q311R, IgGl-N434W, IgGl-N434Y, IgGl-Q311H, IgGl-H433R, IgGl-E345R,

IgG3_AHinge_exonl_2, IgG3_AHinge_exonl_2_3, IgG3_AHinge_exonl_2_3_4, IgG3- AHinge_exon2, IgG3_AHinge_exon2_3, IgG3_AHinge_exon2_3_4, and IgG3- C 131 S/R435H-IgGlHinge.

34. The composition of claim 33, wherein the constant region of said immunoglobulin is described by an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51 and sequences that are at least 90% identical to SEQ ID NOs: SEQ ID NOs: 5, 6, 7, 8, 9, 10, 12, 13, 14, 18, 19, 20, 21, 22, 23, and 26-51.

35. A composition, comprising:

an immunoglobulin comprising a at least one mutation in the Fc region or hinge region of said immunoglobulin, wherein said immunoglobulin modulates TRIM21 signaling, and wherein said mutation is selected from the group consisting of IgGl-Q311R/N434W/M428E, IgGl-Q311R/N434W, IgGl-MST/G385E/M428E, IgGl-Y436W, IgG3(b)-

Q311R/N434F/H433R/R435H, IgGl-Q311R, IgGl-Q311R/M428E, IgGl-Q311H/M428E, IgGl-G385E/M428E, IgGl-Q311F, IgG3(b)-Q311R/G385E/M428E/R435H, IgGl- Q311R/G385E/M428E, IgGl-Q311R/N434Y/M428F, IgGl-Q311R/G385E/M428E/N434Y, IgGl-Q311R/G385E/M428F/N434Y, IgGl-N434F, IgG3-N434F/R435H, IgGl-S131C- IgG3Hinge, IgG3-C131S-IgGlHinge, IgGl-IgG3Fc, IgG3-IgGlFc, IgG3-AHinge_exonl, IgG3_AHinge_exonl_2, IgG3_AHinge_exonl_2_3, IgG3_AHinge_exonl_2_3_4, IgG3- AHinge_exon2, IgG3_AHinge_exon2_3, IgG3_AHinge_exon2_3_4, and IgG3- C131S/R435H-IgGlHinge .