Fc variant compositions and methods of use thereof

Abstract

To provide compositions and methods for augmenting antibody mediate receptor signaling.SOLUTION: Provided is an engineered polypeptide comprising an Fc variant of a wild-type human IgG Fc region, the Fc variant comprising at least two amino acid substitutions, the amino acid substitutions occurring at residue positions 228, 234, 235, 270, 322, 329, 331, 333, 345, 409, 430, 440, or a combination thereof, and the amino acid residues being numbered according to the EU index of Kabat.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 646,053, filed March 21, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. The disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art at the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction of either the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0004] FIELD OF THE INVENTION The present invention generally relates to therapeutic antibodies with enhanced function. Specifically, the present invention relates to polypeptides comprising variant Fc regions and antibodies comprising the same. More specifically, the present invention relates to Fc region-containing polypeptides having altered effector function as a result of one or more amino acid substitutions in the Fc region of the polypeptide. [Background technology]

[0005] Background of the Invention Monoclonal antibodies have great therapeutic potential and play an important role in today's medical portfolio. Over the past decade, a key trend in the pharmaceutical industry has been the development of monoclonal antibodies (mAbs) as therapeutic agents for the treatment of many diseases, such as cancer, asthma, arthritis, and multiple sclerosis.

[0006] The Fc region of an antibody, i.e., the end of the antibody heavy chain spanning the CH2 and CH3 domains and a portion of the hinge region, has limited variability and is involved in influencing the physiological role played by the antibody. The effector functions attributed to the Fc region of an antibody vary depending on the antibody class and subclass and involve the binding of the antibody via the Fc region to specific Fc receptors ("FcR") on cells, which elicits various biological responses. Summary of the Invention

[0007] The present invention features polypeptides comprising Fc variants of a wild-type human IgG Fc region, e.g., Fc variants having amino acid substitutions E345K, E430G, L234A, and L235A; or E345K, E430G, S228P, and R409K, in combination with one or more of D270A, K322A, P329V, P331V, and E333Q in the Fc of human IgG. Residues are numbered according to the EU index of Kabat (see, e.g., Edelman, et al., Proc Natl Acad Sci USA 63 (1969) 78-85). In addition to reduced CDC activity, the polypeptides exhibit reduced affinity for one or more human Fc receptors and / or increased receptor clustering compared to polypeptides having a wild-type IgG Fc region.

[0008] One aspect of the present invention relates to an engineered polypeptide comprising an Fc variant of a wild-type human IgG Fc region. In one embodiment, the Fc variant comprises one amino acid substitution, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions, at residue positions 228, 234, 235, 270, 322, 329, 331, 333, 345, 409, 430, 440, or a combination thereof, where amino acid residues are numbered according to the EU index of Kabat. In one embodiment, the amino acid at residue position 228 according to the EU index of Kabat is substituted with proline (P) or serine (S). In one embodiment, the amino acid at residue position 234 according to the EU index of Kabat is substituted with alanine (A). In one embodiment, the amino acid at residue position 235 according to the EU index of Kabat is substituted with alanine (A). In one embodiment, the glutamic acid (E) at residue position 345 according to the EU index of Kabat is substituted with lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). In one embodiment, the amino acid at residue position 409 according to the EU index of Kabat is substituted with lysine (K) or arginine (R). In one embodiment, the glutamic acid (E) at residue position 430 according to the EU index of Kabat is substituted with glycine (G), serine (S), phenylalanine (F), or threonine (T). In one embodiment, the serine (S) at residue position 440 according to the EU index of Kabat is substituted with tryptophan (W). In one embodiment, the aspartic acid (D) at residue position 270 according to the EU index of Kabat is substituted with a neutral nonpolar amino acid. In one embodiment, the lysine (K) at residue position 322 according to the EU index of Kabat is substituted with a neutral nonpolar amino acid. In one embodiment, the proline (P) at residue position 329 according to the EU index of Kabat is substituted with a neutral nonpolar amino acid. In one embodiment, the amino acid at residue position 331 according to the EU index of Kabat is substituted with a neutral nonpolar amino acid.In one embodiment, the neutral nonpolar amino acid comprises alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P), or valine (V). In one embodiment, glutamic acid (E) at residue position 333 according to the EU index of Kabat is substituted with a neutral polar amino acid. In one embodiment, the neutral polar amino acid is asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y). In one embodiment, the amino acid substitutions comprise L234A, L235A, E345K, and E430G, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, the amino acid substitutions comprise S228P, E345K, R409K, and E430G, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the amino acid substitutions further include D270A, K322A, and P331G, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the amino acid substitutions further include D270A and P331G, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the amino acid substitutions further include D270A, P331V, and E333Q, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the amino acid substitutions further include P329V, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the amino acid substitutions further include P331V, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the amino acid substitutions further include P329V and P331V, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the amino acid substitutions further comprise P329V and / or P331F, where amino acid residues are numbered according to the EU index of Kabat. In some embodiments, the polypeptide exhibits reduced affinity for one or more human Fc receptors compared to a polypeptide comprising a wild-type IgG Fc region.In other embodiments, the polypeptide further exhibits increased receptor clustering compared to a polypeptide comprising a wild-type IgG Fc region, hi a further embodiment, the polypeptide further exhibits reduced complement-dependent cytotoxicity (CDC).

[0009] An embodiment of the present invention relates to an engineered polypeptide comprising an Fc variant of a wild-type human IgG Fc region, wherein the Fc variant comprises an amino acid sequence comprising at least 90% identity to SEQ ID NO: 4, and wherein the amino acid substitutions are at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, A , X B , X C , X D , X E , or a combination thereof. In one embodiment, the Fc variant comprises an amino acid sequence comprising at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4. In one embodiment, X1 is an amino acid substitution comprising serine (S). In one embodiment, X2 is an amino acid substitution comprising alanine (A). In one embodiment, X3 is an amino acid substitution comprising alanine (A). In one embodiment, X4 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). In one embodiment, X5 is an amino acid substitution comprising lysine (K) or arginine (R). In one embodiment, X6 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). In one embodiment, X7 is an amino acid substitution comprising tryptophan (W). In one embodiment, X A , X B , X C , or X D is an amino acid substitution comprising a neutral nonpolar amino acid. In some embodiments, the neutral nonpolar amino acid comprises alanine (A), glycine (G), leucine (L), methionine (M), phenylalanine (F), proline (P), or valine (V). In another embodiment, X Eis an amino acid substitution comprising a polar neutral amino acid. In some embodiments, the polar neutral amino acid comprises asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y).

[0010] An embodiment of the present invention relates to an engineered polypeptide comprising an Fc variant of a wild-type human IgG Fc region, wherein the Fc variant comprises an amino acid sequence comprising at least 90% identity to SEQ ID NO: 5, and wherein the amino acid substitutions are at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, A , X B , X C , X D , X E , or a combination thereof. In one embodiment, the Fc variant comprises an amino acid sequence comprising at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 5. In one embodiment, X1 is an amino acid substitution comprising serine (S). In one embodiment, X2 is an amino acid substitution comprising alanine (A). In one embodiment, X3 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). In one embodiment, X4 is an amino acid substitution comprising lysine (K) or arginine (R). In one embodiment, X5 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). In one embodiment, X6 is an amino acid substitution comprising tryptophan (W). In one embodiment, X A , X B , X C , or X D is an amino acid substitution comprising a neutral nonpolar amino acid. In some embodiments, the neutral nonpolar amino acid comprises alanine (A), glycine (G), leucine (L), methionine (M), phenylalanine (F), proline (P), or valine (V). In another embodiment, X Eis an amino acid substitution comprising a polar neutral amino acid. In some embodiments, the polar neutral amino acid comprises asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y).

[0011] An embodiment of the present invention relates to an engineered polypeptide comprising an Fc variant of a wild-type human IgG Fc region, wherein the Fc variant comprises an amino acid sequence comprising at least 90% identity to SEQ ID NO: 6, and wherein the amino acid substitutions are at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, A , X B , X C , X D , X E , or a combination thereof. In one embodiment, the Fc variant comprises an amino acid sequence comprising at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6. In one embodiment, X1 is a substitution of an amino acid at residue position 228 according to the EU index of Kabat and comprises a proline (P). In one embodiment, X2 is an amino acid substitution comprising an alanine (A). In one embodiment, X3 is an amino acid substitution comprising an alanine (A). In one embodiment, X4 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). In one embodiment, X5 is an amino acid substitution comprising lysine (K) or arginine (R). In one embodiment, X6 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). In one embodiment, X7 is an amino acid substitution containing tryptophan (W). A , X B , X C , or X Dis an amino acid substitution comprising a neutral nonpolar amino acid. In some embodiments, the neutral nonpolar amino acid comprises alanine (A), glycine (G), leucine (L), methionine (M), phenylalanine (F), proline (P), or valine (V). In another embodiment, X E is an amino acid substitution comprising a polar neutral amino acid. In some embodiments, the polar neutral amino acid comprises asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y).

[0012] The polypeptide may be, for example, an antibody or an Fc fusion protein. The antibody may be a monospecific, bispecific, or multispecific antibody. The polypeptide may have a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the polypeptide may be an antibody specific for an immune modulator, such as CD27, OX40, 4-1BB, CD40L, ICOS, or CD28. In some embodiments, the polypeptide is an antibody specific for an inhibitory molecule on T cells, such as PD1, TIGIT, CTLA4, Lag3, Tim3, or KIR. In some embodiments, the polypeptide is an antibody specific for a stimulatory molecule on T cells, such as GITR, CD27, OX40, 4-BB, CD40L, ICOS, or CD28. In other embodiments, the polypeptide is an antibody specific for a chemokine receptor, such as CCR4, CXCR4, or CCR5. In other embodiments, the polypeptide is an antibody specific to a tumor-associated molecule on tumor cells, such as BCMA, CAIX, an antigen-presenting cell molecule, or a combination thereof. In some embodiments, the antigen-presenting cell molecule comprises PDL1 or PDL2. In further embodiments, the polypeptide is an antibody specific to an infectious agent. In further embodiments, the infectious agent comprises severe acute respiratory syndrome virus (SARS), Middle East respiratory syndrome virus (MERS), an alphavirus, a flavivirus, or an influenza virus. For example, the alphavirus can be Western equine encephalitis virus (WEEV), Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus, or chikungunya virus (CHKV). For example, the flavivirus can be West Nile virus (WNV), Denji virus serotypes 1-4, yellow fever virus, or Zika virus. In some embodiments, the flavivirus is mosquito-borne. In some embodiments, the influenza virus is an emerging influenza virus. In other embodiments, the antibody comprises a chimeric antigen receptor (CAR) targeting domain.In yet other embodiments, the CH1 domain, hinge, CH2 domain, CH3 domain, or a combination thereof of IgG Fc is incorporated into the extracellular domain of a chimeric antigen receptor (CAR). Optionally, the polypeptide is an antibody specific for BCMA, CAIX, CCR4, PDL1, PD-L2, PD1, glucocorticoid-inducible tumor necrosis factor receptor (GITR), TIGIT, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), influenza, or flavivirus.

[0013] In one embodiment, the polypeptide is an antibody specific for glucocorticoid-induced tumor necrosis factor receptor (GITR). In one embodiment, the recombinant GITR antibody comprises a variable region amino acid sequence disclosed in Table 1B and a variant Fc region amino acid sequence disclosed in Table 8B (SEQ ID NOs: 18, 19, 22, 26, 45), Table 9B (SEQ ID NOs: 18, 19, 22, 26, 47), Table 10B (SEQ ID NOs: 18, 19, 22, 26, 49), Table 11B (SEQ ID NOs: 18, 19, 22, 26, 51), Table 12B (SEQ ID NOs: 18, 19, 22, 26, 53), Table 13B (SEQ ID NOs: 18, 19, 22, 26, 55), Table 14B (SEQ ID NOs: 18, 19, 22, 26, 57), or Table 15B (SEQ ID NOs: 18, 19, 24, 26, 59).

[0014] In one embodiment, the polypeptide is an antibody specific for CCR4. In one embodiment, the recombinant CCR4 antibody comprises a variable region amino acid sequence disclosed in Table 1B and a variant Fc region amino acid sequence disclosed in Table 8B (SEQ ID NOs: 18, 19, 22, 26, 45), Table 9B (SEQ ID NOs: 18, 19, 22, 26, 47), Table 10B (SEQ ID NOs: 18, 19, 22, 26, 49), Table 11B (SEQ ID NOs: 18, 19, 22, 26, 51), Table 12B (SEQ ID NOs: 18, 19, 22, 26, 53), Table 13B (SEQ ID NOs: 18, 19, 22, 26, 55), Table 14B (SEQ ID NOs: 18, 19, 22, 26, 57), or Table 15B (SEQ ID NOs: 18, 19, 24, 26, 59).

[0015] In various aspects, the polypeptide is conjugated to a drug, a toxin, a radioactive label, or a combination thereof, as practiced in the art. In some embodiments, the toxin can be Pseudomonas exotoxin, ricin, botulinum toxin, or other toxins used by those skilled in the art, such as those described by Polito et al. (Biomedicines.2016 Jun 1;4(2).pii:E12.doi:10.3390 / biomedicines4020012), which is incorporated by reference in its entirety. In some embodiments, the radioactive label can be yttrium-90, rhenium-188, lutetium-177, strontium-89, radium-223, or the like. In some embodiments, the antibody drug conjugate may be monomethyleustatin E, or, for example, those described by Schumacher et al. (J Clin Immunol. 2016 May;36 Suppl 1:100-7. doi:10.1007 / s10875-016-0265-6. Epub 2016 Mar 22), which is incorporated by reference in its entirety.

[0016] The present invention also includes a method for treating a subject suffering from a disease by administering a polypeptide according to the present invention or a nucleic acid encoding the same to the subject, and a therapeutically effective amount of a composition comprising a polypeptide according to the present invention or a nucleic acid encoding the same and a pharmaceutically acceptable carrier to the subject.

[0017] In one embodiment, the present invention provides a method for enhancing T cell immunity, the method comprising administering to a subject a recombinant GITR antibody described herein or a recombinant CCR4 antibody described herein. In one embodiment, the present invention provides a method for treating a tumor in a subject, the method comprising administering to a subject a recombinant GITR antibody described herein or a recombinant CCR4 antibody described herein. In one embodiment, the present invention provides a method for treating a CCL22 / 17-secreting tumor, the method comprising administering to a subject a recombinant GITR antibody described herein or a recombinant CCR4 antibody described herein. In one embodiment, the CCL22 / 17-secreting tumor is a hematological cancer. In one embodiment, the hematological cancer is lymphoma or leukemia. In one embodiment, the tumor is a solid tumor or a liquid tumor. In one embodiment, the CCL22 / 17-secreting tumor is ovarian cancer. In some embodiments, the liquid tumor can be multiple myeloma, acute myeloid leukemia (AML), or acute lymphoblastic leukemia (ALL). In one embodiment, the present invention provides a method for treating a hematological cancer in a subject, the method comprising administering to the subject a recombinant CCR4 antibody described herein. In one embodiment, the hematological cancer is lymphoma or leukemia.

[0018] In another aspect, the present invention provides a method for enhancing cell signaling or inducing cellular receptor clustering by contacting cells with an antibody capable of binding to a ligand on the cells comprising an Fc variant of a wild-type human IgG Fc region. In another aspect, the present invention provides a method for reducing CDC activity of cells by contacting cells with an antibody capable of binding to a ligand on the cells comprising an Fc variant of a wild-type human IgG Fc region. The Fc variant has amino acid substitutions, such as amino acid substitutions at D270, K322, P329, P331, E345, E430, and / or S440, where residues are numbered according to the EU index of Kabat. In one embodiment, the mutations include one or more of D270A, K322A, P329V, P331G, P331V, P331F, E333Q, E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440W.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains.Methods and materials similar or equivalent to those described herein can be used to implement the present invention, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety.In the event of any discrepancy, the present specification, including definitions, shall prevail.In addition, the materials, methods and examples described herein are only illustrative and are not intended to be limiting.

[0020] [The present invention 1001] 1. An engineered polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising at least two amino acid substitutions, said amino acid substitutions occurring at residue positions 228, 234, 235, 270, 322, 329, 331, 333, 345, 409, 430, 440, or a combination thereof, wherein said amino acid residues are numbered according to the EU index of Kabat. [The present invention 1002] 1001. The polypeptide of claim 10, wherein the amino acid at residue position 228 according to EU index of Kabat is substituted with a proline (P) or a serine (S). [The present invention 1003] 1001. The polypeptide of claim 10, wherein said amino acid at residue position 234 according to EU index of Kabat is substituted with alanine (A). [The present invention 1004] 1001. The polypeptide of claim 10, wherein said amino acid at residue position 235 according to EU index of Kabat is substituted with alanine (A). [The present invention 1005] 1001. A polypeptide of the invention, wherein glutamic acid (E) at residue position 345 according to the EU index of Kabat is substituted with lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [The present invention 1006] 1001. The polypeptide of claim 10, wherein said amino acid at residue position 409 according to EU index of Kabat is substituted with lysine (K) or arginine (R). [The present invention 1007] 1001. A polypeptide of the invention, wherein glutamic acid (E) at residue position 430 according to the EU index of Kabat is substituted with glycine (G), serine (S), phenylalanine (F), or threonine (T). [The present invention 1008] 1001. A polypeptide of the invention, wherein the serine (S) at residue position 440 according to EU index of Kabat is substituted with a tryptophan (W). [The present invention 1009] 1001. A polypeptide of the invention, wherein the aspartic acid (D) at residue position 270 according to the EU index of Kabat is substituted with a neutral nonpolar amino acid. [The present invention 1010] 1001. A polypeptide of the invention, wherein the lysine (K) at residue position 322 according to the EU index of Kabat is substituted with a neutral nonpolar amino acid. [The present invention 1011] 1001. A polypeptide of the invention, wherein the proline (P) at residue position 329 according to the EU index of Kabat is substituted with a neutral nonpolar amino acid. [The present invention 1012] 1001. The polypeptide of claim 10, wherein the amino acid at residue position 331 according to EU index of Kabat is substituted with a neutral nonpolar amino acid. [The present invention 1013] The polypeptide of any one of claims 1009, 1010, 1011, and 1012, wherein the neutral nonpolar amino acid comprises alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P), or valine (V). [The present invention 1014] 1001. A polypeptide of the invention, wherein a glutamic acid (E) at residue position 333 according to the EU index of Kabat is substituted with a neutral polar amino acid. [The present invention 1015] The polypeptide of the present invention, wherein the neutral polar amino acid is asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y). [The present invention 1016] 1001. The polypeptide of claim 10, wherein said amino acid substitutions comprise L234A, L235A, E345K, and E430G, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1017] 1001. The polypeptide of claim 1001, wherein said amino acid substitutions comprise S228P, E345K, R409K, and E430G, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1018] 1016 or 1017, a polypeptide of the invention, wherein said amino acid substitutions further comprise D270A, K322A, and P331G, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1019] 1016 or 1017, a polypeptide of the invention, wherein said amino acid substitutions further comprise D270A and P331G, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1020] 1016 or 1017, a polypeptide of the invention, wherein said amino acid substitutions further comprise D270A, P331V, and E333Q, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1021] 1016 or 1017, a polypeptide of the invention, wherein said amino acid substitution further comprises P329V, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1022] 1016 or 1017, a polypeptide of the invention, wherein said amino acid substitution further comprises P331V, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1023] 1016 or 1017, a polypeptide of the invention, wherein said amino acid substitutions further comprise P329V and P331V, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1024] 1016 or 1017, a polypeptide of the invention, wherein said amino acid substitutions further comprise P329V and / or P331F, said amino acid residues being numbered according to the EU index of Kabat. [The present invention 1025] 1001. The polypeptide of the present invention, which exhibits reduced affinity for one or more human Fc receptors compared to a polypeptide comprising said wild-type IgG Fc region. [The present invention 1026] 1025 polypeptides of the invention, further exhibiting increased receptor clustering compared to polypeptides comprising said wild-type IgG Fc region. [The present invention 1027] Polypeptides of the invention 1025 further exhibit reduced complement dependent cytotoxicity (CDC). [The present invention 1028] 1001. A polypeptide of the present invention comprising a human IgG1, IgG2, IgG3, or IgG4 Fc region. [The present invention 1029] The polypeptide of the present invention 1001, which is an antibody or an Fc fusion protein. [The present invention 1030] The polypeptide of the present invention, wherein said antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. [The present invention 1031] The polypeptide of the present invention 1001 conjugated to a drug, a toxin, a radiolabel, or a combination thereof. [The present invention 1032] The polypeptide of the present invention is an antibody specific to an inhibitory molecule on T cells. [The present invention 1033] The polypeptide of the present invention 1032, wherein the inhibitory molecule on T cells comprises PD1, TIGIT, CTLA4, Lag3, Tim3, or KIR. [The present invention 1034] The polypeptide of the present invention is an antibody specific for a stimulatory molecule on a T cell. [This invention 1035] The polypeptide of the present invention 1034, wherein said stimulatory molecule on T cells comprises GITR, CD27, OX40, 4-BB, CD40L, ICOS, or CD28. [The present invention 1036] 1001. A polypeptide of the present invention which is an antibody specific for a chemokine receptor. [This invention 1037] The polypeptide of the present invention 1036, wherein the chemokine receptor comprises CCR4, CXCR4, or CCR5. [The present invention 1038] The polypeptide of the present invention is an antibody specific to a tumor-associated molecule on tumor cells. [This invention 1039] The polypeptide of the present invention, wherein the tumor-associated molecule on the tumor cell comprises BCMA, CAIX, an antigen-presenting cell molecule, or a combination thereof. [The present invention 1040] The polypeptide of the present invention 1039, wherein the antigen-presenting cell molecule comprises PDL1 or PDL2. [This invention 1041] 1001. A polypeptide of the present invention which is an antibody specific for an infectious agent. [The present invention 1042] 1001. The polypeptide of claim 10, wherein said infectious agent comprises Severe Acute Respiratory Syndrome virus (SARS), Middle East Respiratory Syndrome virus (MERS), an alphavirus, a flavivirus, or an influenza virus. [This invention 1043] The polypeptide of the present invention 1042, wherein said alphavirus comprises Western Equine Encephalitis Virus (WEEV), Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus, or Chikungunya Virus (CHKV). [This invention 1044] The polypeptide of the present invention 1042, wherein the flavivirus is mosquito-borne. [This invention 1045] The polypeptide of the present invention 1042, wherein the flavivirus comprises West Nile virus (WNV), Denzi virus serotypes 1 to 4, yellow fever virus, or Zika virus. [The present invention 1046] The polypeptide of the present invention 1042, wherein the influenza virus is an emerging influenza virus. [This invention 1047] 1001. The polypeptide of claim 10, wherein said antibody comprises a targeting domain of a chimeric antigen receptor (CAR). [This invention 1048] The polypeptide of the present invention 1047, wherein a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a combination thereof is incorporated into the extracellular domain. [This invention 1049] The polypeptide of the present invention is an antibody specific to glucocorticoid-induced tumor necrosis factor receptor (GITR). [The present invention 1050] The polypeptide of the present invention is an antibody specific to CCR4. [This invention 1051] An engineered polypeptide comprising an Fc variant human IgG Fc region, said Fc variant comprising an amino acid sequence comprising at least 90% identity to SEQ ID NO: 4, and wherein the amino acid substitutions are at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, X50, X51, X52, X5 A , X B , X C , X D , X E , or a combination thereof. [This invention 1052] The polypeptide of the present invention 1051, wherein X1 is an amino acid substitution comprising serine (S). [This invention 1053] The polypeptide of the present invention 1051, wherein X2 is an amino acid substitution comprising an alanine (A). [This invention 1054] The polypeptide of the present invention 1051, wherein X3 is an amino acid substitution comprising an alanine (A). [This invention 1055] The polypeptide of claim 1051, wherein X4 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [This invention 1056] The polypeptide of claim 1051, wherein X5 is an amino acid substitution comprising lysine (K) or arginine (R). [This invention 1057] The polypeptide of claim 1051, wherein X6 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). [This invention 1058] The polypeptide of claim 1051, wherein X7 is an amino acid substitution comprising tryptophan (W). [This invention 1059] 1. An engineered polypeptide comprising an Fc variant human IgG Fc region, wherein said Fc variant comprises an amino acid sequence comprising at least 90% identity to SEQ ID NO: 5, and wherein amino acid substitutions are selected from the group consisting of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, X50, X51, X A , X B , XC , X D , X E , or a combination thereof. [The present invention 1060] The polypeptide of the present invention 1059, wherein X1 is an amino acid substitution comprising serine (S). [This invention 1061] The polypeptide of the present invention 1059, wherein X2 is an amino acid substitution comprising alanine (A). [This invention 1062] The polypeptide of claim 1059, wherein X3 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [This invention 1063] The polypeptide of claim 1059, wherein X4 is an amino acid substitution comprising lysine (K) or arginine (R). [This invention 1064] The polypeptide of claim 1059, wherein X5 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). [This invention 1065] The polypeptide of claim 1059, wherein X6 is an amino acid substitution comprising tryptophan (W). [The present invention 1066] An engineered polypeptide comprising an Fc variant human IgG Fc region, said Fc variant comprising an amino acid sequence comprising at least 90% identity to SEQ ID NO: 6, and wherein the amino acid substitutions are at least one of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49, X50, X51, X52, X5 A , X B , X C , X D , X E , or a combination thereof. [This invention 1067] 1066. The polypeptide of the invention, wherein X1 is a substitution of an amino acid at residue position 228 according to the EU index of Kabat, and comprises a proline (P). [The present invention 1068] 1066. The polypeptide of the present invention, wherein X2 is an amino acid substitution comprising an alanine (A). [The present invention 1069] 1066. The polypeptide of the present invention, wherein X3 is an amino acid substitution comprising an alanine (A). [The present invention 1070] 1066. The polypeptide of the present invention, wherein X4 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [This invention 1071] 1066. The polypeptide of the present invention, wherein X5 is an amino acid substitution comprising lysine (K) or arginine (R). [This invention 1072] 1066. The polypeptide of the present invention, wherein X6 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). [This invention 1073] 1066. The polypeptide of the present invention, wherein X7 is an amino acid substitution comprising tryptophan (W). [This invention 1074] X A , X B , X C , or X D is an amino acid substitution comprising a neutral nonpolar amino acid. [This invention 1075] The polypeptide of claim 1074, wherein the neutral nonpolar amino acid comprises alanine (A), glycine (G), leucine (L), methionine (M), phenylalanine (F), proline (P), or valine (V). [This invention 1076] X E The polypeptide of claim 1051, 1059, or 1066, wherein the amino acid substitution comprises a polar neutral amino acid. [This invention 1077] 1076. The polypeptide of claim 1076, wherein said neutral polar amino acid comprises asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y). [This invention 1078] A recombinant GITR antibody comprising a variable region amino acid sequence disclosed in Table 1B and a variant Fc region amino acid sequence disclosed in Table 8B (SEQ ID NOs: 18, 19, 22, 26, 45), Table 9B (SEQ ID NOs: 18, 19, 22, 26, 47), Table 10B (SEQ ID NOs: 18, 19, 22, 26, 49), Table 11B (SEQ ID NOs: 18, 19, 22, 26, 51), Table 12B (SEQ ID NOs: 18, 19, 22, 26, 53), Table 13B (SEQ ID NOs: 18, 19, 22, 26, 55), Table 14B (SEQ ID NOs: 18, 19, 22, 26, 57), or Table 15B (SEQ ID NOs: 18, 19, 24, 26, 59). [This invention 1079] A recombinant CCR4 antibody comprising a variable region amino acid sequence disclosed in Table 1B and a variant Fc region amino acid sequence disclosed in Table 8B (SEQ ID NOs: 18, 19, 22, 26, 45), Table 9B (SEQ ID NOs: 18, 19, 22, 26, 47), Table 10B (SEQ ID NOs: 18, 19, 22, 26, 49), Table 11B (SEQ ID NOs: 18, 19, 22, 26, 51), Table 12B (SEQ ID NOs: 18, 19, 22, 26, 53), Table 13B (SEQ ID NOs: 18, 19, 22, 26, 55), Table 14B (SEQ ID NOs: 18, 19, 22, 26, 57), or Table 15B (SEQ ID NOs: 18, 19, 24, 26, 59). [The present invention 1080] A method for enhancing T cell immunity, comprising the step of administering the recombinant GITR antibody of the present invention 1078 or the recombinant CCR4 antibody of the present invention 1079 to a subject. [This invention 1081] A method for treating a tumor in a subject, comprising administering to the subject a recombinant GITR antibody of the present invention. [This invention 1082] A method for treating CCL22 / 17-secreting tumors, comprising administering to a subject a recombinant CCR4 antibody of the present invention. [This invention 1083] The method of claim 1082, wherein the CCL22 / 17-secreting tumor is a hematological cancer. [This invention 1084] The method of claim 1083, wherein the blood cancer is lymphoma or leukemia. [This invention 1085] 1083. The method of claim 1082, wherein said CCL22 / 17-secreting tumor is ovarian cancer. [The present invention 1086] 1. A method for enhancing cell signaling in a cell, comprising contacting the cell with an antibody that binds a ligand to the cell, wherein the antibody comprises a polypeptide of the invention 1001, 1051, 1059, or 1066 or an Fc variant of a wild-type human IgG Fc region, wherein the Fc variant comprises amino acid substitutions at D270, K322, P329, P331, E333, E345, E430, and / or S440, wherein the residues are numbered according to the EU index of Kabat. [This invention 1087] 1086. The method of claim 1086, wherein said substitution comprises D270A, K322A, P329V, P331G, P331V, P331F, E333Q, E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440W, or a combination thereof. [This invention 1088] 1. A method for inducing receptor clustering in cells, comprising contacting the cells with an antibody that binds a ligand to the cells, wherein the antibody comprises a polypeptide of the invention 1001, 1051, 1059, or 1066 or an Fc variant of a wild-type human IgG Fc region, wherein the Fc variant comprises amino acid substitutions at D270, K322, P329, P331, E333, E345, E430, and / or S440, wherein the residues are numbered according to the EU index of Kabat. [This invention 1089] 1088. The method of claim 1088, wherein the substitution comprises D270A, K322A, P329V, P331G, P331V, P331F, E333Q, E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440W, or a combination thereof. [The present invention 1090] The method of claim 1083, wherein the tumor is a solid tumor or a liquid tumor. [This invention 1091] A method for reducing CDC activity of a cell, comprising contacting the cell with an antibody that binds a ligand to the cell, wherein the antibody comprises a polypeptide of the invention 1001, 1051, 1059, or 1066 or an Fc variant of a wild-type human IgG Fc region, wherein the Fc variant comprises amino acid substitutions at D270, L234, L235, K322, P329, P331, and / or E333, wherein the residues are numbered according to the EU index of Kabat. Other features and advantages of the present invention will be apparent from and encompassed by the following detailed description and claims. [Brief explanation of the drawings]

[0021] [Figure 1] SDS-PAGE analysis of anti-GITR antibodies expressed and purified from 293F cells. 293F cells were transiently transfected with pTCAE plasmids encoding the anti-GITR antibodies E1-3H7 IgG1 LALA (lane 1), E1-3H7-stabilized IgG4 (lane 2), CTI-10-stabilized IgG4 (lane 3), E1-3H7 IgG1 LALA hexamer (lane 4), E1-3H7-stabilized IgG4 hexamer (lane 5), and E1-3H7 IgG1 WT hexamer (lane 6). Cell supernatants were collected after 96 hours and purified with Protein A affinity resin. Approximately 2 μg of each purified antibody (measured by OD280 reading after purification) was analyzed on a 4-20% polyacrylamide gel and visualized by Coomassie blue staining. Lane 7 contains the control CTI-10 IgG1 at a known concentration. Panel A: reducing conditions, panel B: non-reducing conditions. Data show that each antibody was expressed and purified. [Figure 2] FIG. 1 shows that GITR-GITRL interaction activates the NF-kB pathway in the GloResponse NF-kB-luc2P / GITR Jurkat cell assay system produced by Promega and used in our assays. [Figure 3-1]GloResponse NF-kB-luc2P / GITR Jurkat cells are reporter cells that generate a ligand or antibody response based on luciferase activity with the surface-expressed receptor GITR. As a system control, panel A shows that the GITR ligand (GITRL) induced the expected luciferase activity, and panel B shows data showing that an anti-HA antibody further enhanced the luciferase activity induced by 111 ng / ml of GITRL (note that GITRL is fused to a c-terminal HA tag). [Figure 3-2] Panel C shows that our newly discovered anti-GITR antibody E1-3H7-sIgG4 can induce GiTR / NF-kB-dependent luciferase alone or further enhance the luciferase activity induced by 111 ng / ml GITRL, which is different from the behavior of the commercial anti-GITR Ab control, CTI-10, panel D. [Figure 4-1] The hexamerized anti-GITR E1-3H7 antibody has increased sensitivity in mediating GITR / NF-kB-dependent luciferase activity. (A) The anti-GITR antibody E1-3H7 IgG1-LALA and the corresponding hexamer (E1-3H7-LALA Hex) induced luciferase activity from GloResponse NF-kB-luc2P / GITR Jurkat cells in a dose-dependent manner. Note that E1-3H7 hexamer was able to shift luciferase induction approximately 1 log lower in antibody concentration. (B) The anti-GITR E1-3H7 antibody further enhances GITRL-induced luciferase activity. Once again, E1-3H7-LALA hexamer achieved such induction at much lower Ab concentrations. Panels C and D show that E1-3H7 had a similar effect in stabilizing IgG4 and its corresponding sIgG4 hexamer. Anti-GITR E1-3H7 antibody was used in 3-fold dilutions from 5000 ng / ml to 20.58 ng / ml in the absence (panels A and C) or presence (panels B and D) of 111 ng / ml of GITR ligand. [Figure 4-2] See description of Figure 4-1. [Figure 5-1] The hexamerized anti-GITR E1-3H7 antibody has increased sensitivity in mediating GITR / NF-kB-dependent luciferase activity. To confirm the full range of luciferase induction, experiments were similar to those shown in Figure 4, except that anti-GITR E1-3H7-IgG1 LALA or IgG4 antibody concentrations were used in three-fold dilutions ranging from 15,000 ng / ml to 61.73 ng / ml in the absence (panels A and C) or presence (panels B and D) of 111 ng / ml of GITR ligand, whereas their corresponding hexamer forms remained constant, ranging from 5,000 ng / ml to 20.58 ng / ml. An irrelevant IgG control showed no significant effect on the basal level of luciferase induction by 111 ng / ml of GITRL. [Figure 5-2] See description of Figure 5-1. [Figure 6-1] The IgG1 Fc wild-type, IgG1 Fc LALA mutant, or stabilized IgG4 hexamer of anti-GITR E1-3H7 antibody have similar activity in mediating GITR / NF-kB-dependent luciferase activity. Anti-GITR E1-3H7-IgG1 WT, IgG1 LALA, or sIgG4 hexamer antibody concentrations were used in 3-fold dilutions ranging from 5,000 ng / ml to 20.58 ng / ml in the absence or presence of 111 ng / ml of GITR ligand, while the control IgG1 had concentrations ranging from 15,000 ng / ml to 61.73 ng / ml. Note that the E1-3H7 IgG1 WT hexamer results in panel A are from a separate experiment than those presented in panels B and C or panels D and E. The x- and y-axes are the same for panels A–E. [Figure 6-2] See description of Figure 6-1. [Figure 7] ADCC assay using a reporter system from Promega. [Figure 8-1] Nucleic acid and amino acid sequences of the Fc region of WT and LALA hexamer mutant of IgG1. Figure 8 discloses SEQ ID NOs: 92 to 95, respectively, in order of appearance. [Figure 8-2]See description of Figure 8-1. [Figure 8-3] See description of Figure 8-1. [Figure 8-4] See description of Figure 8-1. [Figure 8-5] See description of Figure 8-1. [Figure 9-1] Nucleic acid and amino acid sequences of the Fc region of stabilized hexameric IgG4. Figure 9 discloses SEQ ID NOS: 96-97, respectively, in order of appearance. [Figure 9-2] See description of Figure 9-1. [Figure 9-3] See description of Figure 9-1. [Figure 10] Expression vector maps for vectors that can be used for mammalian expression of IgG antibodies. [Figure 11] Expression vector maps for vectors that can be used for mammalian expression of IgG antibodies. [Figure 12-1] 1 is the amino acid sequence for the wild-type Fc region of IgG1 (SEQ ID NO: 1) and the corresponding amino acid residue numbers according to the EU index of Kabat. [Figure 12-2] See description of Figure 12-1. [Figure 12-3] See description of Figure 12-1. [Figure 12-4] See description of Figure 12-1. [Figure 12-5] See description of Figure 12-1. [Figure 12-6] See description of Figure 12-1. [Figure 12-7] See description of Figure 12-1. [Figure 13-1] 1 is the amino acid sequence for the wild-type Fc region of IgG2 (SEQ ID NO: 2) and the corresponding amino acid residue numbers according to the EU index of Kabat. [Figure 13-2] See description of Figure 13-1. [Figure 13-3] See description of Figure 13-1. [Figure 13-4] See description of Figure 13-1. [Figure 13-5] See description of Figure 13-1. [Figure 13-6] See description of Figure 13-1. [Figure 13-7] See description of Figure 13-1. [Figure 14-1] 1 is the amino acid sequence for the wild-type Fc region of IgG4 (SEQ ID NO: 3) and the corresponding amino acid residue numbers according to the EU index of Kabat. [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1. [Figure 14-4] See description of Figure 14-1. [Figure 14-5] See description of Figure 14-1. [Figure 14-6] See description of Figure 14-1. [Figure 14-7] See description of Figure 14-1. [Figure 15] Figure 1 shows that CDC activity remains in all anti-GITR Ab constructs except for the sIgG4 monomer. The graphs represent similar experiments using different reagents to quantify the amount of cell killing. The assay in the left graph uses the CellTiter-Glo system, which determines the number of viable cells in culture, while the assay in the right graph uses CytoTox-Glo, which counts only dead cells. [Figure 16] Ribbon structures of several mutations introduced into the CH2 region of the LALA-hexamer construct to generate reduced complement-dependent cytotoxicity (CDC). Panel A of Figure 16 shows the key residues in CH2 that are involved in C1q binding and are targeted for mutation. Panels B-H show the residue(s) mutated in each construct and the predicted effect(s) of the mutations. Panels I and J show two CL fusions, one with anti-PDL1 scFv and one with the GFP analog zsGreen. [Figure 17-1]Photographic images of SDS-PAGE gels (non-reducing gel, reducing gel (10% BME)) of GITR mutants. Expi293F cells were transfected with ExpiFectamine and cultured for 5 days before harvesting and purifying via Protein A-conjugated Sepharose. 1 μg of each purified protein was run on a Bolt™ 4-12% Bis-Tris Plus Gel. The sample in the right gel was not reduced, while the gel on the left was reduced with 10% β-mercaptoethanol. Lane 1. Ladder (Biorad Precision Plus), lane 2. mAb2-3 IgG1 WT monomer, lane 3. mAb2-3 IgG1 WT hexamer, lane 4. E1-3H7 IgG1 WT monomer, lane 5. E1-3H7 IgG1 WT hexamer, lane 6. E1-3H7 IgG1 LALA monomer, lane 7. E1-3H7 IgG1 LALA hexamer, lane 8. Mt 1, lane 9. Mt 2, lane 10. Mt 3, lane 11. PV, lane 12. VP, lane 13. VV, lane 14. aPDL1, and lane 15. PF (P329P P331F). [Figure 17-2] See description of Figure 17-1. [Figure 18] Binding curves of anti-GITR Ab binding to GITR+ cells, analyzed by flow cytometry for % cells positive for binding. Key to GITR hexamer mutants tested: (a) Mt1:D270A K322A P331G, (b) Mt2:D270A P331G, (c) Mt3:D270A P331V E333Q, (d) VP:P329V P331P, (e) PV:P329P P331V, (f) VV:P329V P331V, and (g) PF:P329P P331F. [Figure 19]Binding curves for anti-GITR Ab binding to GITR+ cells, analyzed by flow cytometry for MFI (mean fluorescence intensity). Key of GITR hexamer mutants tested: (a) Mt1:D270A K322A P331G, (b) Mt2:D270A P331G, (c) Mt3:D270A P331V E333Q, (d) VP:P329V P331P, (e) PV:P329P P331V, (f) VV:P329V P331V, and (g) PF:P329P P331F. [Figure 20] Figure 1 depicts a bar graph of CDC of mutants showing reduced CDC compared to the Wt and LALA constructs (RLU). Key of mutants tested: (a) Mt1:D270A K322A P331G, (b) Mt2:D270A P331G, (c) Mt3:D270A P331V E333Q, (d) VP:P329V P331P, (e) PV:P329P P331V, (f) VV:P329V P331V, and (g) PF:P329P P331F. All antibody heavy and light chain variable regions are derived from the parental E1-3H7 anti-GITR antibody. [Figure 21] Figure 1 depicts a bar graph of CDC of mutants showing reduced CDC compared to Wt and LALA constructs (% kill). Key of mutants tested: (a) Mt1:D270A K322A P331G, (b) Mt2:D270A P331G, (c) Mt3:D270A P331V E333Q, (d) VP:P329V P331P, (e) PV:P329P P331V, (f) VV:P329V P331V, and (g) PF:P329P P331F. All antibodies are derived from the parental E1-3H7 anti-GITR antibody. The introduced mutations significantly reduce the amount of CDC activity compared to the original antibody. [Figure 22]Graphs of the GITR bioassay (RLU) are shown. The left graph is antibody only, while the right graph has 111 ng / ml of GITR added to each sample (results for left and right graphs were performed on different days). The data show that the mutations made to reduce CDC activity do not affect antibody hexamerization. Compared to the monomer, all of the hexamers show a significant shift to the left in the dose-response curve. [Figure 23] Graphs of fold induction by antibody and constant GITRL in the GITR bioassay are shown. The left graph is antibody only, while the right graph has 111 ng / ml GITRL added to each sample (comparison between experiments performed on different days). The data show that the mutations made to reduce CDC activity do not affect antibody hexamerization. Compared to the monomer, all of the hexamers show a significant shift to the left in the dose-response curve. When costimulated with GITR ligand (GITRL), the antibody has an additive effect (Figures 22 and 23), whereas the commercially available GTI-10 anti-GITR antibody does not. [Figure 24] Figure 1 shows a graph of fold induction by antibody when normalized to GITRL in the GITR bioassay. The graph deconvolves the effect of GITRL from the antibody by normalizing the fold induction to GITRL (at 111 ng / ml). The normalized fold induction is calculated as follows: RLU of sample / RLU of GITRL (111 ng / ml) alone. This analysis of the bioactivity assay also shows that the mutated hexamer continues to have a significant shift to the left in the dose-response curve compared to the monomer. [Figure 25] 1 shows a graph of observed ADCC activity. The mutants listed on the graph do not have any measurable ADCC activity. The negative control IgG did not show specific ADCC activity. [Figure 26] 1 is a summary of variants contemplated by the present invention. [Figure 27-1] 1 is the amino acid sequence for the wild-type Fc region of IgG3 (SEQ ID NO: 60) and the corresponding amino acid residue numbers according to the EU index of Kabat. [Figure 27-2] See description of Figure 27-1. [Figure 27-3] See description of Figure 27-1. [Figure 27-4] See description of Figure 27-1. [Figure 27-5] See description of Figure 27-1. [Figure 27-6] See description of Figure 27-1. [Figure 27-7] See description of Figure 27-1. [Figure 28] Figure 1 shows the binding (%) of IgG Lc fusions. A GITR-PDL1 Lc fusion IgG was incubated at various concentrations with CHO-GITR+ cells or Expi293F cells transiently transfected with PDL1 (approximately 75-80% transfection efficiency). After 25 min of incubation at RT, cells were washed, and binding of the fusion antibody was detected with anti-His-PE via the His tag on the C-terminus of the PDL1-scFv fusion. This graph demonstrates that each arm of the bispecific IgG can independently bind to its target, as determined by the % of PE-positive cells detected. [Figure 29] This graph shows the binding (MFI) of IgG Lc fusions. aGITR-PDL1 Lc fusions were incubated at various concentrations with CHO-GITR+ cells or Expi293F cells transiently transfected with PDL1 (approximately 75-80% transfection efficiency). After 25 min of incubation at RT, cells were washed, and binding of the fusion antibodies was detected via the His tag on the C-terminus of the PDL1-scFv fusion. This graph demonstrates that each arm of the bispecific IgG can independently bind to its target, as determined by mean fluorescence intensity (MFI). [Figure 30]Figure 1 shows a graph showing the % simultaneous binding of IgG Lc fusions. 1E6 CHO-GITR cells were used for each sample. aGITR IgG1 LALA Hex Lc fusion (aPDL1) was added in two-fold serial dilutions and incubated at RT for 25 minutes. The samples were then washed, and 1.5 μg of PD-L1-rbFc was added to each tube, and the tubes were incubated at RT for 25 minutes. After another wash, Biolegend's anti-rabbit IgG FITC (2 μg / ml) was added to the wells for detection. [Figure 31] Figure 1 shows the simultaneous binding (MFI) of IgG Lc fusions. 1E6 CHO-GITR cells were used for each sample. aGITR IgG1 LALA Hex Le fusion (aPDL1) was added in two-fold serial dilutions and incubated at RT for 25 minutes. The samples were then washed, and 1.5 μg of PD-L1-rbFc was added to each tube, and the tubes were incubated at RT for 25 minutes. After another wash, Biolegend's anti-rabbit IgG FITC (2 μg / ml) was added to the wells for detection. [Figure 32] Figure 32 shows an amino acid sequence alignment of CH2 of IgG1 and IgG4. Mutations were made in the IgG4 construct similar to IgG1LALA mut3 to eliminate CDC activity from the IgG4 hexamer. IgG1 LALA Mut3 is D270A P331V E333Q. In sIgG4, residue 331 is S. In the first Mut3 analog, only D270A and E33Q were changed, which are identical in IgG1 and IgG4. To create the second construct, residues 330 and 331 were also changed to be identical to IgG1 LALA, as they are part of the C1q binding pocket. Figure 32 discloses SEQ ID NOs: 20 and 38, respectively, in order of appearance. [Figure 33] 1 is a graph showing the CDC activity (1 hour) of sIgG4 mutants. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention A detailed description of one or more embodiments is provided herein. However, it is understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be construed as limiting, but as a basis for the claims and as a representative basis for teaching one skilled in the art how to use the present invention in any suitable manner.

[0023] Fc receptors can have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that can mediate several intracellular signaling events. These receptors are expressed on a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. Formation of the Fc / FcγR complex recruits these effector cells to the site of bound antigen, typically triggering intracellular signaling events and important subsequent immune responses, such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

[0024] In many situations, binding and stimulation of effector functions mediated by the Fc region of an immunoglobulin is highly beneficial, for example, for CD20 antibodies, but in certain instances it may be more advantageous to reduce or even eliminate effector functions.

[0025] In other instances, for example, when the goal is to block the interaction of a widely expressed receptor with its cognate ligand, it would be advantageous to reduce or eliminate all antibody effector functions to reduce undesirable toxicity.

[0026] It would also be advantageous to enhance signaling by increasing receptor clustering.

[0027] It would also be advantageous to significantly reduce complement dependent cytotoxicity (CDC) activity.

[0028] There is an unmet need for antibodies with greatly reduced effector functions, such as ADCC and / or ADCP and / or CDC, and enhanced receptor cell signaling and / or induced receptor cell clustering. Therefore, an object of the present invention was to synthesize and / or engineer polypeptides of immunoglobulin Fc regions with introduced mutations to promote such effects and ultimately identify antibodies comprising the engineered Fc regions. In one embodiment, antibodies can be developed for cancer therapy with the variant Fc regions described herein (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 variant Fc regions). In one embodiment, antibodies can be generated that can hexamerize while avoiding complement activation. In another embodiment, antibodies can be generated that can hexamerize while also avoiding effector function (e.g., antibody-dependent cellular cytotoxicity (ADCC)). In one embodiment, the antibody is specific for GITR. In one embodiment, the antibody is specific for CCR4. In some embodiments, the variant Fc region can comprise a variant hinge, CH1, and / or CH2 domain of an IgD or IgE Fc region, the amino acid sequence of which is described in WO 2007 / 121354, the entire contents of which are incorporated by reference.

[0029] The present invention is based, in part, on the discovery that mutations in the Fc region of antibodies known to promote antibody hexamerization and increased complement-dependent cytotoxicity (CDC) also have the unexpected ability to significantly enhance effector cell signaling. All of the polypeptide variants, including antibody variants, of the present invention comprise a binding region and a full-length or partial Fc domain of an immunoglobulin that contains one or more mutation(s) known to promote antibody hexamerization and reduce effector function.

[0030] SEQ ID NO: 1 provides the amino acid sequence of the wild-type Fc region of IgG1 (UniProtKB-P01857(IGHG1_HUMAN), 330 amino acids), with the CH1 domain in bold, the hinge region underlined, the CH2 domain in italics, the CH3 domain in dashed, and the shaded boxes are amino acids that may be substituted according to the invention. Figure 12 is a table correlating SEQ ID NO: 1 with amino acid residues numbered according to the EU index of Kabat. TIFF0007804715000001.tif20157

[0031] SEQ ID NO: 4 provides the amino acid sequence of the variant Fc region of IgG1 (UniProtKB-P01857(IGHG1_HUMAN), 330 amino acids), where the CH1 domain is in bold, the hinge region is underlined, the CH2 domain is in italics, the CH3 domain is dashed, and the shaded boxes represent amino acid residues that may be substituted in accordance with the invention, where X1 is a substitution of the amino acid at residue position 228 according to the EU index of Kabat and contains a proline (P), X2 is a substitution of the amino acid at residue position 234 according to the EU index of Kabat and contains an alanine (A), and X3 is a substitution of the amino acid at residue position 235 according to the EU index of Kabat and contains an alanine (A). X is a substitution of an amino acid at residue position 345 according to the EU index of Kabat and comprises lysine (K), glutamine (Q), arginine (R), or tyrosine (Y), X is a substitution of an amino acid at residue position 409 according to the EU index of Kabat and comprises arginine (R), X is a substitution of an amino acid at residue position 430 according to the EU index of Kabat and comprises glycine (G), serine (S), phenylalanine (F), or threonine (T), and X is a substitution of an amino acid at residue position 440 according to the EU index of Kabat and comprises tryptophan (W). A is an amino acid substitution at residue position 270 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X Bis an amino acid substitution at residue position 322 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X C is an amino acid substitution at residue position 329 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X D is an amino acid substitution at residue position 331 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X E is a substitution of an amino acid at residue position 333 according to the EU index of Kabat, and includes a neutral polar amino acid. In some embodiments, the neutral nonpolar amino acid includes alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P), or valine (V). In some embodiments, the neutral nonpolar amino acid is an amino acid that does not have a ring structure (e.g., alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), valine (V)). In some embodiments, the neutral polar amino acid includes asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y). TIFF0007804715000002.tif20158

[0032] SEQ ID NO: 2 provides the amino acid sequence of the wild-type Fc region of IgG2 (UniProtKB-P01859 (IGHG2_HUMAN), 326 amino acids), with the CH1 domain in bold, the hinge region underlined, the CH2 domain in italics, the CH3 domain in dashed, and the shaded boxes are amino acids that may be substituted according to the invention. Figure 13 is a table correlating SEQ ID NO: 2 with amino acid residues numbered according to the EU index of Kabat. TIFF0007804715000003.tif20157

[0033] SEQ ID NO: 5 provides the amino acid sequence of a variant Fc region of IgG2 (UniProtKB-P01859 (IGHG2_HUMAN), 326 amino acids), in which the CH1 domain is in bold, the hinge region is underlined, the CH2 domain is in italics, the CH3 domain is dashed, and shaded boxes represent amino acid residues that may be substituted in accordance with the invention, where X1 is a substitution of the amino acid at residue position 228 according to the EU index of Kabat and contains a proline (P), X2 is a substitution of the amino acid at residue position 235 according to the EU index of Kabat and contains an alanine (A), and X X3 is a substitution of an amino acid at residue position 345 according to the EU index of Kabat which contains lysine (K), glutamine (Q), arginine (R), or tyrosine (Y), X4 is a substitution of an amino acid at residue position 409 according to the EU index of Kabat which contains arginine (R), X5 is a substitution of an amino acid at residue position 430 according to the EU index of Kabat which contains glycine (G), serine (S), phenylalanine (F), or threonine (T), and X6 is a substitution of an amino acid at residue position 440 according to the EU index of Kabat which contains tryptophan (W). A is an amino acid substitution at residue position 270 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X B is an amino acid substitution at residue position 322 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X C is an amino acid substitution at residue position 329 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X D is an amino acid substitution at residue position 331 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X Eis a substitution of an amino acid at residue position 333 according to the EU index of Kabat, and includes a neutral polar amino acid. In some embodiments, the neutral nonpolar amino acid includes alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P), or valine (V). In some embodiments, the neutral nonpolar amino acid is an amino acid that does not have a ring structure (e.g., alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), valine (V)). In some embodiments, the neutral polar amino acid includes asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y). TIFF0007804715000004.tif20157

[0034] SEQ ID NO: 3 provides the amino acid sequence of the wild-type Fc region of IgG4 (UniProtKB-P01861 (IGHG4_HUMAN), 327 amino acids), with the CH1 domain in bold, the hinge region underlined, the CH2 domain in italics, the CH3 domain in dashed, and the shaded boxes are amino acids that may be substituted in accordance with the invention. Figure 14 is a table correlating SEQ ID NO: 3 with amino acid residues numbered according to the EU index of Kabat. TIFF0007804715000005.tif20157

[0035] SEQ ID NO: 6 provides the amino acid sequence of a variant Fc region of IgG4 (UniProtKB-P01861 (IGHG4_HUMAN), 327 amino acids), in which the CH1 domain is in bold, the hinge region is underlined, the CH2 domain is in italics, the CH3 domain is dashed, and shaded boxes represent amino acid residues that may be substituted in accordance with the invention, where X1 is a substitution of the amino acid at residue position 228 according to the EU index of Kabat and contains a proline (P), X2 is a substitution of the amino acid at residue position 234 according to the EU index of Kabat and contains an alanine (A), and X3 is a substitution of the amino acid at residue position 234 according to the EU index of Kabat and contains an alanine (A), and X4 is a substitution of the amino acid at residue position 240 according to the EU index of Kabat and contains an alanine (A). X is a substitution of an amino acid at residue position 345 according to the EU index of Kabat and comprises lysine (K), glutamine (Q), arginine (R), or tyrosine (Y), X is a substitution of an amino acid at residue position 409 according to the EU index of Kabat and comprises lysine (K), X is a substitution of an amino acid at residue position 430 according to the EU index of Kabat and comprises glycine (G), serine (S), phenylalanine (F), or threonine (T), and X is a substitution of an amino acid at residue position 440 according to the EU index of Kabat and comprises tryptophan (W). A is an amino acid substitution at residue position 270 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X B is an amino acid substitution at residue position 322 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X C is an amino acid substitution at residue position 329 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X D is an amino acid substitution at residue position 331 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X Eis a substitution of an amino acid at residue position 333 according to the EU index of Kabat, and includes a neutral polar amino acid. In some embodiments, the neutral nonpolar amino acid includes alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P), or valine (V). In some embodiments, the neutral nonpolar amino acid is an amino acid that does not have a ring structure (e.g., alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), valine (V)). In some embodiments, the neutral polar amino acid includes asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y). TIFF0007804715000006.tif22157

[0036] SEQ ID NO: 98 provides the amino acid sequence of the wild-type Fc region of IgG3 (UniProtKB-P01860 (IGHG3_HUMAN), 377 amino acids), with the CH1 domain in bold, the hinge region underlined, the CH2 domain in italics, the CH3 domain in dashed, and the shaded boxes are amino acids that may be substituted in accordance with the invention. Figure 27 is a table correlating SEQ ID NO: 60 with amino acid residues numbered according to the EU index of Kabat. TIFF0007804715000007.tif24157

[0037] SEQ ID NO: 61 provides the amino acid sequence of a variant Fc region of IgG3 (UniProtKB-P01860 (IGHG3_HUMAN), 377 amino acids), where the CH1 domain is in bold, the hinge region is underlined, the CH2 domain is in italics, the CH3 domain is dashed, and shaded boxes represent amino acid residues that may be substituted in accordance with the invention, where X1 is a substitution of the amino acid at residue position 228 according to the EU index of Kabat and contains a proline (P), X2 is a substitution of the amino acid at residue position 234 according to the EU index of Kabat and contains an alanine (A), and X3 is a substitution of the amino acid at residue position 234 according to the EU index of Kabat and contains an alanine (A), and X4 is a substitution of the amino acid at residue position 240 according to the EU index of Kabat and contains an alanine (A). X is a substitution of an amino acid at residue position 345 according to the EU index of Kabat and comprises lysine (K), glutamine (Q), arginine (R), or tyrosine (Y), X is a substitution of an amino acid at residue position 409 according to the EU index of Kabat and comprises arginine (R), X is a substitution of an amino acid at residue position 430 according to the EU index of Kabat and comprises glycine (G), serine (S), phenylalanine (F), or threonine (T), and X is a substitution of an amino acid at residue position 440 according to the EU index of Kabat and comprises tryptophan (W). A is an amino acid substitution at residue position 270 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X B is an amino acid substitution at residue position 322 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X C is an amino acid substitution at residue position 329 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X D is an amino acid substitution at residue position 331 according to the EU index of Kabat, containing a neutral nonpolar amino acid, X Eis a substitution of an amino acid at residue position 333 according to the EU index of Kabat, and includes a neutral polar amino acid. In some embodiments, the neutral nonpolar amino acid includes alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P), or valine (V). In some embodiments, the neutral nonpolar amino acid is an amino acid that does not have a ring structure (e.g., alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), valine (V)). In some embodiments, the neutral polar amino acid includes asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y). TIFF0007804715000008.tif24157

[0038] SEQ ID NO: 62 provides the amino acid sequence of the wild-type Fc region of IgA1 (UniProtKB-P01876 (IGHA1_HUMAN), 353 amino acids), with the CH1 domain in bold, the hinge region underlined, the CH2 domain in italics, the CH3 domain in dashed lines, and the shaded boxes representing amino acids that may be substituted according to the present invention. See also WO2007 / 121354 and Rogers et al., (2008) J Immunol., 180:4816-24, each of which is incorporated by reference in its entirety. TIFF0007804715000009.tif20157

[0039] SEQ ID NO: 63 provides the amino acid sequence of the wild-type Fc region of IgA2 (UniProtKB-P01877 (IGHA2_HUMAN), 340 amino acids), with the CH1 domain in bold, the hinge region underlined, the CH2 domain in italics, the CH3 domain in dashed lines, and the shaded boxes indicating amino acids that may be substituted according to the present invention. See also WO2007 / 121354 and Rogers et al., (2008) J Immunol., 180:4816-24, each of which is incorporated by reference in its entirety. TIFF0007804715000010.tif20157

[0040] Fc mutations capable of promoting antibody hexamerization include one or more mutation(s) in a segment corresponding to amino acid residues approximately 345-440 of the Fc region of an immunoglobulin. In one embodiment, Fc mutations capable of promoting antibody hexamerization include one or more mutation(s) in a segment corresponding to E345-S440 in IgG1. Such one or more mutation(s) may also include mutations corresponding to amino acid residues at amino acid residue positions 345, 430, and / or 440 (e.g., E345, E430, and / or S440 in IgG1). In some embodiments, mutations may include E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440W. In some embodiments, mutations include E345K and E430G. These mutations are known in the context of the present invention as "hexamerization-enhancing mutations."

[0041] Fc mutations capable of reducing effector function include one or more mutation(s) at amino acid residues L234 and / or L235-S440 in IgG1. In one embodiment, effector function mutations in the Fc region include L234A and L235A in IgG1. Fc mutations capable of stabilizing IgG4 include, but are not limited to, S228, L235, and / or R409 in IgG4. In one embodiment, Fc mutations capable of stabilizing IgG4 include S228P and L235E or R409K in IgG4. (See also Vidarsson et al., Front Immunol 2014;5-520 for a general discussion of IgG subclass structure and effector function.) Fc mutations capable of reducing complement-dependent cytotoxicity (CDC) include one or more mutation(s) at amino acid residues at positions 270, 322, 329, 331, 333 (according to the EU index of Kabat) in IgG1, IgG2, IgG3, or IgG4.

[0042] In one embodiment, a polypeptide according to the invention is an engineered polypeptide comprising an Fc variant of a wild-type human IgG Fc region, wherein the Fc variant comprises an amino acid substitution at residue position 228, 234, 235, 345, 409, 430, 440, or a combination thereof, where the amino acid residues are numbered according to the EU index of Kabat. In a further embodiment, the Fc variant further comprises an amino acid substitution at residue position 270, 322, 329, 331, 333, or a combination thereof, where the amino acid residues are numbered according to the EU index of Kabat. In some embodiments, at least 2, 3, 4, 5, 6, or 7 amino acid substitutions are made at residue positions 228, 234, 235, 345, 409, 430, 440. In some embodiments, at least 2, 3, 4, or 5 amino acid substitutions are made at residue positions 270, 322, 329, 331, 333. In one embodiment, the amino acid at residue position 228 according to the EU index of Kabat is substituted with proline (P) or serine (S). In one embodiment, the amino acid at residue position 234 according to the EU index of Kabat is substituted with alanine (A). In one embodiment, the amino acid at residue position 235 according to the EU index of Kabat is substituted with alanine (A). In one embodiment, the glutamic acid (E) at residue position 345 according to the EU index of Kabat is substituted with lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). In one embodiment, the amino acid at residue position 409 according to the EU index of Kabat is substituted with lysine (K) or arginine (R). In one embodiment, the glutamic acid (E) at residue position 430 according to the EU index of Kabat is substituted with glycine (G), serine (S), phenylalanine (F), or threonine (T). In one embodiment, the serine (S) at residue position 440 according to the EU index of Kabat is substituted with a tryptophan (W). In one embodiment, the amino acids at residue positions 270, 322, 329, and / or 331 according to the EU index of Kabat are substituted with a neutral nonpolar amino acid.In some embodiments, the neutral nonpolar amino acid comprises alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P), or valine (V). In some embodiments, the neutral nonpolar amino acid is an amino acid that does not have a ring structure (e.g., alanine (A), glycine (G), leucine (L), isoleucine (I), methionine (M), valine (V)). In one embodiment, the amino acid at residue position 333 according to the EU index of Kabat is substituted with a neutral polar amino acid. In some embodiments, the neutral polar amino acid comprises asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), or tyrosine (Y).

[0043] In this specification and claims, the numbering of residues in immunoglobulin heavy chains is that of the EU index in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference. "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0044] Thus, the present invention provides antibody variants having a binding region and a full-length or partial immunoglobulin Fc domain with one or more hexamerization-enhancing mutations and one or more effector function-reducing mutations, which have enhanced receptor clustering and / or effector cell signaling compared to antibodies with wild-type Fc domains.

[0045] The invention described herein further relates to antibodies comprising a variant Fc domain. In one embodiment, the antibody is an anti-GITR antibody comprising a variant Fc domain. Tables 1A-1B provide the nucleic acid sequences (SEQ ID NOS: 7-8) and amino acid sequences (SEQ ID NOS: 9-10) of the heavy and light chain variable regions of anti-GITR antibodies, respectively. In one embodiment, the variant Fc regions described herein can be used to graft the variable regions of antibodies to engineer an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0046] Table 1A: Ab#E1-3H7 variable region nucleic acid sequence TIFF0007804715000011.tif73161

[0047] Table 1B: Ab#E1-3H7 variable region amino acid sequence TIFF0007804715000012.tif37161

[0048] Table 1C below shows the framework and CDR boundaries of the heavy and light chain variable regions of anti-GITR antibodies based on SEQ ID NOs: 9-10.

[0049] Table 1C: Anti-GITR E1-3H7 amino acid sequence TIFF0007804715000013.tif87169

[0050] In one embodiment, the antibody is an anti-CCR4 antibody comprising a variant Fc domain. Table 1D provides the amino acid sequences of the heavy and light chain variable regions of anti-CCR4 antibodies (SEQ ID NOS: 11-12). In one embodiment, the variant Fc regions described herein can be grafted onto antibody variable regions to engineer an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0051] Table 1D. Anti-CCR4 mAb 2.3 variable region amino acid sequence (= affinity-matured humanized mAb1567) TIFF0007804715000014.tif37161

[0052] Table 1E below shows the framework and CDR boundaries of the heavy and light chain variable regions for anti-CCR4 antibodies based on SEQ ID NOs: 11-12.

[0053] Table 1E: Anti-CCR4 mAb 2.3 amino acid sequence TIFF0007804715000015.tif87169

[0054] Table 2A provides the nucleic acid sequences (SEQ ID NOS: 13-17) for the constant regions (Fc) of the IgG1 heavy and light chains. For example, the Fc regions described herein can be used to engineer the Fc region of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0055] Table 2A: Ab#E1-3H7 constant region nucleic acid sequence—wild-type IgG1 monomer (C L (same as the anti-CCR4 mAb2.3 construct described herein, except for TIFF0007804715000016.tif145161

[0056] In one embodiment, the Fc region of the light chain described herein can be used to engineer the Fc region of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody. In one embodiment, the Fc region of the light chain (C L(カッパ) ) comprises the nucleic acid sequence of SEQ ID NO: 43: TIFF0007804715000017.tif33157 In one embodiment, the Fc region of the light chain (C L(カッパ) ) comprises the amino acid sequence of SEQ ID NO: 64: TIFF0007804715000018.tif9156

[0057] Table 2B provides the amino acid sequences for the IgG1 heavy and light chain constant regions (Fc) (SEQ ID NOS: 18-22). For example, the Fc regions described herein can be used to engineer the Fc region of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0058] (Table 2B) Ab#E1-3H7 constant region amino acid sequence—wild-type IgG1 monomer (C L Same as anti-CCR4 mAb2.3 construct except for the bolded residues in CH2 and CH3 are wild-type residues that can be mutated, for example, to create different IgG1 variants (residues highlighted in yellow in Tables 3-5). TIFF0007804715000019.tif69161

[0059] Table 3A provides the nucleic acid sequences (SEQ ID NO: 23) for the variant constant regions (Fc) of the IgG1 heavy and light chains. The residues highlighted in yellow indicate the mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0060] Table 3A: Ab#E1-3H7 constant region nucleic acid sequence—IgG1 LALA mutant monomer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000020.tif78161

[0061] Table 3B provides the amino acid sequences (SEQ ID NO: 24) for the variant constant regions (Fc) of the IgG1 heavy and light chains. The residues highlighted in yellow indicate the mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0062] Table 3B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA mutant monomer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000021.tif55161

[0063] Table 4A provides the nucleic acid sequences (SEQ ID NO: 25) for the variant constant regions (Fc) of the IgG1 heavy and light chains. The residues highlighted in yellow indicate the mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0064] (Table 4A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 WT hexamer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000022.tif78161

[0065] Table 4B provides the amino acid sequences (SEQ ID NO: 26) for the variant constant regions (Fc) of the IgG1 heavy and light chains. The residues highlighted in yellow indicate the mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0066] (Table 4B) Ab#E1-3H7 constant region amino acid sequence - IgG1 WT hexamer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000023.tif55161

[0067] Table 5A provides the nucleic acid sequences (SEQ ID NOS: 27-28) for the variant constant regions (Fc) of the IgG1 heavy and light chains. The residues highlighted in yellow indicate the mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0068] (Table 5A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hexamer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000024.tif105161

[0069] Table 5B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOs: 29-30). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0070] Table 5B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hexamer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000025.tif64161

[0071] Table 6A provides the nucleic acid sequences (SEQ ID NOS: 31-35) for the constant regions (Fc) of stabilized IgG4 heavy and light chains. Residues highlighted in yellow are mutations introduced to stabilize IgG4. For example, the Fc regions described herein can be used to engineer the Fc region of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0072] Table 6A: Ab#E1-3H7 constant region nucleic acid sequence—sIgG4 monomer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000026.tif145161

[0073] Table 6B provides the amino acid sequences (SEQ ID NOS: 36-40) for the constant regions (Fc) of stabilized IgG4 heavy and light chains. Residues highlighted in yellow are mutations introduced to stabilize IgG4. Residues highlighted in bold / light blue are wild-type residues that can be mutated to create the sIgG4 hexamers in Table 7. For example, the Fc regions described herein can be used to engineer the Fc region of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0074] Table 6B: Ab#E1-3H7 constant region amino acid sequence—sIgG4 monomer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000027.tif87161

[0075] Table 7A provides the nucleic acid sequences (SEQ ID NOS: 31-33, 35, and 41) for the variant constant regions (Fc) of stabilized IgG4 heavy and light chains. Residues highlighted in yellow are mutations introduced to stabilize IgG4. Bolded residues are wild-type residues that can be mutated to create the sIgG4 hexamers in Table 7. For example, the Fc regions described herein can be used to engineer the Fc region of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0076] Table 7A: Ab#E1-3H7 constant region nucleic acid sequence—sIgG4 hexamer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000028.tif158161

[0077] Table 7B provides the amino acid sequences (SEQ ID NOS: 36-40) for the stabilized IgG4 heavy and light chain constant regions (Fc). Residues highlighted in yellow are mutations introduced to stabilize the IgG4. Residues highlighted in bold / light blue are wild-type residues that can be mutated to create the sIgG4 hexamers in Table 7. For example, the Fc regions described herein can be used to engineer the Fc region of an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0078] Table 7B: Ab#E1-3H7 constant region amino acid sequence—sIgG4 hexamer (C L (same as anti-CCR4 mAb2.3 construct except TIFF0007804715000029.tif82161

[0079] Table 8A provides the nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 17, 25, and 44). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0080] (Table 8A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hex Mt1 TIFF0007804715000030.tif105161

[0081] Table 8B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 22, 26, and 45). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0082] Table 8B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hex Mt1 TIFF0007804715000031.tif64161

[0083] Table 9A provides the nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 17, 25, and 46). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0084] (Table 9A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hex Mt2 TIFF0007804715000032.tif105161

[0085] Table 9B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 22, 26, and 47). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0086] Table 9B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hex Mt2 TIFF0007804715000033.tif64161

[0087] Table 10A provides the nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 17, 25, and 48). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0088] (Table 10A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hex Mt3 TIFF0007804715000034.tif109161

[0089] Table 10B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 22, 26, and 49). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0090] Table 10B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hex Mt3 TIFF0007804715000035.tif69161

[0091] Table 11A provides the nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 17, 25, and 50). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0092] (Table 11A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hex-VP TIFF0007804715000036.tif100161

[0093] Table 11B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 22, 26, and 51). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0094] Table 11B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hex-VP TIFF0007804715000037.tif64161

[0095] Table 12A provides the nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 17, 25, and 52). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0096] (Table 12A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hex-PV TIFF0007804715000038.tif105161

[0097] Table 12B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 22, 26, and 53). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0098] Table 12B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hex-PV TIFF0007804715000039.tif64161

[0099] Table 13A provides the nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 17, 25, and 54). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0100] (Table 13A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hex-PF TIFF0007804715000040.tif105161

[0101] Table 13B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 22, 26, and 55). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0102] Table 13B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hex-PF TIFF0007804715000041.tif64161

[0103] Table 14A provides the nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 17, 25, and 56). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variants. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0104] (Table 14A) Ab#E1-3H7 constant region nucleic acid sequence - IgG1 LALA hex-VV TIFF0007804715000042.tif109161

[0105] Table 14B provides the amino acid sequences for the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 22, 26, and 57). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR antibodies or anti-CCR4 antibodies.

[0106] Table 14B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA hex-VV TIFF0007804715000043.tif73161

[0107] Table 15A provides nucleic acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 13-14, 23, 25, and 58). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR or anti-CCR4 antibodies. In one embodiment, Table 15A provides in-frame fusions with scFvs, such as anti-PDL1.

[0108] Table 15A: Ab#E1-3H7 constant region nucleic acid sequence—IgG1 LALA-aPDL1 TIFF0007804715000044.tif212161

[0109] Table 15B provides the amino acid sequences for variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 18-19, 24, 26, and 59). Residues highlighted in yellow indicate mutations introduced into the Fc region to create the IgG1 Fc variant. For example, the Fc regions described herein can be used to engineer variant Fc regions of antibodies of interest, such as anti-GITR or anti-CCR4 antibodies. In one embodiment, Table 15B provides in-frame fusions with scFvs, such as anti-PDL1.

[0110] Table 15B: Ab#E1-3H7 constant region amino acid sequence—IgG1 LALA-aPDL1 TIFF0007804715000045.tif113161

[0111] Antibody variants with one or more hexamerization-enhancing mutations, one or more effector function-reducing mutations, and one or more CDC activity-reducing mutations may have improved therapeutic potential. In particular, antibodies that act as agonists or antagonists after binding to the target cell surface may have increased biological activity. Without being bound by theory, this is true when cell surface receptor clustering is required for their biological function. The enhanced receptor clustering and / or effector cell signaling of the antibody variants of the present invention may lead to practical clinical benefits, such as reducing the effective dose of a human monoclonal antibody to achieve a therapeutic effect and using an antibody with lower affinity.

[0112] For example, enhancing receptor signaling through antibody-induced clustering using antibodies with Fc variant regions described herein can be used to exploit druggable targets on the cell surface where agonistic activity is desired. In one embodiment, a chemokine receptor can serve as a druggable target on the cell surface where increased agonistic activity is desired by using an antibody with an Fc variant region described herein to enhance signaling capacity through chemokine receptor clustering. In another embodiment, an antibody with an Fc variant region described herein can be used to enhance the agonistic or antagonistic activity of a cytokine, hormone, or ligand while bound to that receptor by targeting a region of the cytokine, hormone, or ligand that is still exposed as it binds to that receptor. For example, an antibody with an Fc variant region described herein can be directed against IL-2 to promote T cell proliferation. In yet other embodiments, increasing agonistic activity can be exploited more broadly by targeting proteins spanning seven transmembrane domains (such as G protein-coupled receptors, discussed below), which serve as targets for small molecule drugs.

[0113] One aspect of the present invention relates to enhanced signal transduction of the Wnt pathway through cell surface receptor clustering of Wnt signaling receptor proteins. For example, Wnt proteins belong to a large family of secreted signaling glycoproteins that control various developmental processes, including, but not limited to, the specification of cell fate, cell proliferation, survival, and migration. Thus, Wnt signaling is an important developmental signaling pathway that controls cell fate determination and tissue patterning during early embryonic development and later development. In one embodiment, a Wnt agonist antibody (e.g., an antibody specific for a protein member of the R-spondin family or specific for Norrin) having an Fc variant region described herein (e.g., that induces clustering of wnt proteins and / or their ligands) can be used to induce (e.g., stimulate) stem cell differentiation.

[0114] Another aspect of the present invention generally relates to enhanced agonist and / or antagonist signaling of type I, type II, or type III receptors through receptor clustering of these cell surface molecules. Type I receptors are nicotinic or GABAergic receptors, which are targets of the neurotransmitters acetylcholine and GABA, respectively. Nicotinic receptors can also bind to the ligand nicotine. Type II receptors are metabotropic receptors, such as G protein-coupled receptors, serotonin receptors, and glutamate receptors. Ligands for these receptors include, for example, various hormones (epinephrine, glucagon, calcitonin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), neurokinins, thyrotropin-releasing hormone (TRH), cannabinoids, and oxytocin), and neurotransmitters (dopamine, serotonin, and metabotropic glutamate, etc.). Type III receptors include, for example, receptor tyrosine kinases and enzyme-linked receptors. The insulin receptor is a type III surface molecule that binds to the ligand insulin. Other type III receptors include, but are not limited to, epidermal growth factor receptor, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, and colon cancer kinase 4. In one embodiment, antibody-mediated clustering of type I, type II, or type III cell surface molecules and / or their ligands using an antibody with an Fc variant region described herein can be used to enhance agonist and / or antagonist activity. For example, an antibody specific for PCSK9 (e.g., an antibody with an Fc variant region described herein that induces clustering of proteins and / or their ligands) can bind to PCSK9 and more efficiently inhibit its binding to LDL receptors.

[0115] For example, signaling of seven-transmembrane domain (7-TMD) surface receptors can be amplified by using antibodies with the Fc variant regions described herein specific for these proteins to enhance signaling capacity through clustering of 7-TMD proteins. In some embodiments, increased antagonist activity can also be exploited by targeting inhibitory 7-TMD proteins (e.g., G protein-coupled receptors) that also serve as targets for small molecule drugs. For example, 7-TMD cell surface receptor inhibitory signaling can be enhanced using antibodies with the Fc variant regions described herein, as these targeted 7-TMD inhibitory proteins are clustered on the cell surface in the presence of antagonist antibodies with the Fc variant regions described herein. Thus, antibody-mediated clustering of 7-TMD receptors and / or their ligands can be used to enhance agonist or antagonist activity. In one embodiment, administering to a subject an antibody with the Fc variant region described herein can be used to block calcitonin gene-related peptide (CGRP) ligand / 7-TMD receptor interaction to treat migraine.

[0116] In a further embodiment, antibody-mediated clustering (e.g., inducing receptor clustering by using antibodies with Fc variant regions described herein) can be used to convert low avidity antibodies into antibodies with apparently high affinity, which can be used to increase binding activity and subsequent biological activity.

[0117] Accordingly, the present invention also provides methods of using the antibody variants of the present invention in therapeutic methods for treating cancer, autoimmune disorders, inflammatory disorders, neurological diseases, cardiovascular diseases, infectious diseases, and inducing stem cell lineage pathways. The term "treating" can refer to partially or completely alleviating, ameliorating, improving, mitigating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms, characteristics, or clinical manifestations of a particular disease, disorder, and / or condition. Treatment can be administered to subjects who do not exhibit signs of the disease, disorder, and / or condition (e.g., before an identifiable disease, disorder, and / or condition) and / or who exhibit only early signs of the disease, disorder, and / or condition, with the aim of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment involves enhancing cell signaling or inducing cellular receptor clustering.

[0118] The antibody variants of the present invention may be specific for any target of interest, including but not limited to, tumor-associated surface antigens such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigens, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein (P501), PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor I (IGF1)-I, IGF-II, IGF1 receptor, mesothelium, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, fibronectin extra domain A (EDA) and extra domain B (EDB), and tenascin C (TnC) A1) and the A1 domain of fibroblast-associated protein (fap); lineage-specific or tissue-specific antigens such as CD3, CD4, CD8, CD24, CD25, CD28, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF 17), or virus-specific surface antigens, such as HIV-specific antigens (HIV gp120, etc.); EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lasse virus-specific antigens, influenza virus-specific antigens, and any derivatives or variants of these surface markers.

[0119] The antibody variants of the invention described herein (e.g., having a variant Fc region from IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 as disclosed herein) can be specific for any target of interest, e.g., a protein target as described herein. In one embodiment, the antibody is specific for an inhibitory molecule on T cells. Non-limiting examples of inhibitory molecules on T cells include programmed cell death protein 1 (PD-1 (GenPept Accession No. NP_005009), also known as CD279), T cell immunoreceptor with Ig and ITIM domain protein (TIGIT (GenPept Accession No. NP_776160)), CTLA4 (also known as CD152, GenPept Accession No. NP_005205), lymphocyte activation gene 3 protein (LAG3 (GenPept Accession No. NP_002277)), TIM3 (also known as hepatitis A virus cellular receptor 2 (GenPept Accession No. NP_116171)), and KIR (also known as killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1, GenPept Accession No. NP_001309097)). In one embodiment, the inhibitory molecule on T cells recognized by the antibody of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0120] In one embodiment, the antibody is specific for a stimulatory molecule on T cells. Non-limiting examples of stimulatory molecules on T cells include glucocorticoid-inducible tumor necrosis factor receptor (GITR, GenPept Accession No. NP_683700), CD27 (GenPept Accession No. NP_001233), OX40 (also known as TNFRSF, GenPept Accession No. NP_003318), 4-1BB (also known as TNF receptor superfamily member 9 (TNFRSF9), GenPept Accession No. NP_001552), CD40L (CD154 and and GenPept Accession No. NP_000065), inducible T cell costimulatory protein (ICOS, GenPept Accession No. NP_036224), CD3 (GenPept Accession Nos. for delta chain, NP_000723, epsilon chain, NP_000724, and gamma chain, NP_000064), and CD28 (GenPept Accession No. NP_006130). In one embodiment, the stimulatory molecules on T cells recognized by the antibodies of the invention are about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequences available under the accession numbers provided herein. In another embodiment, antibodies specific for CD3 and / or CD28 can be used to enhance T cell proliferation for ex vivo expansion of cells for cell therapy, such as chimeric antigen receptor (CAR) T cell immunotherapy.

[0121] In one embodiment, the antibody is specific for a chemokine receptor. Non-limiting examples of chemokine receptors include CC motif chemokine receptor 4 (CCR4, GenPept accession number NP_005499), CC motif chemokine receptor 5 (CCR5, GenPept accession number NP_000570), and CXC motif chemokine receptor 4 (CXCR4, GenPept accession number for isoform c, NP_001334985). In one embodiment, the chemokine receptor recognized by the antibody of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0122] In one embodiment, the antibody is specific to a tumor-associated molecule on a tumor cell. Non-limiting examples of tumor-associated molecules include TNF receptor superfamily member 17 (TNFRSF17, also known as BCMA, GenPept Accession No. NP_001183), carbonic anhydrase 9 (CAIX, GenPept Accession No. NP_001207), and antigen-presenting cell molecules such as PDL1 (also known as programmed cell death ligand 1, CD274, GenPept Accession No. NP_054862 for isoform a) or PD-L2 (also known as programmed cell death ligand 2 (PDCD1LG2), CD273, GenPept Accession No. NP_079515). In one embodiment, the tumor-associated molecule recognized by the antibody of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession number provided herein.

[0123] In one embodiment, the antibody is specific for an infectious agent. A non-limiting example of an infectious agent is the severe acute respiratory syndrome (SARS) virus (see https: / / www.ncbi.nlm.nih.gov / genomes / SARS / SARS.html, e.g., an antibody specific for the S (spike) protein of the SARS virus (GenPept Accession No. NP_828851, incorporated by reference in its entirety, J Mol Biol 2003;331:991-1004), influenza viruses (e.g., when the antibodies are specific for influenza A (such as group 1 and group 2), influenza B, influenza C, or influenza D viruses, e.g., when the antibodies are specific for the hemagglutinin (HA) protein of influenza virus (GenPept accession number NP_040980, or NP_056660) or the neuraminidase (NA) protein of influenza virus (GenPept accession number NP_040980 or NP_056663)), flaviviruses, alphaviruses, and Middle East Respiratory Syndrome (MERS) virus (GenBank accession number AKL59399, e.g., when the antibodies are specific for the S (sequence number) of MERS virus (GenBank accession number AHX71946). pike) protein). In some embodiments, the influenza virus is an emerging influenza virus. In one embodiment, the SARS virus recognized by the antibody of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence accessible at www.ncbi.nlm.nih.gov / genomes / SARS / SARS.html or the accession numbers provided herein. In one embodiment, the MERS virus recognized by the antibody of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available at the accession numbers provided herein.

[0124] Non-limiting examples of alphaviruses include Western equine encephalitis virus (WEEV, GenPept accession number NP_640330, e.g., strain BFN3060 (GenBank accession number AAC56453), strain BFS932 (GenBank accession number AIC81861), strain AG80-646 (GenBank accession number: ACT75287)), Eastern equine encephalitis virus (EEEV, GenPept accession number NP_632021, Gen GenBank Accession No. AJP13624, e.g., strain FL93-939 (GenBank Accession No. ABL84686), Venezuelan equine encephalitis virus (GenPept Accession No. NP_040822, e.g., strain TC-83 (GenBank Accession No. AAB02516)), and chikungunya virus (CHKV, GenPept Accession No. NP_690588, GenBank Accession No. AFP43243).

[0125] GenBank accession numbers for various strains of Western equine encephalitis virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=57240&decorator=toga. In one embodiment, the WEEV virus recognized by the antibodies of the invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein. GenBank accession numbers for various strains of Eastern equine encephalitis virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga. In one embodiment, the EEEV virus recognized by the antibodies of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein. GenBank accession numbers for various strains of Venezuelan equine encephalitis virus can be obtained at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=2681&decorator=toga. In one embodiment, the Venezuelan equine encephalitis virus recognized by the antibodies of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein. GenBank accession numbers for various strains of Chikungunya virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=728&decorator=toga.In one embodiment, the Chikungunya virus recognized by the antibodies of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0126] Non-limiting examples of flaviviruses include West Nile virus (WNV, GenPept Accession No. NP_041724, e.g., Kerala strain, GenBank Accession No. AGI16461), dengue virus serotypes 1-4 (GenPept Accession No. NP_059433 (e.g., DENV1 BR / SJRP / 287 / 2011 strain, GenBank Accession No. AKQ00011, DENV1 BR / SJRP / 484 / 2012 strain, GenBank Accession No. AKQ00014), GenPept Accession No. NP_056776, GenBank Accession No. AFU65934.1 (e.g., Dengue virus 2 / Homo sapiens / Haiti-1 / 2016 strain, GenBank accession number AOE23002), GenPept accession number YP_001621843, GenBank accession number AAA99437 (e.g., Dengue virus 3 isolate Jeddah-2014, GenBank accession number AIH13925), GenPept accession number NP_073286 (e.g., DENV-4 strain Br264RR / 10, GenBank accession number: AEX91754.1), https: / / www.viprbrc.org / brc / home.See also spg?decorator=flavi_dengue), yellow fever virus (GenPept accession number NP_041726 (e.g., strain BeAn754036 (PR4408), GenBank accession number ARQ19026), DAK AR B490 strain, GenPept accession number YP_009344961, YMP 48 strain, GenPept accession number YP_009256192, Uganda S strain, GenPept accession number YP_009344968, Wesselsbron strain, GenPept accession number YP_002922020), Zika virus (GenPept accession number YP_009428568, GenPept accession number YP_002790881, e.g., MR with GenBank accession number AAV34151). 766), Powassan virus (POW, GenPept accession number NP_620099), St. Louis encephalitis virus (SLE, UniProtKB / Swiss-Prot:P09732), and Japanese encephalitis virus (JEV, GenPept accession number NP_059434). In some embodiments, the flavivirus is mosquito-borne (e.g., dengue virus serotypes 1-4 (DENV1-4), West Nile virus (WNV), yellow fever virus (YFV), Zika virus (ZIKV), St. Louis encephalitis virus (SLE), Japanese encephalitis virus (JEV), etc.).

[0127] GenBank accession numbers for various strains of West Nile Virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=2694&decorator=flavi. In one embodiment, the West Nile Virus recognized by the antibodies of the invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0128] GenBank accession numbers for various strains of dengue virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=730-731-732-733-734-735-736-843-844-845-846-847-848-849-850&decorator=flavi. In one embodiment, the dengue virus recognized by the antibodies of the present invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0129] GenBank accession numbers for various strains of yellow fever virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=2713&decorator=flavi. In one embodiment, the yellow fever virus recognized by the antibodies of the invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0130] GenBank accession numbers for various strains of Zika virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=2721&decorator=flavi. In one embodiment, the Zika virus recognized by the antibodies of the invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0131] GenBank accession numbers for various strains of Powassan virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=2280&decorator=flavi. In one embodiment, the Powassan virus recognized by the antibodies of the invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0132] GenBank accession numbers for various strains of St. Louis encephalitis virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=2588&decorator=flavi. In one embodiment, the St. Louis encephalitis virus recognized by the antibodies of the invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0133] GenBank accession numbers for various strains of Japanese encephalitis virus are available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=1695&decorator=flavi. In one embodiment, the St. Louis encephalitis virus recognized by the antibodies of the invention is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the amino acid sequence available under the accession numbers provided herein.

[0134] Exemplary antibodies useful in constructing antibody variants according to the present invention include, for example, those disclosed in WO / 2005 / 060520, WO / 2006 / 089141, WO / 2007 / 065027, WO / 2009 / 086514, WO / 2009 / 079259, WO / 2011 / 153380, WO / 2014 / 055897, WO2015 / 143194, WO2015 / 164865, WO2013 / 166500, and WO2014 / 144061, PCT / US2015 / 054202, PCT / US2015 / 054010, and 62 / 144,729, the contents of each of which are incorporated herein by reference in their entirety.

[0135] The antibodies and fragments thereof of the present invention can be synthesized, engineered, and / or produced using nucleic acids such as those listed in the tables herein. In one embodiment, the nucleic acid has a sequence comprising a nucleotide set forth in Table 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, SEQ ID NO: 43, or a combination thereof. In another embodiment, the nucleic acid has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical sequence to the nucleic acid sequence disclosed in Table 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, SEQ ID NO: 43, or a combination thereof.It will be understood that the present invention includes portions and variants of the sequences specifically disclosed herein.For example, codon-optimized sequence forms can be used in embodiments.

[0136] The antibodies and fragments thereof of the present invention can also be synthesized, engineered, and / or produced using polypeptides comprising the amino acid sequences listed in the tables herein. In one embodiment, the polypeptide has an amino acid sequence comprising consecutive amino acids disclosed in Table 1B, 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B, 11B, 12B, 13B, 14B, 15B, an amino acid sequence encoded by SEQ ID NO: 64, or a combination thereof. In another embodiment, the polypeptide has an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence disclosed in Table 1B, 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B, 11B, 12B, 13B, 14B, 15B, the amino acid sequence encoded by SEQ ID NO:64, or a combination thereof.

[0137] The coding sequence can be present, for example, in a replicating or non-replicating adenovirus vector, an adeno-associated virus vector, an attenuated Mycobacterium tuberculosis vector, a Bacillus Calmette-Guerin (BCG) vector, a vaccinia or modified vaccinia Ankara (MVA) vector, another poxvirus vector, a recombinant polio and other enteric virus vector, a Salmonella species bacterial vector, a Shigella species bacterial vector, a Venezuelan equine encephalitis virus (VEE) vector, a Semliki Forest virus vector, or a tobacco mosaic virus vector. The coding sequence can also be expressed as a DNA plasmid with an active promoter, such as a CMV promoter. Other live vectors can also be used to express the sequences of the invention. Expression of the antibodies of the invention can be induced in the subject's own cells by introducing nucleic acid encoding the antibody into those cells, preferably using codons and promoters optimized for expression in human cells.

[0138] Embodiments of the present invention include cells expressing the antibody variants of the present invention (i.e., CARTs). The cells can be of any type, including immune cells capable of expressing antibody variants for cancer therapy, or cells such as bacterial cells harboring an expression vector encoding a CAR. As used herein, the terms "cell," "cell line," and "cell culture" can be used interchangeably. All of these terms include their progeny, that is, any subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, a "host cell" refers to a eukaryotic cell capable of replicating the vector and / or expressing the heterologous gene encoded by the vector. Host cells can and have been used as recipients of vectors. Host cells may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" or host cells can refer to cells into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Recombinant cells are therefore distinguishable from naturally occurring cells that do not contain a recombinantly introduced nucleic acid. In embodiments of the invention, the host cells are T cells, including cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells); CD4+ T cells, NK cells, and NKT cells are also encompassed by the invention.

[0139] Some vectors may use control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions for incubating all of the above host cells to maintain them and allow vector replication. Also understood and known are the techniques and conditions that allow large-scale production of vectors and the production of nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0140] The cells may be autologous, syngeneic, allogeneic, or in some cases xenogeneic.

[0141] In many situations, one may wish to be able to kill modified CTLs if their absence after their presence is of interest or for other reasons, in studies where the cells become tumorigenic and one wishes to terminate treatment. To this end, one can provide for the expression of a specific gene product capable of killing modified cells under controlled conditions, such as an inducible suicide gene.

[0142] The present invention further includes a CART modified to secrete one or more polypeptides. The polypeptide can be, for example, an antibody or a cytokine. For example, the antibody can be specific for CAIX, GITR, PDL1, PD-L2, PD-1, CCR4, or TIGIT.

[0143] Armed CARTs have the advantage of simultaneously secreting the polypeptide at the targeted site, e.g., the tumor site.

[0144] An armed CART can be constructed by including a nucleic acid encoding a polypeptide of interest after an intracellular signaling domain. Preferably, an internal ribosome entry site (IRES) is located between the intracellular signaling domain and the polypeptide of interest. Those skilled in the art will appreciate that multiple IRES sequences can be used in tandem to express two or more polypeptides.

[0145] Antibodies, including engineered polypeptides, can be purified, for example, from cells or recombinant systems, using a variety of well-known techniques for isolating and purifying proteins. See, for example, the antibody purification methods in Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives (Basics: From Background to Bench), Springer-Verlag Ltd., New York, 2000; Basic Methods in Antibody Production and Characterization, Chapter 11, "Antibody Purification Methods," Howard and Bethell, Eds., CRC Press, 2000; Antibody Engineering (Springer Lab Manual), Kontermann and Dubel, Eds., Springer-Verlag, 2001, each of which is incorporated herein by reference in its entirety.

[0146] The antibodies, fragments, and antibody derivatives described herein, e.g., chimeric or humanized antibodies, can be formulated as compositions (e.g., pharmaceutical compositions), such as for use in a subject. Suitable compositions can include the antibody or fragment (or derivative thereof) dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium).

[0147] Pharmaceutically acceptable carriers can include any solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. Any conventional media or agent compatible with antibodies can be used. Supplementary active agents can also be incorporated into the composition. Non-limiting examples of pharmaceutically acceptable carriers include solid or liquid fillers, diluents, and encapsulating substances, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil.

[0148] Pharmaceutical compositions of the invention can be sterile and formulated to be compatible with their intended route of administration, including parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0149] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, glycerol, propylene glycol, a pharmaceutically acceptable polyol such as liquid polyethylene glycol, and suitable mixtures thereof. 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 dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.In many cases, it can be useful to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride.The sustained absorption of injectable compositions can be achieved by including absorption-delaying agents, for example, aluminum monostearate and gelatin in the composition.

[0150] Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein.

[0151] As another example, oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, antibodies can be incorporated with excipients and used in the form of tablets, troches, or capsules.

[0152] Pharmaceutically compatible binders, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, primogel, or corn starch; lubricants such as magnesium stearate or sterols; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0153] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and for example, for transmucosal administration, include detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, active compounds are formulated into ointments, salves, gels, or creams generally known in the art.

[0154] The antibodies or fragments (or derivatives thereof) can also be formulated into compositions suitable for topical administration to the skin or mucous membranes (e.g., rectal or vaginal administration). Such compositions can take the form of liquids, ointments, creams, gels, and pastes. The antibodies or fragments (or derivatives thereof) can also be formulated into compositions suitable for intranasal administration. Standard formulation techniques can be used to prepare suitable compositions.

[0155] The antibodies and / or compositions of the invention can be administered to a subject once (e.g., as a single injection or deposition). Alternatively, administration can be once or twice daily for a period of about 2 to about 28 days, or about 7 to about 10 days, or about 7 to about 15 days to a subject in need thereof. They can also be administered once or twice daily to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0156] The therapeutically effective dose range may depend on the antibody or fragment (or derivative thereof), as well as the nature of the formulation and the route of administration. The optimal dose can be determined by one of ordinary skill in the art without undue experimentation and may vary depending on known factors such as the pharmacodynamic properties of the active ingredient and its mode and route of administration; the time of administration of the active ingredient; the recipient's age, sex, health, and weight; the nature and severity of symptoms; the type of concurrent treatment, the frequency of treatment, and the desired effect; and the excretion rate. For example, a therapeutically effective dose of an antibody in the range of about 0.1 to 1000 mg / kg of body weight can be used. Preferably, a dose of an antibody in the range of about 1 to 50 mg / kg can be used.

[0157] The antibody or nucleic acid of the present invention can also be provided in a kit. In one embodiment, the kit includes (a) a container containing a composition containing the antibody, and optionally (b) informational material. The informational material can be explanatory, instructional, marketing, or other material regarding the methods described herein and / or the use of the agent for therapeutic benefit. In one embodiment, the kit also includes a second agent for treating a subject suffering from a disease or condition. For example, the kit includes a first container containing a composition comprising the polypeptide and a second container containing the second agent.

[0158] The informational material of the kit is not limited in its form. In one embodiment, the informational material can include information about the production of the antibody, the antibody's molecular weight, concentration, expiration date, batch or production site information, etc. In one embodiment, the informational material relates to methods for administering the polypeptide or a nucleic acid encoding it, e.g., at an appropriate dose, dosage form, or method of administration (e.g., a dose, dosage form, or method of administration described herein), to treat a subject. The information can be provided in a variety of formats, including printed text, computer-readable material, video or audio recordings, or information providing a link or address to substantive material.

[0159] In addition to the antibody or the nucleic acid encoding it, the composition in the kit can contain other components such as solvent or buffer, stabilizer, or preservative.The antibody or nucleic acid can be provided in any form, for example, preferably substantially pure and / or sterile, liquid, dry, or lyophilized form.When provided in liquid solution, the liquid solution is preferably an aqueous solution.When provided in dry form, reconstitution is generally by adding a suitable solvent.A solvent, for example, sterile water or buffer, can optionally be provided in the kit.

[0160] The kit can include one or more containers for the antibody, nucleic acid, or composition comprising the same. In some embodiments, the kit contains separate containers, dividers, or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe having informational material attached thereto in the form of a label. In some embodiments, the kit includes multiple (e.g., packs) of individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the antibody or nucleic acid. The container can include a combination unit dosage, e.g., a unit containing both the antibody and the second agent, e.g., in a desired ratio. For example, the kit includes multiple syringes, ampoules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be airtight, waterproof (e.g., impervious to moisture changes or evaporation), and / or light-tight. The kits optionally include a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device can be provided pre-loaded or can be empty but suitable for loading.

[0161] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0162] Whenever the phrases "for example," "such as," "including," and the like are used herein, unless expressly stated otherwise, it is understood that they are accompanied by the phrase "without limitation." Similarly, "one example," "exemplary," and the like are understood to be non-limiting.

[0163] The term "substantially" permits deviations from the descriptor that do not adversely affect the intended purpose. It is understood that a descriptor is modified by the term "substantially" even if the word "substantially" is not expressly recited.

[0164] The terms "comprising" and "including," as well as "having" and "involving" (and similarly, "comprises," "includes," "has," and "involves"), etc., are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising," and therefore should be interpreted as having the open term meaning "at least the following," and not excluding additional features, limitations, embodiments, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" and "an" are used, they should be understood to mean "one or more," unless such interpretation is meaningless in the context.

[0165] The term "about" as used herein means approximately, roughly, roughly, or within a range thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. Generally, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent (up and down).

[0166] In this specification and claims, the numbering of residues in immunoglobulin heavy chains is that of the EU index in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference. "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0167] "Affinity" can refer, for example, to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or Fc receptor). Unless otherwise indicated, "binding affinity" can refer to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody / Fc receptor or an antibody and an antigen). The affinity of molecule X for its partner Y can be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. See also Yang, Danlin, et al., "Determination of High-Affinity Antibody-Antigen Binding Kinetics Using Four Biosensor Platforms," ​​Journal of Visualized Experiments: JoVE 122 (2017), incorporated herein by reference in its entirety. Certain illustrative and exemplary embodiments for measuring binding affinity are described below. See, for example, WO2003056296; Neri, Dario, et al., "Biophysical methods for the determination of antibody-antigen affinities," Trends in biotechnology 14.12 (1996): 465-470; Leonard, Paul et al., "Measuring protein-protein interactions using Biacore," Protein Chromatography. Humana Press, 2011. 403-418; and Karlsson, Robert, et al., "Analyzing a kinetic titration series using affinity biosensors," Analytical biochemistry 349.1 (2006): 136-147, each of which is incorporated herein by reference in its entirety.

[0168] An "affinity matured" antibody can be, for example, an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, which alterations may result in an improvement in the affinity of the antibody for an antigen.

[0169] An "amino acid modification" can be, for example, a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include amino acid substitution, insertion, and / or deletion. A preferred amino acid modification herein is a substitution. For example, an "amino acid modification" at a specified position in the Fc region can refer to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent to the specified residue. By insertion, the "adjacent" specified residue can be, for example, an insertion within one to two residues thereof. The insertion can be N-terminal or C-terminal to the specified residue.

[0170] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another, different "substitute" amino acid residue. The replacement residue(s) may be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) and may be selected from the group consisting of alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In one embodiment, the replacement residue is not cysteine. Substitution with one or more non-naturally occurring amino acid residues may also be referred to as an amino acid substitution herein. A "non-naturally occurring amino acid residue" may be, for example, a residue other than the naturally occurring amino acid residues listed above that can be covalently linked to adjacent amino acid residue(s) in a polypeptide chain. Non-limiting examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as those described in Ellman, et al. (Meth. Enzym. 202 (1991) 301-336). To generate such non-naturally occurring amino acid residues, for example, the procedures of Noren, et al. (Science 244 (1989) 182 and Ellman, et al., supra) can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA.

[0171] An "amino acid insertion" can refer to the incorporation of at least one amino acid into a predetermined amino acid sequence. Insertions typically consist of the insertion of one or two amino acid residues, although the invention described herein can utilize larger "peptide insertions," e.g., insertions of about 3 to about 5 or even up to about 10 amino acid residues. The inserted residue(s) can be naturally or non-naturally occurring, as described above.

[0172] An "amino acid deletion" can refer to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0173] 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), humanized antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. Antibodies of the present invention include those comprising an Fc sequence selected from those described herein. For example, antibodies include Fc variants of wild-type human IgG Fc regions, such as Fc variants with amino acid substitutions E345K, E430G, L234A, and L235A; or E345K, E430G, S228P, and R409K. Residues are numbered according to the EU index of Kabat. In embodiments of the present invention, either intact antibodies, antibody derivatives, or fragments thereof (e.g., antigen-binding fragments) can be used. That is, for example, intact antibodies, Fab fragments, F(ab)2 fragments, minibodies, or bispecific whole antibodies can be used in aspects of the present invention, such as to enhance cell signaling and / or induce receptor clustering.

[0174] Toxins can be attached to the antibodies or antibody fragments described herein. Such toxins can include radioisotopes, biological toxins, boronated dendrimers, and immunoliposomes (Chow et al., Adv. Exp. Biol. Med. 746: 121-41, 2012). Toxins can be conjugated to antibodies or antibody fragments using methods well known in the art (Chow et al., Adv. Exp. Biol. Med. 746: 121-41 (2012)). Combinations of the antibodies disclosed herein, or their fragments or derivatives, can also be used in the methods of the present invention.

[0175] The term "antibody variant" as used herein refers to a variant of a wild-type antibody, characterized in that the antibody variant has a change in amino acid sequence compared to the wild-type antibody, for example, by mutation of a specific amino acid residue in the wild-type antibody. For example, the antibody variant may contain an amino acid substitution in the Fc region that enhances cell signaling and / or induces receptor clustering. Such substitutions include those described herein, such as E345K, E430G, L234A, and L235A in combination with D270, K322, P329, P331, E333, E345, E430, and / or S440; or E345K, E430G, S228P, and R409K in combination with D270, K322, P329, P331, E333, E345, E430, and / or S440 in the Fc of human IgG. Residues are numbered according to the EU index of Kabat.

[0176] As used herein, the term "antibody effector function(s)" or "effector function" can refer to a function provided by the Fc effector domain(s) of an IgG (e.g., the Fc region of an immunoglobulin). Such a function can be provided, for example, by binding of the Fc effector domain(s) to an Fc receptor on an immune cell with phagocytic or lytic activity, or by binding of the Fc effector domain(s) to a component of the complement system. Exemplary effector functions are ADCC, ADCP, and CDC.

[0177] An "antibody fragment" may be a molecule other than an intact antibody that contains a portion of an intact antibody that binds to 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')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0178] An "antibody that binds to the same epitope" as a reference antibody can be, for example, an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0179] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which, for example, nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells and subsequently cause lysis of the target cell. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.

[0180] For example, "antibody-dependent cellular phagocytosis" and "ADCP" are processes in which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc region.

[0181] A "binding domain" can be, for example, a region of a polypeptide that binds to another molecule. In the case of an FcR, a binding domain can comprise the portion of its polypeptide chain (e.g., the α chain) that is involved in binding an Fc region. One useful binding domain is the extracellular domain of the FcR α chain.

[0182] For example, "binding" to an Fc receptor can be, for example, the binding of an antibody to an Fc receptor in a BIAcore® assay (Pharmacia Biosensor AB, Uppsala, Sweden).

[0183] In a BIAcore® assay, Fc receptors are bound to a surface and binding of mutated variants, eg, antibody variants, is measured by surface plasmon resonance (SPR). See, for example, Rich, Rebecca L., and David G. Myszka, "Advances in surface plasmon resonance biosensor analysis," Current opinion in biotechnology 11.1 (2000): 54-61, and Rich, Rebecca L.; Rich, Rebecca L., and David G. Myszka, "Spying on HIV with SPR," Trends in microbiology 11.3 (2003): 124-133; McDonnell, James M., "Surface plasmon resonance: toward an understanding of the mechanisms of biological molecular recognition," Current opinion in chemical biology 5.5 (2001): 572-577, and David G. Myszka, "BIACORE J: a new platform for routine biomolecular interaction analysis," Journal of Molecular Recognition, each of which is incorporated herein by reference in its entirety. 14.4(2001):223-228. The affinity of binding is k a (rate constant for association of antibody from the antibody / Fc receptor complex), k d (dissociation constant), and K D Alternatively, for example, the binding signal of an SPR sensorgram can be directly compared to the response signal of a reference in terms of resonance signal height and dissociation behavior.

[0184] The "CH2 domain" (also referred to as the "Cγ2" domain) of the human IgG Fc region typically spans from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact native IgG molecules. It has been speculated that carbohydrates may provide an alternative interdomain pairing and help stabilize the CH2 domain (Burton, Molec. Immunol. 22 (1985) 161-206). In one embodiment, Figures 8, 9, 11, and 27 show the CH domains of IgG1, IgG2, IgG4, and IgG3, respectively.

[0185] The "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG). In one embodiment, Figures 8, 9, 11, and 27 show the CH domains of IgG1, IgG2, IgG4, and IgG3, respectively.

[0186] "Cancer" and "cancerous" refer to or describe, for example, a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer.

[0187] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and culture from which it is derived, regardless of the number of transfers. It is understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where separate designations are intended, it will be clear from the context.

[0188] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. See, for example, Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions," Frontiers in immunology 5 (2014): 520, and Spiegelberg, Hans L., "Biological Activities of Immunoglobulins of Different Classes and Subclasses 1," Advances in immunology. Vol. 19. Academic Press, 1974, 259-294, each of which is incorporated herein by reference in its entirety. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0189] For example, as used herein, a "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes such as nucleases and fragments thereof; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents discussed herein.

[0190] "Complement-dependent cytotoxicity" or CDC refers to a mechanism for inducing cell death in which, for example, the Fc effector domain(s) of a target-binding antibody activate a series of enzymatic reactions that result in the formation of holes in the target cell membrane. Antigen-antibody complexes, such as those on antibody-coated target cells, bind and activate complement component C1q, which then activates the complement cascade, causing target cell death. Complement activation can also result in the deposition of complement components on the target cell surface, which promotes ADCC by binding complement receptors (e.g., CR3) on leukocytes.

[0191] A "disorder" can be any condition that would benefit from treatment with a polypeptide, such as an antibody comprising an Fc variant. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. In one embodiment, the disorder is cancer.

[0192] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies, for example, with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0193] As used herein, a "reduced effector function" can refer to at least a 20% reduction in a particular effector function, such as, for example, ADCC or CDC, compared to a control (e.g., a polypeptide having a wild-type Fc region), and a "greatly reduced effector function" as used herein can refer to at least a 50% reduction in a particular effector function, such as, for example, ADCC or CDC, compared to a control.

[0194] An "effective amount" of an agent, eg, a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0195] "Fc region" refers to, for example, the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term can include 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, except that the C-terminal lysine (Lys447) of the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as 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).

[0196] A "variant Fc region" comprises an amino acid sequence that differs from that of a "native" or "wild-type" sequence Fc region by virtue of at least one "amino acid modification," as described herein. In one embodiment, a variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions. In one embodiment, a variant Fc region has about one to about five amino acid substitutions in a native-sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region herein can retain at least about 80% homology with a native-sequence Fc region and / or the Fc region of a parent polypeptide, at least about 90% homology therewith, at least about 95% homology therewith, at least about 96% homology therewith, at least about 97% homology therewith, at least about 98% homology therewith, or at least about 99% homology therewith.

[0197] As used herein, "Fc variant" refers to a polypeptide containing modifications in the Fc domain. Fc variants of the present invention are defined according to their constituent amino acid modifications. Thus, for example, P329G is an Fc variant having a proline-to-glycine substitution at position 329 relative to the parent Fc polypeptide, where the numbering is according to the EU index. The identity of the wild-type amino acid may not be specified, in which case the variant is referred to as P329G. For all positions discussed herein, the numbering is according to the EU index. The EU index, or the EU index as in the Kabat or EU numbering scheme, refers to the numbering of EU antibodies (Edelman, et al., Proc Natl Acad Sci USA 63 (1969) 78-85, incorporated herein by reference in its entirety). Modifications can be additions, deletions, or substitutions. Substitutions can include naturally occurring and non-naturally occurring amino acids. Variants can include non-naturally occurring amino acids. Examples include U.S. Pat. No. 6,586,207, WO 98 / 48032, WO 03 / 073238, US 2004 / 0214988 A1, WO 05 / 35727 A2, WO 05 / 74524 A2, Chin, JW, et al., Journal of the American Chemical Society 124 (2002) 9026-9027, Chin, JW and Schultz, PG, ChemBioChem 11 (2002) 1135-1137, Chin, JW, et al., PICAS United States of America 99 (2002) 11020-11024, and Wang, L., and Schultz, PG, Chem. (2002) 1-10, all of which are incorporated by reference in their entirety.

[0198] "Fc region-containing polypeptide" refers to a polypeptide, such as an antibody or immunoadhesin (see discussion herein), that comprises an Fc region.

[0199] "Fc receptor" or "FcR" is used, for example, to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native-sequence human FcR. Another exemplary FcR is one that binds IgG antibodies (gamma receptors), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See review in Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492, Capel et al., Immunomethods 4 (1994) 25-34, and de Haas et al., J. Lab. Clin. Med. 126 (1995) 330-41. Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes FcRn, the neonatal receptor involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117 (1976) 587 and Kim et al., J. Immunol. 24 (1994) 249).

[0200] For example, an "IgG Fc ligand" can be a molecule, e.g., a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγR, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRHs), a family of Fc receptors that are homologous to FcγR (Davis, et al., Immunological Reviews 190 (2002) 123-136, fully incorporated by reference). Fc ligands can also include undiscovered molecules that bind to Fc. Specific IgG Fc ligands are FcRn and Fc gamma receptors. In one embodiment, an "Fc ligand" can be a molecule, e.g., a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0201] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that bind to the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, Fc.γ.RI (CD64), which includes the isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32), which includes the isoforms FcγRIIA (including allotypes H131 and R131), FcγRIIB (including FcγRIIB-1 and FcγRIIB-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIA (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIB-NA1 and FcγRIIB-NA2) (Jefferis, et al., Immunol Lett 82 (2002) 57-65, incorporated by reference in its entirety), as well as unidentified human FcγR or FcγR isoforms or allotypes. FcγRs can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as undetected mouse FcγRs or FcγR isoforms or allotypes.

[0202] "FcRn" or "neonatal Fc receptor" can be, for example, a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2-microglobulin.

[0203] For example, a "wild-type or parent polypeptide" can be an unmodified polypeptide that is subsequently modified to produce a variant. A wild-type polypeptide can be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. A wild-type polypeptide can refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence that encodes it. Thus, a "wild-type immunoglobulin" refers to an unmodified immunoglobulin polypeptide that is modified to produce a variant, and a "wild-type antibody" refers to an unmodified antibody that is modified to produce a variant antibody. Note that "wild-type antibody" includes known, commercially available, recombinantly produced antibodies, as described herein.

[0204] A "fragment crystallizable (Fc) polypeptide" is the portion of an antibody molecule that interacts with effector molecules and cells. It comprises the C-terminal portion of an immunoglobulin heavy chain.

[0205] "Framework" or "FR" refers to variable domain residues other than, for example, hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0206] "Full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0207] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using the various assays disclosed herein.

[0208] The "hinge region" generally refers to the stretch of amino acids from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22 (1985) 161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same positions.

[0209] The "lower hinge region" of an Fc region corresponds, for example, to the residues immediately C-terminal to the hinge region, ie, the stretch of residues 233-239 of the Fc region.

[0210] " Homology " refers to the percentage of residues in amino acid sequence variants that are identical, for example, after aligning sequences and introducing gaps as necessary to achieve maximum homology percentage. Methods and computer programs for alignment are well known in the art. One such computer program is "Align 2" created by Genentech, Inc., which was filed with the U.S. Copyright Office, Washington, DC 20559 on December 10, 1991, together with user documentation.

[0211] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0212] A "human antibody" is one that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. Human antibodies specifically exclude humanized antibodies that comprise non-human antigen-binding residues.

[0213] "Human effector cells" are leukocytes that express one or more FcRs and perform effector function. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells can be isolated from their native source, for example, from blood or PBMCs as described herein.

[0214] A "humanized" antibody can refer, for example, to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody can 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 can optionally 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. For example, a "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, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0215] As used herein, "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or "complementarity-determining regions" (CDRs), the latter of which are most highly sequence variable and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, and Lesk, J. Mol. Biol. 196 (1987) 901-917). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). With the exception of CDR1 in VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs referred to as abbreviated-CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (see Almagro, and Fransson, Front. Biosci. 13 (2008) 1619-1633).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0216] "Immune complex" refers to a relatively stable structure formed when at least one target molecule and at least one heterologous Fc region-containing polypeptide bind to each other to form a complex of larger molecular weight. Examples of immune complexes are antigen-antibody aggregates and target molecule-immunoadhesin aggregates. As used herein, the term "immune complex" refers to an ex vivo complex (i.e., a form or setting other than that in which it can be found in nature) unless otherwise specified. However, for example, to evaluate the clearance of the immune complex in a mammal, the immune complex can be administered to a mammal.

[0217] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0218] An "individual" or "subject" can be a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0219] The terms "subject" or "patient" can refer to any organism to which embodiments of the present invention can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects to which compounds of the present disclosure can be administered would be mammals, particularly primates, and especially humans. For veterinary applications, a wide variety of subjects would be suitable, including livestock such as cattle, sheep, goats, cows, pigs, etc., poultry such as chickens, ducks, geese, turkeys, etc., and domestic animals, particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals would be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, including inbred pigs. The term "living subject" refers to the subjects described above or another living organism. The term "living subject" refers to the entire subject or organism, not just excised parts (e.g., liver or other organs) from a living subject. [Example]

[0220] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be used to obtain similar results.

[0221] Example 1 - ADCC Assay We performed an ADCC assay using the reporter system from Promega. A pool of CHO-GITR cells was sorted to achieve a cell population with a purity of >99% GITR+ cells. Cells were seeded at 15k cells / well and incubated with various concentrations of different aGITR antibodies. Promega ADCC bioassay effector cells were added at a 5:1 E:T ratio, and the plate was incubated for 6 hours at 37°C and 5% CO2. After incubation, Bio-Glo Lucifierase Assay reagent was added, and the luminescent signal was detected using a BMG PolarStart Multilabel plate reader. The data show that only the IgG1 WT monomer and hexamer constructs exhibited significant ADCC activity, as expected. Interestingly, hexamerization appears to reduce the magnitude of ADCC in WT IgG1. The negative control IgG did not exhibit any specific ADCC activity.

[0222] Example 2 - Fc variants as CDC active mutants The P329-P331 motif in IgG1 forms a loop that fits into a pocket between the C1q side chains [Schneider et al., Molecular Immunology 51 (2012) 66-72]. Without being bound by theory, the side chain ring structure of proline (P) significantly contributes to Fc interaction with C1q. Without being bound by theory, by modifying the structure and altering the side chain of proline, we can create either a repulsive interaction or a steric clash between the side chain of the loop and the binding pocket on C1q. Using amino acids at positions D270, K322, P329, and P331, we designed single, double, and triple mutants to eliminate CDC activity while maintaining the hexamer structure and ADCC null function of the final construct. Figure 15 shows several mutations we introduced into the CH2 region of the LALA-hexamer construct.

[0223] We also investigated testing alternative methods for blocking C1q binding. When antibodies hexamerize on the cell surface, they form flat disks onto which C1q constructs can dock. Without being bound by theory, a second construct can be tethered to the light chain constant region (CL) to sterically interfere with C1q binding at a more macroscopic level. To test this, we generated two CL fusions, one with an anti-PDL1 scFv and one with the GFP analog zsGreen, as shown in panels I and J of Figure 15.

[0224] In one embodiment, point mutation analysis was performed using human IgG1 Fc fragment, glycoform (G0F)2 (PDB code -1h3x). Point mutants were manually modeled in COOT (Crystallographic Object-Oriented Tool).

[0225] Example 3 - CDC activity remains in all anti-GITR Ab constructs except for the sIgG4 monomer The target cells in this experiment (Figure 16) were sorted CHO-GITR cells. Target cells were seeded at 50,000 / well for CellTiter and 10,000 cells / well for CytoTox, and the antibody of interest was added in 3-fold serial dilutions in a final concentration of 10% human serum (Quidel). All samples were analyzed in triplicate. After 1 hour of incubation at 37°C, Promega's CellTiter Glo (viable cell count) or CytoTox-Glo (dead cell count) reagent was added, and the plates were read on a BMG PolarStar Omega. All samples were normalized using wells containing both cells and 10% serum (no antibody).

[0226] Example 4 - Binding Analysis FACS was performed using sorted CHO-GITR cells. 200k cells / well were incubated with increasing amounts of antibody as indicated, washed once with MACS buffer, and then resuspended in MACS buffer containing 2ul / well of FITC-labeled anti-human Lc lambda (BioLegend 316606). After washing with MACS buffer, cells were read on a Fortessa HTS FACS machine, gated on live cells, and FITC+ percent and MFI were calculated and plotted (see Figures 18 and 19, respectively). The negative control IgG showed no specific binding activity.

[0227] Example 5 - CDC Activity Assay The target cells in Figures 20 and 21 were sorted CHO-GITR cells (P2 after sorting). Target cells were seeded at 10,000 cells / well, and the antibody of interest was added in 3-fold serial dilutions in a final concentration of 10% human serum (Quidel). All samples were analyzed in triplicate. After 2 hours of incubation at 37°C, Promega's CytoTox-Glo reagent was added, and the plates were read on a BMG PolarStar Omega. All samples were normalized using wells containing both cells and 10% serum (no antibody). The selected mutations shown in Figure 20 significantly reduce CDC activity compared to the original antibody. The negative control IgG (mA2.3) showed no specific CDC activity.

[0228] Example 6 - GITR Bioassay Promega's GITR Bioassay reporter assay was used for the experiments (Figures 22-24). Frozen and thawed GITR Jurkat cells were incubated with either GITRL (GITR ligand) alone, antibody alone, or antibody plus 111 ng / ml GITRL for 6 hours at 37°C. Promega's Bio-Glo luciferase substrate was then added, and luminescence was read on a Polarstar Omega plate reader. Values ​​were normalized by subtracting the unstimulated cell signal for Figures 22-23.

[0229] Example 7 - ADCC Assay ADCC was performed using the Promega ADCC reporter assay (Figure 25). A pool of CHO-GITR cells was sorted to achieve a cell population with a purity of >99% GITR+ cells. Cells were seeded at 15k cells / well and incubated with different αGITR antibodies at various concentrations. Promega ADCC bioassay effector cells were added at an E:T ratio of 5:1, and the plates were incubated at 37°C for 6 hours. After incubation, Bio-Glo Lucifierase Assay reagent was added and luminescent signal was detected.

[0230] Example 8 Referring to Figure 33, CDC was performed using Promega's CellTiter-Glo kit (live cell assay). 50k cells were seeded and mixed with 10% human serum (final concentration) and various antibodies. They were incubated at 37°C for 1 or 2 hours and then equilibrated at RT for 30 minutes. CellTiter Glo reagent was then added, and after equilibration, the plate was read on a Polarstar Omega.

[0231] The data show that the selected sIgG4 mutations significantly increase CDC activity compared to sIgG4 hex WT.

[0232] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0233] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures specifically described herein which equivalents are considered to be within the scope of this invention and covered by the following claims.

[0234] Sequence information SEQUENCE LISTING <110> DANA-FARBER CANCER INSTITUTE, INC. <120> FC VARIANT COMPOSITIONS AND METHODS OF USE THEREOF <150> US 62 / 646,053 <151> 2018-03-21 <160> 98 <170> PatentIn version 3.5 <210> 1 <211> 330 <212> PRT <213> Homo sapiens <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 2 <211> 326 <212> PRT <213> Homo sapiens <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 3 <211> 327 <212> PRT <213> Homo sapiens <400> 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 4 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (153)..(153) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (205)..(205) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (212)..(212) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (214)..(214) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (216)..(216) <223> Any neutral polar amino acid <220> <221> MOD_RES <222> (228)..(228) <223> Lys, Gln, Arg or Tyr <220> <221> MOD_RES <222> (313)..(313) <223> Gly, Ser, Phe or Thr <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Xaa Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Xaa Val Ser Asn 195 200 205 Lys Ala Leu Xaa Ala Xaa Ile Xaa Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Xaa Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Xaa Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Trp Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 5 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (149)..(149) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (201)..(201) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (208)..(208) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (210)..(210) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (212)..(212) <223> Any neutral polar amino acid <220> <221> MOD_RES <222> (224)..(224) <223> Lys, Gln, Arg or Tyr <220> <221> MOD_RES <222> (309)..(309) <223> Gly, Ser, Phe or Thr <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Ala Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Xaa Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Xaa Val Ser Asn Lys Gly Leu Xaa 195 200 205 Ala Xaa Ile Xaa Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Xaa 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Xaa Ala Leu His Asn His Tyr Thr Gln Lys Trp Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 6 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (150)..(150) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (202)..(202) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (209)..(209) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (211)..(211) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (213)..(213) <223> Any neutral polar amino acid <220> <221> MOD_RES <222> (225)..(225) <223> Lys, Gln, Arg or Tyr <220> <221> MOD_RES <222> (310)..(310) <223> Gly, Ser, Phe or Thr <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 6 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Xaa Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Xaa Val Ser Asn Lys Gly Leu 195 200 205 Xaa Ser Xaa Ile Xaa Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Xaa Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Xaa Ala Leu His Asn His Tyr Thr Gln Lys Trp 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 7 <211> 355 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 7 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agccatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaatcggt 300 acggcggatg cttttgatat ctggggccaa gggaccacgg tcaccgtctc ctcag 355 <210> 8 <211> 331 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 8 cagtctgccc tgactcagcc accctcagtg tctgggaccc ccggacagag ggtcaccatc 60 tcttgttctg gaggcgtccc siacatcgga agtaatcctg taaactgta cctccaccgc 120 ccaggaacgg ccccaacct cctcatctat atagcaatc agtggccctc aggggtccct 180 gaccgatttt ctggctccag gtctggcacc tcagcctccc tggcattcag tggctccag 240 tctgaggatg aggctgatta ttactgtgca gcatggtg acagcctgga tggtctggtt 300 ttcggcggag ggaccaagtt gaccgtccta g 331 <210> 9 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser On Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Gly Thr Ala Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 10 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Gly Val Pro Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn Trp Tyr Leu His Arg Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asn Ser Asn Gln Trp Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asp Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Ala 20 25 30 Trp Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr Tyr Arg Pro Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 12 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Gln Is Gln Only Glu Asp Only On Tyr Tyr Cys His Gln 85 90 95 Tyr Met Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 13 <211> 285 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 13 accaagggcc catcggctt cccctggca cccctcca agagcacctc tggggcaca 60 gcggccctgg gctgcctggt caggactac ttccccgac cggtgacggt gtcgtggac 120 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 180 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 240 tgcaacgtga atcacaagcc cagcacacc aaggtggaca agaaa 285 <210> 14 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 14 gcagagccca aatcttgtga caaaactcac acatgcccac cgtgccca 48 <210> 15 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 15 gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 cccatcgaga aaaccatctc caaagccaaa 330 <210> 16 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 16 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 60 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 240 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 300 ctctccctgt ctccgggtaa atga 324 <210> 17 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 17 ggtcagccca aggctgcccc ctcggtcact ctgttcccgc cctcctctga ggagcttcaa 60 gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120 gcctggaagg cagatggcag cccctcaag gcgggagtgg agaccaccac accctccaaa 180 caaagcaca acagtacgc ggccagcagc tatctgagcc tgacgcctga gcagtggaag 240 tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300 gccctacag aatgttcatg a 321 <210> 18 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 18 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 20 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 21 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 22 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 22 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 23 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 23 gcacctgaag ccgccggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 cccatcgaga aaaccatctc caaagccaaa 330 <210> 24 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 24 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 25 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 25 gggcagcccc gaaagccaca ggtgtacacc ctgccccat cccgggatga gctgaccaag 60 aaccaggtca gcctgacctg cctggtcaaa ggctctatc cgccgtggag 120 tgggagagca atgggcagcc ggagacaac tacaagacca cgcctcccgt gctggactcc 180 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 240 aacgtcttct catgctccgt gatgcatgga gctctgcaca accactac gcagaagagc 300 ctctccctgt ctccgggtaa atga 324 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 26 Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Thr Lys Asn Gln Will Be Thr Cys Thr Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 27 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 27 gcacctgaag ccgccggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 cccatcgaga aaaccatctc caaagccaaa 330 <210> 28 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 28 gggcagcccc gaaagccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 60 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 240 aacgtcttct catgctccgt gatgcatgga gctctgcaca accactacac gcagaagagc 300 ctctccctgt ctccgggtaa atga 324 <210> 29 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 29 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 30 Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 31 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 31 gctagcacca agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agtt 294 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 32 gagtccaaat atggtccccc atgcccacca tgccca 36 <210> 33 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 33 gcacctgagt tcctgggggg accatcagtc ttcctgttcc ccccaaaacc caaggacact 60 ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac 120 cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagttcaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 300 tccatcgaga aaaccatctc caaagccaaa 330 <210> 34 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 34 gggcagcccc gagagccaca ggtgtacacc ctgcccccat ccccggagga gatgaccaag 60 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 120 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180 gacggctcct tcttcctcta cagcaagcta accgtggaca agagcaggtg gcaggagggg 240 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300 ctctccctgt ctctgggtaa atga 324 <210> 35 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 35 ggtcagccca aggctgcccc ctcggtcact ctgttcccgc cctcctctga ggagcttcaa 60 gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120 gcctggaagg cagatggcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180 caaagcaaca acaagtacgc ggccagcagc tatctgagcc tgacgcctga gcagtggaag 240 tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300 gcccctacag aatgttcatg a 321 <210> 36 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 36 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 38 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 38 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 39 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 39 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Pro Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 100 105 <210> 40 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 40 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 41 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 41 gggcagcccc gaaagccaca ggtgtacacc ctgcccccat ccccggagga gatgaccaag 60 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 120 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180 gacggctcct tcttcctcta cagcaagcta accgtggaca agagcaggtg gcaggagggg 240 aatgtcttct catgctccgt gatgcatgga gctctgcaca accactacac acagaagagc 300 ctctccctgt ctctgggtaa atga 324 <210> 42 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 42 Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Pro Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 100 105 <210> 43 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 43 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttga 324 <210> 44 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 44 gcacctgaag ccgccggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagcc 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcgccgtct ccaacaaagc cctcccagcc 300 ggcatcgaga aaaccatctc caaagccaaa 330 <210> 45 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Ala Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Gly Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 46 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 46 gcacctgaag ccgccggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagcc 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 ggcatcgaga aaaccatctc caaagccaaa 330 <210> 47 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 47 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Ala Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Will Be Asn Lys 85 90 95 Leu Pro Wing Gly Wing Glu Lys Thr Wing Being Lys Wing Lys Wing 100 105 110 <210> 48 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 48 gcacctgaag ccgccgggggg accgtcagtc ttcctctcc cccaaaacc CAggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtg ccacgaagcc 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc cagacaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggca ggagtacaag tgcaggtct ccaaaagc cctcccagcc 300 gtgatccaga aaaccatctc caaagccaaa 330 <210> 49 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Ala Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Val Ile Gln Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 50 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 50 gcacctgaag ccgccggggg accgtcagtc ttcctctttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcgtggcc 300 cccatcgaga aaaccatctc caaagccaaa 330 <210> 51 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 51 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Val Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 52 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 52 gcacctgaag ccgccggggg accgtcagtc ttcctctttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 gtgatcgaga aaaccatctc caaagccaaa 330 <210> 53 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Will Be Asn Lys 85 90 95 Leu Pro Wing Val Ile Glu Lys Thr Wing To Be Lys Wing Lys 100 105 110 <210> 54 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 54 gcacctgaag ccgccgggggg accgtcagtc ttcctctcc cccaaaacc CAggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtg ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc cagacaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300 ttcatcgaga aaaccatctc caaagccaaa 330 <210> 55 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 55 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Phe Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 56 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 56 gcacctgaag ccgccggggg accgtcagtc ttcctctttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcgtggcc 300 gtgatcgaga aaaccatctc caaagccaaa 330 <210> 57 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 57 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Val Ala Val Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <210> 58 <211> 1137 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 58 ggtcagccca aggctgcccc ctcggtcact ctgttcccgc cctcctctga ggagcttcaa 60 gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120 gcctggaagg cagatggcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180 caaagcaaca acaagtacgc ggccagcagc tatctgagcc tgacgcctga gcagtggaag 240 tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300 gcccctacag aatgttcagg tggcggcggt tccggaggtg gtggttcatc gatggcccag 360 gtgcagctgg tgcagtctgg ggctgaggtg aagaagcctg ggtcctcggt gaaggtctcc 420 tgcaaggctt ctggaggcac cttcagcagc tatgctatca gctgggtgcg acaggcccct 480 ggacaagggc ttgagtggat gggagggatc atccctatct ttggtacagc aaactacgca 540 cagaagttcc agggcagagt cacgattacc gcggacaaat ccacgagcac agcctacatg 600 gagctgagca gcctgagatc tgaggacacg gccgtctatt actgtgcgag agggcgtcaa 660 atgttcggtg cgggaattga tttctggggc ccgggcaccc tggtcaccgt ctcctcaggt 720 ggcggcggtt ccggaggtgg tggttctggc ggtggtggca tcaattttat gctgactcag 780 ccccactctg tgtcggagtc tccggggaag acggtaacca tctcctgcac ccgcagcagt 840 ggcagcattg acagcaacta tgtgcagtgg taccagcagc gcccgggcag cgcccccacc 900 actgtgatct atgaggataa ccaaagaccc tctggggtcc ctgatcggtt ctctggctcc 960 atcgacagct cctccaactc tgcctccctc accatctctg gactgaagac tgaggacgag 1020 gctgactact actgtcagtc ttatgatagc aacaatcgtc atgtgatatt cggcggaggg 1080 accaagctga ccgtcctagg tggatccgga aaggctagcc atcatcatca tcatcat 1137 <210> 59 <211> 378 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 59 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu 115 120 125 Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly 130 135 140 Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala 165 170 175 Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys 180 185 190 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 195 200 205 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Gln Met Phe Gly Ala Gly 210 215 220 Ile Asp Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gly 225 230 235 240 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asn Phe 245 250 255 Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr Val 260 265 270 Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Asp Ser Asn Tyr Val 275 280 285 Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val Ile Tyr 290 295 300 Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 305 310 315 320 Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu Lys 325 330 335 Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Asn Asn 340 345 350 Arg His Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly 355 360 365 Ser Gly Lys Ala Ser His His His His His 370 375 <210> 60 <211> 377 <212> PRT <213> Homo sapiens <400> 60 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 61 <211> 377 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (200)..(200) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (252)..(252) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (259)..(259) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (261)..(261) <223> Any neutral non-polar amino acid <220> <221> MOD_RES <222> (263)..(263) <223> Any neutral polar amino acid <220> <221> MOD_RES <222> (275)..(275) <223> Lys, Gln, Arg or Tyr <220> <221> MOD_RES <222> (360)..(360) <223> Gly, Ser, Phe or Thr <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 61 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Pro Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Xaa Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Xaa Val Ser Asn Lys 245 250 255 Ala Leu Xaa Ala Xaa Ile Xaa Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Xaa Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Xaa Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Trp Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 62 <211> 353 <212> PRT <213> Homo sapiens <400> 62 Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys Ser Thr 1 5 10 15 Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Gly Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro 100 105 110 Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser 115 120 125 Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn 130 135 140 Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe 145 150 155 160 Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu 165 170 175 Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys 180 185 190 Ala Glu Pro Trp Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr 195 200 205 Pro Glu Ser Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn 210 215 220 Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu 225 230 235 240 Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser 245 250 255 Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro 260 265 270 Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly 275 280 285 Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp 290 295 300 Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu 305 310 315 320 Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro 325 330 335 Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys 340 345 350 Tyr <210> 63 <211> 340 <212> PRT <213> Homo sapiens <400> 63 Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr 1 5 10 15 Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Ser Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro Pro Cys Cys His Pro 100 105 110 Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125 Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140 Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly 145 150 155 160 Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170 175 Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 180 185 190 Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys 195 200 205 Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser 210 215 220 Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg 225 230 235 240 Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255 Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265 270 Ser Gln Gly Thr Thr Thr Tyr Ala Val Thr Ser Ile Leu Arg Val Ala 275 280 285 Ala Glu Asp Trp Lys Lys Gly Glu Thr Phe Ser Cys Met Val Gly His 290 295 300 Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Met Ala 305 310 315 320 Gly Lys Pro Thr His Ile Asn Val Ser Val Val Met Ala Glu Ala Asp 325 330 335 Gly Thr Cys Tyr 340 <210> 64 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 64 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 65 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Gly Phe Thr Phe Ser Ser His Ala 1 5 <210> 67 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 67 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Ala <210> 68 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 69 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 69 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 70 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 Ala Lys Ile Gly Thr Ala Asp Ala Phe Asp Ile 1 5 10 <210> 71 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 71 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 72 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 72 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Gly 20 25 <210> 73 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 73 Val Pro Asn Ile Gly Ser Asn Pro 1 5 <210> 74 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 74 Val Asn Trp Tyr Leu His Arg Pro Gly Thr Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 75 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 75 Gln Trp Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Arg Ser Gly 1 5 10 15 Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 76 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 76 Ala Ala Trp Asp Asp Ser Leu Asp Gly Leu Val 1 5 10 <210> 77 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 77 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 78 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 79 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Gly Tyr Thr Phe Ala Ser Ala Trp 1 5 <210> 80 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Trp <210> 81 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 81 Ile Asn Pro Gly Asn Val Asn Thr 1 5 <210> 82 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 82 Lys Tyr Asn Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Thr 1 5 10 15 Ser Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys Ala Arg 35 40 <210> 83 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Ser Thr Tyr Tyr Arg Pro Leu Asp Tyr 1 5 <210> 84 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 85 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser 20 25 <210> 86 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 86 Gln Ser Ile Leu Tyr Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 87 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 87 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 88 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Trp Ala Ser Thr Arg Glu 1 5 <210> 89 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 89 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 1 5 10 15 Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr 20 25 30 Cys <210> 90 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 His Gln Tyr Met Ser Ser Tyr Thr 1 5 <210> 91 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 92 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> CDS <222> (1)..(993) <400> 92 cta gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc 48 Leu Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 1 5 10 15 aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac 96 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25 30 tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc 144 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45 agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac 192 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60 tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag 240 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 65 70 75 80 acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac 288 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95 aag aaa gca gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg 336 Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 100 105 110 tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc 384 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 115 120 125 cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca 432 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 130 135 140 tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac 480 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 145 150 155 160 tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 528 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 165 170 175 gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc 576 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 180 185 190 ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc 624 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 195 200 205 aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa 672 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 210 215 220 ggg cag ccc cga aag cca cag gtg tac acc ctg ccc cca tcc cgg gat 720 Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 225 230 235 240 gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 768 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 245 250 255 tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 816 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 260 265 270 aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 864 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285 ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg 912 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295 300 aac gtc ttc tca tgc tcc gtg atg cat gga gct ctg cac aac cac tac 960 Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr 305 310 315 320 acg cag aag agc ctc tcc ctg tct ccg ggt aaa tgaggatccg cg 1005 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 93 <211> 331 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 93 Leu Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 1 5 10 15 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25 30 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 65 70 75 80 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95 Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 100 105 110 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 115 120 125 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 130 135 140 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 145 150 155 160 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 165 170 175 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 180 185 190 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 195 200 205 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 210 215 220 Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 225 230 235 240 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 245 250 255 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 260 265 270 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295 300 Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr 305 310 315 320 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 94 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> CDS <222> (1)..(993) <400> 94 cta gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc 48 Leu Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 1 5 10 15 aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac 96 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25 30 tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc 144 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45 agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac 192 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60 tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag 240 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 65 70 75 80 acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac 288 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95 aag aaa gca gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg 336 Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 100 105 110 tgc cca gca cct gaa gcc gcc ggg gga ccg tca gtc ttc ctc ttc ccc 384 Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 115 120 125 cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca 432 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 130 135 140 tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac 480 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 145 150 155 160 tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 528 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 165 170 175 gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc 576 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 180 185 190 ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc 624 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 195 200 205 aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa 672 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 210 215 220 ggg cag ccc cga aag cca cag gtg tac acc ctg ccc cca tcc cgg gat 720 Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 225 230 235 240 gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 768 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 245 250 255 tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 816 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 260 265 270 aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 864 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285 ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg 912 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295 300 aac gtc ttc tca tgc tcc gtg atg cat gga gct ctg cac aac cac tac 960 Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr 305 310 315 320 acg cag aag agc ctc tcc ctg tct ccg ggt aaa tgaggatccg cg 1005 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 95 <211> 331 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 95 Leu Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 1 5 10 15 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 20 25 30 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 65 70 75 80 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95 Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 100 105 110 Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 115 120 125 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 130 135 140 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 145 150 155 160 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 165 170 175 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 180 185 190 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 195 200 205 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 210 215 220 Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 225 230 235 240 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 245 250 255 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 260 265 270 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295 300 Asn Val Phe Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr 305 310 315 320 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 96 <211> 984 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> CDS <222> (1)..(981) <400> 96 gct agc acc aag ggc cca tcc gtc ttc ccc ctg gcg ccc tgc tcc agg 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 agc acc tcc gag agc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac 96 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acg aag acc 240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 tac acc tgc aac gta gat cac aag ccc agc aac acc aag gtg gac aag 288 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 aga gtt gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca gca cct 336 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 gag ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag 384 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 gac act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg 432 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 gac gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat 480 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 528 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 576 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc 624 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 672 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 aag cca cag gtg tac acc ctg ccc cca tcc ccg gag gag atg acc aag 720 Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Pro Glu Glu Met Thr Lys 225 230 235 240 aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 768 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 816 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 864 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 aag cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 912 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 tgc tcc gtg atg cat gga gct ctg cac aac cac tac aca cag aag agc 960 Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 ctc tcc ctg tct ctg ggt aaa tga 984 Leu Ser Leu Ser Leu Gly Lys 325 <210> 97 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 97 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Pro Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 98 <211> 377 <212> PRT <213> Homo sapiens <400> 98 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Trp Leu Ser Leu Ser Pro Gly Lys 370 375

Claims

1. 1. An engineered polypeptide comprising an Fc variant of a wild-type human IgGl Fc region, said Fc variant comprising amino acid substitutions at residue positions 234, 235, 329, 345, and 430, said amino acid residues being numbered according to the EU index of Kabat; the amino acid at residue position 234 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 235 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 329 according to the EU index of Kabat is substituted with a valine (V); the amino acid at residue position 345 according to the EU index of Kabat is substituted with a lysine (K); the amino acid at residue position 430 according to the EU index of Kabat is substituted with a glycine (G); the polypeptide exhibits reduced affinity for one or more human Fc receptors, increased receptor clustering, and reduced complement-dependent cytotoxicity (CDC) compared to a polypeptide comprising the wild-type IgG1 Fc region; The Fc variant comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the wild-type Fc region of IgG1 set forth in SEQ ID NO:

1. Polypeptide.

2. The polypeptide of claim 1 which is an antibody or an Fc fusion protein.

3. The polypeptide of claim 2 , wherein the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

4. The polypeptide of any one of claims 1 to 3, conjugated to a drug, a toxin, a radiolabel, or a combination thereof.

5. The polypeptide according to any one of claims 1 to 4, which is an antibody specific to an inhibitory molecule on a T cell.

6. The polypeptide of claim 5 , wherein the inhibitory molecule on a T cell comprises PD1, TIGIT, CTLA4, Lag3, Tim3, or KIR.

7. The polypeptide according to any one of claims 1 to 4, which is an antibody specific for a stimulatory molecule on a T cell.

8. 8. The polypeptide of claim 7, wherein the stimulatory molecule on a T cell comprises GITR, CD27, OX40, 4-1BB, CD40L, ICOS, or CD28.

9. The polypeptide according to any one of claims 1 to 4, which is an antibody specific to a chemokine receptor.

10. The polypeptide of claim 9 , wherein the chemokine receptor comprises CCR4, CXCR4, or CCR5.

11. The polypeptide according to any one of claims 1 to 4, which is an antibody specific to a tumor-associated molecule on a tumor cell.

12. The polypeptide of claim 11 , wherein the tumor-associated molecule on a tumor cell comprises BCMA, CAIX, an antigen-presenting cell molecule, or a combination thereof.

13. The polypeptide of claim 12, wherein the antigen-presenting cell molecule comprises PDL1 or PDL2.

14. The polypeptide of any one of claims 1 to 4, which is an antibody specific to an infectious agent.

15. 15. The polypeptide of claim 14, wherein the infectious agent comprises Severe Acute Respiratory Syndrome virus (SARS), Middle East Respiratory Syndrome virus (MERS), an alphavirus, a flavivirus, or an influenza virus.

16. 16. The polypeptide of claim 15, wherein the alphavirus comprises Western Equine Encephalitis Virus (WEEV), Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus, or Chikungunya Virus (CHKV).

17. The polypeptide of claim 15 , wherein the flavivirus is mosquito-borne.

18. 16. The polypeptide of claim 15, wherein the flavivirus comprises West Nile virus (WNV), dengue virus serotypes 1-4, yellow fever virus, or Zika virus.

19. The polypeptide of claim 15, wherein the influenza virus is an emerging influenza virus.

20. The polypeptide of claim 2, wherein the antibody comprises a chimeric antigen receptor (CAR) targeting domain.

21. 21. The polypeptide of claim 20, wherein a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a combination thereof is incorporated into the extracellular domain.

22. The polypeptide according to any one of claims 1 to 4, which is an antibody specific to glucocorticoid-induced tumor necrosis factor receptor (GITR).

23. The polypeptide according to any one of claims 1 to 4, which is an antibody specific to CCR4.

24. 1. An engineered polypeptide comprising an Fc variant human IgGl Fc region, said Fc variant comprising an amino acid sequence having at least 90% identity to SEQ ID NO:4, said amino acid sequence comprising amino acid substitutions at least at residue positions 234, 235, 329, 345, and 430, said amino acid residues being numbered according to the EU index of Kabat; the amino acid at residue position 234 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 235 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 345 according to the EU index of Kabat is substituted with a lysine (K); the amino acid at residue position 430 according to the EU index of Kabat is substituted with a glycine (G); the amino acid at residue position 329 according to the EU index of Kabat is substituted with a valine (V); the polypeptide exhibits reduced affinity for one or more human Fc receptors, increased receptor clustering, and reduced complement-dependent cytotoxicity (CDC) compared to a polypeptide comprising the wild-type IgG1 Fc region. Polypeptide.

25. A recombinant GITR antibody comprising a variable region amino acid sequence disclosed in Table 1B and a variant Fc region amino acid sequence disclosed in Table 11B (SEQ ID NOs: 18, 19, 22, 26, 51).

26. A recombinant CCR4 antibody comprising a variable region amino acid sequence disclosed in Table 1D and a variant Fc region amino acid sequence disclosed in Table 11B (SEQ ID NOs: 18, 19, 22, 26, 51).

27. A pharmaceutical composition for enhancing T cell immunity, comprising the recombinant GITR antibody of claim 25 or the recombinant CCR4 antibody of claim 26.

28. A pharmaceutical composition for treating a tumor in a subject, comprising the recombinant GITR antibody of claim 25.

29. A pharmaceutical composition for treating CCL22 / 17-secreting tumors, comprising the recombinant CCR4 antibody of claim 26.

30. 30. The pharmaceutical composition of claim 29, wherein the CCL22 / 17-secreting tumor is a hematological cancer.

31. 31. The pharmaceutical composition of claim 30, wherein the hematological cancer is lymphoma or leukemia.

32. 30. The pharmaceutical composition of claim 29, wherein the CCL22 / 17-secreting tumor is ovarian cancer.

33. A pharmaceutical composition for enhancing cell signaling in a cell, comprising an antibody comprising a polypeptide according to any one of claims 1 to 23.

34. A pharmaceutical composition for inducing receptor clustering in cells, comprising an antibody comprising the polypeptide of any one of claims 1 to 23.

35. 30. The pharmaceutical composition of claim 28 or 29, wherein the tumor is a solid tumor or a liquid tumor.

36. A pharmaceutical composition for reducing CDC activity of a cell, comprising an antibody comprising the polypeptide according to any one of claims 1 to 23.

37. a nucleic acid encoding an engineered polypeptide comprising an Fc variant of a wild-type human IgGl Fc region, said Fc variant comprising amino acid substitutions at residue positions 234, 235, 329, 345, and 430, said amino acid residues being numbered according to the EU index of Kabat; the amino acid at residue position 234 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 235 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 329 according to the EU index of Kabat is substituted with a valine (V); the amino acid at residue position 345 according to the EU index of Kabat is substituted with a lysine (K); the amino acid at residue position 430 according to the EU index of Kabat is substituted with a glycine (G); the polypeptide exhibits reduced affinity for one or more human Fc receptors, increased receptor clustering, and reduced complement-dependent cytotoxicity (CDC) compared to a polypeptide comprising the wild-type IgG1 Fc region; The Fc variant comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the wild-type Fc region of IgG1 set forth in SEQ ID NO:

1. Nucleic acid.

38. a nucleic acid encoding an engineered polypeptide comprising an Fc variant of a wild-type human IgGl Fc region, said Fc variant comprising amino acid substitutions at residue positions 234, 235, 331, 345, and 430, said amino acid residues being numbered according to the EU index of Kabat; the amino acid at residue position 234 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 235 according to the EU index of Kabat is substituted with alanine (A); the amino acid at residue position 331 according to the EU index of Kabat is substituted with glycine (G), valine (V), or phenylalanine (F); the amino acid at residue position 345 according to the EU index of Kabat is substituted with a lysine (K); the amino acid at residue position 430 according to the EU index of Kabat is substituted with a glycine (G); the polypeptide exhibits reduced affinity for one or more human Fc receptors, increased receptor clustering, and reduced complement-dependent cytotoxicity (CDC) compared to a polypeptide comprising the wild-type IgG1 Fc region; The Fc variant comprises an amino acid sequence having at least 90% identity to the amino acid sequence of the wild-type Fc region of IgG1 set forth in SEQ ID NO:

1. Nucleic acid.

39. 39. The nucleic acid of claim 37 or 38, comprising a sequence disclosed in Table 8A (SEQ ID NOs: 13, 14, 17, 25, 44), Table 9A (SEQ ID NOs: 13, 14, 17, 25, 46), Table 10A (SEQ ID NOs: 13, 14, 17, 25, 48), Table 11A (SEQ ID NOs: 13, 14, 17, 25, 50), Table 12A (SEQ ID NOs: 13, 14, 17, 25, 52), Table 13A (SEQ ID NOs: 13, 14, 17, 25, 54), or Table 14A (SEQ ID NOs: 13, 14, 17, 25, 56).

40. A nucleic acid encoding a recombinant GITR antibody, comprising a variable region nucleic acid sequence disclosed in Table 1A (SEQ ID NOs:7, 8) and a variant Fc region nucleic acid sequence disclosed in Table 8A (SEQ ID NOs:13, 14, 17, 25, 44), Table 9A (SEQ ID NOs:13, 14, 17, 25, 46), Table 10A (SEQ ID NOs:13, 14, 17, 25, 48), Table 11A (SEQ ID NOs:13, 14, 17, 25, 50), Table 12A (SEQ ID NOs:13, 14, 17, 25, 52), Table 13A (SEQ ID NOs:13, 14, 17, 25, 54), or Table 14A (SEQ ID NOs:13, 14, 17, 25, 56).

41. A nucleic acid encoding a recombinant CCR4 antibody, comprising a variable region nucleic acid sequence encoding an amino acid sequence disclosed in Table ID and a variant Fc region nucleic acid sequence disclosed in Table 8A (SEQ ID NOs: 13, 14, 17, 25, 44), Table 9A (SEQ ID NOs: 13, 14, 17, 25, 46), Table 10A (SEQ ID NOs: 13, 14, 17, 25, 48), Table 11A (SEQ ID NOs: 13, 14, 17, 25, 50), Table 12A (SEQ ID NOs: 13, 14, 17, 25, 52), Table 13A (SEQ ID NOs: 13, 14, 17, 25, 54), or Table 14A (SEQ ID NOs: 13, 14, 17, 25, 56).

42. A vector comprising the nucleic acid of any one of claims 37 to 41.

43. A cell comprising the vector of claim 42.

44. 44. The cell of claim 43, which is a prokaryotic or eukaryotic cell.

45. 44. The cell of claim 43, which is an immune cell or a bacterial cell.

46. 46. ​​The cell of claim 45, wherein the immune cell is a T cell.

47. 47. The cell of claim 46, wherein the T cell is a CD8+ T cell, a CD4+ T cell, a NK cell, a NKT cell, or any combination thereof.

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