Compositions and methods for enhancing antibody-mediated receptor signaling - Patents.com

Abstract

Compositions and methods for enhancing antibody-mediated receptor signaling are provided. [Solution] The present invention provides 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 positions 228, 234, 235, 345, 409, 430, 440, or a combination thereof, and the amino acid residues are numbered according to the EU index of Kabat.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 455,245, filed February 6, 2017, the entire disclosure of which is incorporated by reference in its entirety.

[0002] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled in the art as of 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 files or records, but otherwise reserves all copyright rights whatsoever.

[0004] FIELD OF THE INVENTION The present invention relates generally to therapeutic antibodies with enhanced function. Specifically, the present invention relates to polypeptides comprising variant Fc regions and antibodies comprising the same. More particularly, the present invention relates to Fc region-containing polypeptides with 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 last decade, a major trend in the pharmaceutical industry has been to develop monoclonal antibodies (mAbs) as therapeutic agents for the treatment of many diseases, including cancer, asthma, arthritis, and multiple sclerosis.

[0006] The Fc region of an antibody, i.e., the heavy chain ends of the antibody-spanning domains CH2, CH3, and part 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 include 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 the amino acid substitutions E345K, E430G, L234A, and L235A; or E345K, E430G, S228P, and R409K. 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). 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 engineered polypeptides comprising Fc variants of a wild-type human IgG Fc region. In one embodiment, the Fc variants comprise one amino acid substitution or at least two, three, four, five, six, or seven substitutions at residue positions 228, 234, 235, 345, 409, 430, 440, or a combination thereof, wherein the 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 by 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 by 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 by tryptophan (W). In one embodiment, the amino acid substitutions comprise L234A, L235A, E345K, and E430G, wherein 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, wherein the 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.

[0009] One aspect of the present invention relates to engineered polypeptides comprising Fc variants of a wild-type human IgG Fc region, wherein the Fc variants comprise an amino acid sequence comprising at least 90% identity to SEQ ID NO:4, and wherein the amino acid substitution occurs at X1, X2, X3, X4, X5, X6, X7, or a combination thereof. In one embodiment, the Fc variants comprise 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 containing lysine (K) or arginine (R). In one embodiment, X6 is an amino acid substitution containing glycine (G), serine (S), phenylalanine (F), or threonine (T). In one embodiment, X7 is an amino acid substitution containing tryptophan (W).

[0010] One aspect of the present invention relates to engineered polypeptides comprising Fc variants of a wild-type human IgG Fc region, wherein the Fc variants comprise an amino acid sequence comprising at least 90% identity to SEQ ID NO: 5, and wherein the amino acid substitution occurs at X1, X2, X3, X4, X5, X6, 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).

[0011] One aspect of the present invention relates to engineered polypeptides comprising Fc variants 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, wherein the amino acid substitution occurs at X1, X2, X3, X4, X5, X6, X7, 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 comprising tryptophan (W).

[0012] The polypeptide can be, for example, an antibody or an Fc fusion protein. The antibody can be a monospecific, bispecific, or multispecific antibody. The polypeptide can have the Fc region of human IgG1, IgG2, or IgG4. In some embodiments, the polypeptide can be antibody-specific for an immune modulator, such as CD27, OX40, 4-1BB, CD40L, ICOS, and CD28. Optionally, the polypeptide is an antibody specific for BCMA, CAIX, CCR4, PD-L1, 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 3B (SEQ ID NOs: 18, 19, 21, 22, 24), Table 4B (SEQ ID NOs: 18, 19, 20, 22, 26), Table 5B (SEQ ID NOs: 18, 19, 22, 29, and 30), or Table 6B (SEQ ID NOs: 36, 37, 38, 40, and 42).

[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 3B (SEQ ID NOs: 18, 19, 21, 24), Table 4B (SEQ ID NOs: 18, 19, 20, 26), Table 5B (SEQ ID NOs: 18, 19, 29, and 30), Table 6B (SEQ ID NOs: 36, 37, 38, and 42), or SEQ ID NO: 44.

[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 monomethyl auristatin E or others described, for example, by Schumacher et al. (J Clin Immunol. 2016 May;36 Suppl 1:100-7. doi:10.1007 / s10875-016-0265-6. Epub 2016 Mar22), 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. The present invention also includes a method for treating a subject suffering from a disease by administering to the subject 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. In one embodiment, the present invention provides a method for treating a tumor in a subject, comprising administering to the subject a recombinant GITR antibody described herein. In one embodiment, the tumor is a solid tumor or a liquid tumor. In some embodiments, the liquid tumor may be multiple myeloma, acute myeloid leukemia (AML), or acute lymphoblastic leukemia (ALL). In one embodiment, the present invention provides a method for treating a blood-borne cancer in a subject, comprising administering to the subject a recombinant CCR4 antibody described herein. In one embodiment, the blood-borne cancer is lymphoma or leukemia.

[0017] In another aspect, the present invention provides a method for enhancing intracellular signaling or inducing cellular receptor clustering by contacting a cell with an antibody capable of binding to a ligand on the cell, the antibody 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 E345, E430, and / or S440, where the residues are numbered according to the EU index of Kabat. In one embodiment, the mutations include one or more of E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, or S440W.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of this invention, 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 case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0019] [The present invention 1001] 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 positions 228, 234, 235, 345, 409, 430, 440, or a combination thereof, wherein the amino acid residues are numbered according to the EU index of Kabat. [The present invention 1002] 1001. A polypeptide of the invention, wherein the amino acid at residue position 228 according to the EU index of Kabat is substituted by a proline (P) or a serine (S). [The present invention 1003] 1001. A polypeptide of the invention, wherein the amino acid at residue position 234 according to the EU index of Kabat is substituted by an alanine (A). [The present invention 1004] 1001. A polypeptide of the invention, wherein the amino acid at residue position 235 according to the EU index of Kabat is substituted by an 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 by lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [The present invention 1006] 1001. A polypeptide of the invention, wherein the amino acid at residue position 409 according to the EU index of Kabat is substituted by 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 EU index of Kabat is substituted by 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 by a tryptophan (W). [The present invention 1009] 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 1010] 1001. The polypeptide of claim 10, wherein said amino acid substitutions comprise S228P, E345K, R409K, and E430G, and said amino acid residues are numbered according to the EU index of Kabat. [The present invention 1011] 1001. A polypeptide of the present invention that exhibits reduced affinity for one or more human Fc receptors compared to a polypeptide comprising a wild-type IgG Fc region. [The present invention 1012] 1011. A polypeptide of the invention further exhibiting increased receptor clustering compared to a polypeptide comprising a wild-type IgG Fc region. [The present invention 1013] 1001. A polypeptide of the present invention comprising an Fc region of human IgG1, IgG2, or IgG4. [The present invention 1014] The polypeptide of the present invention 1001, which is an antibody or an Fc fusion protein. [The present invention 1015] The polypeptide of the present invention, wherein the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. [The present invention 1016] The polypeptide of the present invention 1001 conjugated to a drug, a toxin, a radiolabel, or a combination thereof. [The present invention 1017] 1001. The polypeptide of the present invention, which is an antibody specific for BCMA, CAIX, CCR4, PD-L1, PD-L2, PD1, glucocorticoid-induced tumor necrosis factor receptor (GITR), TIGIT, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), influenza, or flavivirus. [The present invention 1018] The polypeptide of the present invention is an antibody specific to glucocorticoid-induced tumor necrosis factor receptor (GITR). [The present invention 1019] The polypeptide of the present invention is an antibody specific to CCR4. [The present invention 1020] 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 amino acid substitutions occur at X1, X2, X3, X4, X5, X6, X7, or a combination thereof. [The present invention 1021] The polypeptide of the present invention 1020, wherein X1 is an amino acid substitution comprising serine (S). [The present invention 1022] The polypeptide of the present invention 1020, wherein X2 is an amino acid substitution comprising alanine (A). [The present invention 1023] The polypeptide of the present invention 1020, wherein X3 is an amino acid substitution comprising an alanine (A). [The present invention 1024] The polypeptide of the present invention 1020, wherein X4 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [The present invention 1025] The polypeptide of the present invention 1020, wherein X5 is an amino acid substitution comprising lysine (K) or arginine (R). [The present invention 1026] The polypeptide of the present invention 1020, wherein X6 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). [The present invention 1027] The polypeptide of the present invention 1020, wherein X7 is an amino acid substitution comprising tryptophan (W). [The present invention 1028] 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 amino acid substitutions occur at X1, X2, X3, X4, X5, X6, or a combination thereof. [The present invention 1029] The polypeptide of the present invention 1028, wherein X1 is an amino acid substitution comprising serine (S). [The present invention 1030] The polypeptide of the present invention, wherein X2 is an amino acid substitution comprising alanine (A). [The present invention 1031] The polypeptide of claim 1028, wherein X3 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [The present invention 1032] The polypeptide of claim 1028, wherein X4 is an amino acid substitution comprising lysine (K) or arginine (R). [The present invention 1033] The polypeptide of claim 1028, wherein X5 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). [The present invention 1034] The polypeptide of claim 1028, wherein X6 is an amino acid substitution containing tryptophan (W). [This invention 1035] 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 amino acid substitutions occur at X1, X2, X3, X4, X5, X6, X7, or a combination thereof. [The present invention 1036] 1035. 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). [This invention 1037] The polypeptide of the present invention 1035, wherein X2 is an amino acid substitution comprising alanine (A). [The present invention 1038] The polypeptide of the present invention 1035, wherein X3 is an amino acid substitution comprising an alanine (A). [This invention 1039] The polypeptide of the present invention 1035, wherein X4 is an amino acid substitution comprising lysine (K), glutamine (Q), arginine (R), or tyrosine (Y). [The present invention 1040] The polypeptide of the present invention 1035, wherein X5 is an amino acid substitution comprising lysine (K) or arginine (R). [This invention 1041] The polypeptide of the present invention 1035, wherein X6 is an amino acid substitution comprising glycine (G), serine (S), phenylalanine (F), or threonine (T). [The present invention 1042] The polypeptide of the present invention 1035, wherein X7 is an amino acid substitution comprising tryptophan (W). [This invention 1043] 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 3B (SEQ ID NOs: 18, 19, 21, 22, 24), Table 4B (SEQ ID NOs: 18, 19, 20, 22, 26), Table 5B (SEQ ID NOs: 18, 19, 22, 29, and 30), or Table 6B (SEQ ID NOs: 36, 37, 38, 40, and 42). [This invention 1044] 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 3B (SEQ ID NOs: 18, 19, 21, 24), Table 4B (SEQ ID NOs: 18, 19, 20, 26), Table 5B (SEQ ID NOs: 18, 19, 29, and 30), or Table 6B (SEQ ID NOs: 36, 37, 38, and 42). [This invention 1045] A method for treating a tumor in a subject, comprising administering to the subject a recombinant GITR antibody of the present invention. [The present invention 1046] A method for treating blood-borne cancer, comprising administering the recombinant CCR4 antibody of the present invention 1044 to a subject. [This invention 1047] 1046. The method of claim 1046, wherein said blood-borne cancer is lymphoma or leukemia. [This invention 1048] A method for enhancing intracellular signaling in a cell, comprising contacting the cell with an antibody that binds to a ligand on the cell, wherein the antibody comprises an Fc variant of a wild-type human IgG Fc region, the Fc variant comprising amino acid substitutions at E345, E430, and / or S440, the residues being numbered according to the EU index of Kabat. [This invention 1049] 1048. The method of claim 1048, wherein said substitutions include E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440W, or a combination thereof. [The present invention 1050] 1049. The method of claim 1049, wherein said substitutions are E345K and E430G. [This invention 1051] A method for inducing receptor clustering in cells, comprising contacting the cells with an antibody that binds to a ligand on the cells, wherein the antibody comprises an Fc variant of a wild-type human IgG Fc region, the Fc variant comprising amino acid substitutions at E345, E430, and / or S440, the residues being numbered according to the EU index of Kabat. [This invention 1052] 1051. The method of claim 1051, wherein said substitutions include E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440W, or a combination thereof. [This invention 1053] 1052. The method of claim 1052, wherein said substitutions are E345K and E430G. [This invention 1054] The method of claim 1045, wherein the tumor is a solid tumor or a liquid tumor. Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims. [Brief explanation of the drawings]

[0020] [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 wild-type hexamer (lane 6). Cell supernatants were collected 96 hours later and purified using Protein A affinity resin. Approximately 2 μg of each purified antibody (determined by OD280 reading after purification) was analyzed on a 4-20% polyacrylamide gel and visualized by Coomassie blue staining. Lane 7 contains a known concentration of control CTI-10 IgG1. Panel A: Reduced condition; Panel B: Non-reduced condition. The 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 from Promega used in our assay. [Figure 3-1]GloResponse NF-kB-luc2P / GITR Jurkat cells are reporter cells that generate ligand or antibody responses based on luciferase activity with the surface-expressed receptor GITR. As a system control, panel A shows that the GITR ligand (GITRL) induced luciferase activity as expected. Panel B presents data showing that an anti-HA antibody further enhanced luciferase activity induced by 111 ng / ml of GITRL (note that GITRL is fused to a C-terminal HA tag). Panel C shows that our newly discovered anti-GITR antibody, E1-3H7-sIgG4, can induce GiTR / NF-kB-dependent luciferase alone or further enhance luciferase activity induced by 111 ng / ml of GITRL, which differs from the behavior of the commercially available anti-GITR antibody control, CTI-10 (panel D). [Figure 3-2] See description of Figure 3-1. [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 hexamer) induced luciferase activity from GloResponse NF-kB-luc2P / GITR Jurkat cells in a dose-dependent manner. Note that the 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. Again, the E1-3H7-LALA hexamer achieved such induction at a much lower antibody concentration. Panels C and D show a similar effect by E1-3H7 stabilizing IgG4 and its corresponding sIgG4 hexamer. Anti-GITR E1-3H7 antibody was used at 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 exhibits increased sensitivity in mediating GITR / NF-kB-dependent luciferase activity. Similar experiments are 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 to confirm the full range of luciferase induction, whereas their corresponding hexamer formats remained at 5,000 ng / ml to 20.58 ng / ml. An irrelevant IgG control demonstrated 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 wild-type, 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 results for the E1-3H7 IgG1 wild-type hexamer in panel A were 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] This is an ADCC assay using Promega's reporter system. [Figure 8-1] Nucleic acid and amino acid sequences of the Fc region of IgG1 wild-type and LALA hexamer mutant. [Figure 8-2] See description of Figure 8-1. [Figure 9]Nucleic acid and amino acid sequences of the Fc region of stabilized hexameric IgG4. [Figure 10] 1 is an expression vector map of vectors that can be used for mammalian expression of IgG antibodies. [Figure 11] 1 is an expression vector map of vectors that can be used for mammalian expression of IgG antibodies. [Figure 12-1] 1 shows the amino acid sequence of 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 shows the amino acid sequence of 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 shows the amino acid sequence of 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. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Invention A detailed description of one or more preferred embodiments is provided herein. However, it should be understood that the present invention can be embodied in various forms. Therefore, the details disclosed herein should not be construed as limiting, but rather as a basis for the claims and as a representative basis for teaching those skilled in the art how to use the present invention in any suitable manner.

[0022] 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 resulting in intracellular signaling events and subsequent important immune responses, such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

[0023] While in many situations the binding and stimulation of effector functions mediated by the Fc region of immunoglobulins is highly beneficial, for example for CD20 antibodies, in certain cases it may be more advantageous to reduce or even eliminate effector functions.

[0024] In other cases, for example when the goal is to block the interaction of a widely expressed receptor with its cognate ligand, it may be advantageous to reduce or eliminate all antibody effector functions to reduce undesired toxicity.

[0025] It may also be advantageous to enhance signaling by increasing receptor clustering.

[0026] There is an unmet need for antibodies that have significantly reduced effector functions such as ADCC and / or ADCP and / or CDC, enhanced receptor cell signaling, and / or induced receptor cell clustering. Accordingly, it is an object of the present invention to synthesize and / or engineer immunoglobulin Fc region polypeptides with mutations introduced to enhance such effects, and ultimately to identify antibodies comprising the engineered Fc region.

[0027] The present invention is based in part on the discovery of mutations in the Fc region of antibodies known to promote antibody hexamerization, increased complement-dependent cytotoxicity (CDC), and also have the unexpected ability to significantly enhance effector cell signaling. All polypeptide variants, including antibody variants of the present invention, comprise an immunoglobulin binding region and a full-length or partial Fc domain that contain one or more mutation(s) known to promote antibody hexamerization and reduced effector function.

[0028] SEQ ID NO: 1 provides the amino acid sequence of the wild-type Fc region of IgG1 (UniProtKB-P01857(IGHG1_HUMAN); 330 amino acids) (CH1 domain is in bold; hinge region is underlined; CH2 domain is in italics; CH3 domain is dashed; shaded regions 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. TIFF0007606501000001.tif18148

[0029] SEQ ID NO: 4 is the amino acid sequence of a variant Fc region of IgG1 (UniProtKB-P01857(IGHG1_HUMAN); 330 amino acids) (the CH1 domain is in bold; the hinge region is underlined; the CH2 domain is italic; the CH3 domain is dashed; the shaded regions 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. X4 is a substitution of an amino acid at residue position 345 according to the EU index of Kabat and includes lysine (K), glutamine (Q), arginine (R), or tyrosine (Y); X5 is a substitution of an amino acid at residue position 409 according to the EU index of Kabat and includes arginine (R); X6 is a substitution of an amino acid at residue position 430 according to the EU index of Kabat and includes glycine (G), serine (S), phenylalanine (F), or threonine (T); and X7 is a substitution of an amino acid at residue position 440 according to the EU index of Kabat and includes tryptophan (W). TIFF0007606501000002.tif19148

[0030] SEQ ID NO: 2 provides the amino acid sequence of the wild-type Fc region of IgG2 (UniProtKB-P01859(IGHG2_HUMAN); 326 amino acids) (CH1 domain is in bold; hinge region is underlined; CH2 domain is in italics; CH3 domain is dashed; shaded regions 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. TIFF0007606501000003.tif18148

[0031] SEQ ID NO: 5 is the amino acid sequence of a variant Fc region of IgG2 (UniProtKB-P01859(IGHG2_HUMAN); 326 amino acids) (CH1 domain is in bold; hinge region is underlined; CH2 domain is in italics; CH3 domain is dashed; shaded regions 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 comprises a proline (P); X2 is a substitution of the amino acid at residue position 235 according to the EU index of Kabat and comprises an alanine (A); X3 is a substitution of the amino acid at residue position 240 according to the EU index of Kabat and comprises an alanine (A); X1 is a substitution of an amino acid at residue position 345 according to the EU index of Kabat and includes lysine (K), glutamine (Q), arginine (R), or tyrosine (Y); X2 is a substitution of an amino acid at residue position 409 according to the EU index of Kabat and includes arginine (R); X3 is a substitution of an amino acid at residue position 430 according to the EU index of Kabat and includes glycine (G), serine (S), phenylalanine (F), or threonine (T); and X4 is a substitution of an amino acid at residue position 440 according to the EU index of Kabat and includes tryptophan (W). TIFF0007606501000004.tif19148

[0032] SEQ ID NO: 3 provides the amino acid sequence of the wild-type Fc region of IgG4 (UniProtKB-P01861(IGHG4_HUMAN); 327 amino acids) (CH1 domain is in bold; hinge region is underlined; CH2 domain is in italics; CH3 domain is dashed; shaded regions 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. TIFF0007606501000005.tif19148

[0033] SEQ ID NO: 6 is the amino acid sequence of a variant Fc region of IgG4 (UniProtKB-P01861 (IGHG4_HUMAN); 327 amino acids) (the CH1 domain is in bold; the hinge region is underlined; the CH2 domain is italicized; the CH3 domain is dashed; the shaded regions 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 comprises a proline (P); X2 is a substitution of the amino acid at residue position 234 according to the EU index of Kabat and comprises an alanine (A); and X3 is a substitution of the amino acid at residue position 235 according to the EU index of Kabat). X4 is a substitution of an amino acid at residue position 345 according to the EU index of Kabat and includes lysine (K), glutamine (Q), arginine (R), or tyrosine (Y); X5 is a substitution of an amino acid at residue position 409 according to the EU index of Kabat and includes lysine (K); X6 is a substitution of an amino acid at residue position 430 according to the EU index of Kabat and includes glycine (G), serine (S), phenylalanine (F), or threonine (T); and X7 is a substitution of an amino acid at residue position 440 according to the EU index of Kabat and includes tryptophan (W). TIFF0007606501000006.tif19148

[0034] Fc mutations capable of promoting hexamerization of antibodies include one or more mutation(s) in the segment corresponding to amino acid residues at about positions 345-440 of the Fc region of an immunoglobulin. In one embodiment, Fc mutations capable of promoting hexamerization of antibodies include one or more mutation(s) in the 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 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-promoting mutations."

[0035] Fc mutations that can reduce 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 that can stabilize IgG4 include, but are not limited to, S228, L235, and / or R409 in IgG4. In one embodiment, Fc mutations that can stabilize IgG4 include S228P and L235E or R409K in IgG4 (see also Vidarsson et al., Front Immunol 2014;5-520 for a general discussion of the structure and effector function of IgG subclasses).

[0036] In one embodiment, a polypeptide according to the present 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, wherein 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 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 by 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 by 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 by 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 by tryptophan (W).

[0037] In this specification and claims, the numbering of residues in immunoglobulin heavy chains is that of the EU index as 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 as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0038] Thus, the present invention provides antibody variants having an immunoglobulin binding region and a full-length or partial Fc domain with one or more hexamerization-promoting 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.

[0039] 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 grafted onto the variable regions of antibodies to engineer an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0040] Table 1A: Antibody No. E1-3H7 variable region nucleic acid sequence TIFF0007606501000007.tif69152

[0041] (Table 1B) Amino acid sequence of the variable region of antibody number E1-3H7 TIFF0007606501000008.tif35152

[0042] Table 1C below shows the frameworks and CDRs of the heavy and light chain variable regions of anti-GITR antibodies based on SEQ ID NOs: 9-10.

[0043] Table 1C: Anti-GITR E1-3H7 amino acid sequence TIFF0007606501000009.tif82130

[0044] 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 the variable region of an antibody to engineer an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0045] Table 1D. Anti-CCR4 mAb2.3 variable region amino acid sequence (= affinity matured humanized mAb1567) TIFF0007606501000010.tif35152

[0046] Table 1E below shows the frameworks and CDRs of the heavy and light chain variable regions of anti-CCR4 antibodies based on SEQ ID NOs: 11-12.

[0047] Table 1E: Anti-CCR4 mAb 2.3 amino acid sequence TIFF0007606501000011.tif82130

[0048] Table 2A provides the nucleic acid sequences of the wild-type IgG1 heavy and light chain constant regions (Fc) (SEQ ID NOS: 13-17). 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.

[0049] Table 2A: Antibody No. E1-3H7 constant region nucleic acid sequence—wild-type IgG1 monomer (C L (Identical to the anti-CCR4 mAb2.3 construct described herein, except for: TIFF0007606501000012.tif136152

[0050] 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. L(カッパ) ) is represented by SEQ ID NO: 43: Contains the nucleic acid sequence of TIFF0007606501000013.tif31148.

[0051] In one embodiment, the light chain (C L(カッパ) ) is represented by SEQ ID NO: 44: Contains the amino acid sequence of TIFF0007606501000014.tif9147.

[0052] Table 2B provides the amino acid sequences of the wild-type 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.

[0053] (Table 2B) Antibody No. E1-3H7 constant region amino acid sequence - wild-type IgG1 monomer (C L (These are the same as the anti-CCR4 mAb2.3 constructs except for the residues in CH2 and CH3 in bold. For example, the bolded residues in CH2 and CH3 are wild-type residues that can be mutated to generate different IgG1 mutants (residues highlighted in yellow in Tables 3-5). TIFF0007606501000015.tif65152

[0054] Table 3A provides the nucleic acid sequences of the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NO: 23). The residues highlighted in yellow indicate the 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 an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0055] Table 3A: Antibody No. E1-3H7 constant region nucleic acid sequence - IgG1 LALA mutant monomer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000016.tif73152

[0056] Table 3B provides the amino acid sequences of the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NO: 24). 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.

[0057] (Table 3B) Antibody No. E1-3H7 constant region amino acid sequence - IgG1 LALA mutant monomer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000017.tif52152

[0058] Table 4A provides the nucleic acid sequences of the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NO: 25). The residues highlighted in yellow indicate the 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 an antibody of interest, such as an anti-GITR antibody or an anti-CCR4 antibody.

[0059] (Table 4A) Antibody number E1-3H7 constant region nucleic acid sequence - IgG1 wild type hexamer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000018.tif73152

[0060] Table 4B provides the amino acid sequences of the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NO: 26). 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.

[0061] (Table 4B) Antibody No. E1-3H7 constant region amino acid sequence - IgG1 wild type hexamer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000019.tif52152

[0062] Table 5A provides the nucleic acid sequences of the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOs: 27-28). Residues highlighted in yellow indicate the 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.

[0063] (Table 5A) Antibody number E1-3H7 constant region nucleic acid sequence - IgG1 LALA hexamer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000020.tif99152

[0064] Table 5B provides the amino acid sequences of the variant constant regions (Fc) of the IgG1 heavy and light chains (SEQ ID NOS: 29-30). Residues highlighted in yellow indicate the 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.

[0065] (Table 5B) Antibody No. E1-3H7 constant region amino acid sequence - IgG1 LALA hexamer (C L(same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000021.tif61152

[0066] Table 6A provides the nucleic acid sequences of the stabilized IgG4 heavy and light chain constant regions (Fc) (SEQ ID NOS: 31-35). 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.

[0067] Table 6A: Antibody No. E1-3H7 constant region nucleic acid sequence - sIgG4 monomer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000022.tif136152

[0068] Table 6B provides the amino acid sequences of the stabilized IgG4 heavy and light chain constant regions (Fc) (SEQ ID NOS: 36-40). Residues highlighted in yellow are mutations introduced to stabilize IgG4. Bold 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.

[0069] (Table 6B) Antibody No. E1-3H7 constant region amino acid sequence - sIgG4 monomer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000023.tif82152

[0070] Table 7A provides the nucleic acid sequences of variant constant regions (Fc) of stabilized IgG4 heavy and light chains (SEQ ID NOS: 31-33, 35, and 41). Residues highlighted in yellow are mutations introduced to stabilize IgG4. Bold 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.

[0071] Table 7A: Antibody No. E1-3H7 constant region nucleic acid sequence - sIgG4 hexamer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000024.tif149152

[0072] Table 7B provides the amino acid sequences of the stabilized IgG4 heavy and light chain constant regions (Fc) (SEQ ID NOS: 36-40). Residues highlighted in yellow are mutations introduced to stabilize IgG4. Bold 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.

[0073] (Table 7B) Antibody No. E1-3H7 constant region amino acid sequence - sIgG4 hexamer (C L (same as anti-CCR4 mAb2.3 construct except for TIFF0007606501000025.tif77152

[0074] Antibody variants with one or more hexamerization-promoting mutations and one or more effector function-reducing mutations 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. This is particularly true when cell surface receptor clustering is required for their biological function. Enhanced receptor clustering and / or effector cell signaling, or antibody variants of the present invention, can lead to practical clinical benefits, such as a reduction in the effective dose of a human monoclonal antibody to achieve a therapeutic effect and the use of antibodies with low affinity.

[0075] Accordingly, the present invention also provides methods of using the antibody variants of the invention in therapeutic methods for treating cancer, autoimmune diseases, inflammatory diseases, neurological diseases, cardiovascular diseases, infectious diseases, and for directing stem cell lineage pathways. The term "treating" can refer to the partial or complete alleviation, remission, improvement, amelioration, delay in onset, inhibition of progression, reduction in severity, and / or reduction in 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., prior to the onset of a discernible disease, disorder, and / or condition) and / or to subjects who exhibit only early signs of the disease, disorder, and / or condition, with the goal of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment involves enhancing intracellular signaling or inducing cellular receptor clustering.

[0076] The antibody variants of the invention can be specific for any target of interest, for example, targets of interest include tumor-associated surface antigens, e.g., 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, prostase-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 (IGF1)-I, IGF-II, IGF1 receptor, mesothelium, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC) A1) and fibroblast-associated proteins (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, TNFRSF17), or virus-specific surface antigens, such as HIV-specific antigens (e.g., HIV gp120); EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lassa Virus-specific antigens, influenza virus-specific antigens, and derivatives or variants of any of these surface markers.

[0077] Preferably, the antibody is specific to BCMA, CAIX, CCR4, PD-L1, PD-L2, PD1, glucocorticoid-induced tumor necrosis factor receptor (GITR), TIGIT, severe acute respiratory syndrome (SARS), influenza, flavivirus, or Middle East respiratory syndrome (MERS).

[0078] Exemplary antibodies useful for 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, WO 2015 / 143194, WO 2015 / 164865, WO 2013 / 166500, and WO 2014 / 144061; PCT / US2015 / 054202, PCT / US2015 / 054010, and 62 / 144,729, the contents of which are incorporated by reference herein in their entireties.

[0079] 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 nucleotides disclosed in Table 1A, 2A, 3A, 4A, 5A, 6A, 7A, SEQ ID NO:43, or a combination thereof. In another embodiment, the nucleic acid has a 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 a nucleic acid sequence disclosed in Table 1A, 2A, 3A, 4A, 5A, 6A, 7A, SEQ ID NO:43, or a combination thereof. It is understood that the present invention encompasses portions and variants of the sequences specifically disclosed herein. For example, in embodiments, codon-optimized sequence forms can be used.

[0080] 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.

[0081] Embodiments of the present invention include cells expressing the antibody variants of the present invention (i.e., CART). The cells can be of any type, including immune cells capable of expressing the antibody variants for cancer therapy, or 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, which are 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 a vector and / or expressing a heterologous gene encoded by the vector. A host cell can be, and has been, used as a recipient for a vector. A host cell may be "transfected" or "transformed," which refers to the process by which foreign nucleic acid is transferred or introduced into a host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" or host cell can refer to a cell into which a foreign nucleic acid sequence, such as a vector, has been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain recombinantly introduced nucleic acids. In embodiments of the invention, the host cells are T cells, including cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytotoxic T cells, CD8+ T cells, or killer T cells); CD4+ T cells, NK cells, and NKT cells are also encompassed by the invention.

[0082] 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, maintaining them, and allowing the replication of vectors. Techniques and conditions that will allow large-scale production of vectors, and production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides, are also understood and known.

[0083] The cells can be autologous, syngeneic, allogeneic, and in some cases xenogeneic.

[0084] In many situations, it may be desirable to be able to kill modified CTLs when one wishes to terminate treatment, when cells become neoplastic, in studies where the absence of the cells after their presence is of interest, or other times. To this end, expression of a specific gene product, e.g., an inducible suicide gene, capable of killing the modified cells under controlled conditions may be provided.

[0085] The present invention further includes CARTs modified to secrete one or more polypeptides. The polypeptides can be, for example, antibodies or cytokines. Preferably, the antibodies are specific for CAIX, GITR, PD-L1, PD-L2, PD-1, CCR4, or TIGIT.

[0086] Armed CARTs have the advantage of simultaneously secreting the polypeptide at the target site, e.g., the tumor site.

[0087] 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 placed between the intracellular signaling domain and the polypeptide of interest. Those skilled in the art can understand that two or more polypeptides can be expressed by using multiple IRES sequences in series.

[0088] Antibodies, including engineered polypeptides, can be purified, for example, from cells or from recombinant systems, using a variety of well-known techniques for protein isolation and purification. See, e.g., 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).

[0089] The antibodies, fragments, and antibody derivatives described herein, such as 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).

[0090] Pharmaceutically acceptable carriers may include any solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically 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 materials, 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.

[0091] 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.

[0092] 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 dispersions. 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 should be 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, a pharmaceutically acceptable polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), 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 the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it will be useful to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, and the like in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0093] Sterile injectable solutions can be prepared by mixing the required amount of antibody in an appropriate solvent containing one or a combination of ingredients enumerated herein, followed by filtered sterilization, as required. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated herein.

[0094] For 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 mixed with excipients and used in the form of tablets, troches, or capsules.

[0095] 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: a binder such as microcrystalline cellulose, tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0096] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is generally formulated into ointments, salves, gels, or creams as known in the art.

[0097] The antibody or fragment (or derivative thereof) can also be formulated as a composition suitable for topical administration to the skin or mucosa (e.g., rectal or vaginal administration). Such compositions can take the form of liquids, ointments, creams, gels, and pastes. The antibody or fragment (or derivative thereof) can also be formulated as a composition suitable for intranasal administration. Standard formulation techniques can be used in preparing suitable compositions.

[0098] The antibodies and / or compositions of the invention can be administered once to a subject (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. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0099] The therapeutically effective dose range can depend on the antibody or fragment (or derivative thereof) and the nature of the formulation and the route of administration. The optimal dose can be determined by one skilled in the art without undue experimentation and can vary depending on known factors such as the pharmacodynamic properties of the active ingredient and its method and route of administration; the timing of administration of the active ingredient; the recipient's age, sex, health condition, and weight; the nature and severity of symptoms; the type of concurrent treatment, the frequency of treatment, and the desired effect; and excretion rate. For example, a therapeutically effective dose of antibody in the range of about 0.1 to 1000 mg / kg body weight can be used. Preferably, an antibody in a dose range of about 1 to 50 mg / kg can be used.

[0100] The antibodies or nucleic acids of the present invention may also be provided in a kit. In one embodiment, the kit includes (a) a container containing a composition comprising the antibody, and, optionally, (b) informational material. The informational material may be descriptive, explanatory, marketing, or other material relating to the methods described herein and / or the use of the agent to achieve a therapeutic effect. 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.

[0101] The informational material of the kit is not limited in form. In one embodiment, the informational material can include information regarding 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 treating a subject by administering the polypeptide or a nucleic acid encoding it, e.g., in an appropriate dose, dosage form, or method of administration (e.g., a dose, dosage form, or method of administration described herein). The information can be provided in a variety of formats, including printed text, computer-readable material, video recording, or audio recording, or information providing a link or address to substantive material.

[0102] In addition to the antibody or the nucleic acid encoding it, the composition in the kit can contain other components, such as a solvent or buffer, a stabilizer, or a preservative. The antibody or nucleic acid can be provided in any form, for example, in a liquid, dry, or lyophilized form, preferably substantially pure and / or sterile. When provided in a solution, the solution is preferably an aqueous solution. When provided in a dry form, reconstitution is generally by adding a suitable solvent. A solvent, such as sterile water or a buffer, can be provided in the kit as needed.

[0103] The kit can include one or more containers for the antibody, nucleic acid, or composition comprising same. In some embodiments, the kit contains different 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 different components of the kit are contained in a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe that has informational material in the form of a label attached to it. In some embodiments, the kit contains 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 contain a combination unit dose, e.g., a unit containing both the antibody and the second agent, e.g., in a desired ratio. For example, the kit can include multiple syringes, ampoules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kit can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight. The kit optionally includes a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device may be provided pre-filled or may be empty, but suitable for filling.

[0104] 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 term "comprising" in the claims and / or specification may mean "one," but it is also consistent with "one or more," "at least one," and "one or more."

[0105] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, they are understood to be followed by the phrase "without limitation." Similarly, "example," "exemplary," etc. are understood to be non-limiting.

[0106] The term "substantially" permits deviations from the descriptor that do not adversely affect the intended purpose. Even if the word "substantially" is not explicitly recited, the descriptive term will be understood to be modified by the term "substantially."

[0107] The terms "comprising," "including," "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, is interpreted as an open term meaning "at least the following," and is also interpreted as not excluding additional features, limitations, embodiments, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the term "a" or "an" is used, it is understood to mean "one or more," unless such interpretation is not meaningful in the context.

[0108] As used herein, the term "about" is used herein to mean approximately, roughly, roughly, or in the region of. 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. In general, the term "about" is used herein to modify the stated numerical value up or down by a variance (higher or lower) of 20%.

[0109] In this specification and claims, the numbering of residues in immunoglobulin heavy chains is that of the EU index as 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 as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0110] "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 exemplary and representative embodiments for measuring binding affinity are described below. See, e.g., 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 by reference herein in its entirety).

[0111] An "affinity matured" antibody may 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 may result in an improvement in the affinity of the antibody for antigen.

[0112] For example, an "amino acid modification" can be a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include amino acid substitution, insertion, and / or deletion. Preferred amino acid modifications herein are substitutions. An "amino acid modification" at a specific position, for example, in the Fc region, can refer to the substitution or deletion of a specific residue, or the insertion of at least one amino acid residue adjacent to the specific residue. An insertion "adjacent to" a specific residue can be, for example, an insertion within 1 to 2 residues of the specific residue. The insertion can be on the N-terminal or C-terminal side of the specific residue.

[0113] 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 "replacement" amino acid residue. The replacement residue may be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) and is 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 herein as an amino acid substitution. A "non-naturally occurring amino acid residue" may be, for example, a residue other than the naturally occurring amino acid residues listed above that is capable of covalently bonding to an 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.

[0114] An "amino acid insertion" can refer to the incorporation of at least one amino acid into a predetermined amino acid sequence. Insertions can typically consist of the insertion of one or two amino acid residues, although the invention as 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 natural or non-natural, as described above.

[0115] An "amino acid deletion" can refer to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0116] The term "antibody" herein is used in the broadest sense and encompasses a variety of 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 a wild-type human IgG Fc region, 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 embodiments of the present invention, for example, to enhance cell signaling and / or induce receptor clustering.

[0117] 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 fragments or derivatives thereof, can also be used in the methods of the present invention.

[0118] The term "antibody variant" as used herein refers to a variant of a wild-type antibody, characterized, for example, by the amino acid sequence changes made in the antibody variant relative to the wild-type antibody (e.g., specific amino acid residues in the wild-type antibody are introduced by mutation). For example, the antibody variant may contain amino acid substitutions in the Fc region that enhance cell signaling and / or induce receptor clustering. Such substitutions include those described herein, such as E345K, E430G, L234A, and L235A in the Fc of human IgG; or E345K, E430G, S228P, and R409K. Residues are numbered according to the EU index of Kabat.

[0119] 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 achieved, for example, by binding of the Fc effector domain(s) to Fc receptors on immune cells that have phagocytic or lytic functions, or by binding of the Fc effector domain(s) to components of the complement system. Exemplary effector functions are ADCC, ADCP, and CDC.

[0120] An "antibody fragment" may be a molecule other than an intact antibody that contains a portion of the 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.

[0121] 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 competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0122] "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 cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, 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.

[0123] "Antibody-dependent cellular phagocytosis" and "ADCP" are, for example, the processes by which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

[0124] 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 include the portion of that polypeptide chain (e.g., that chain) that is involved in binding an Fc region. One useful binding domain is the extracellular domain of the FcR alpha chain.

[0125] For example, "binding" to an Fc receptor can be the binding of an antibody to an Fc receptor in a BIAcore™ assay, for example (Pharmacia Biosensor AB, Uppsala, Sweden).

[0126] In a BIAcore™ assay, Fc receptors are bound to a surface and binding of variants, eg, mutated antibody variants, is measured by surface plasmon resonance (SPR). 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 microbiology11.3(2003):124-133;McDonnell, James M. “Surface plasmon resonance:towards an understanding of the mechanisms of biological molecular recognition.”Current opinion in chemical biology5.5(2001):572-577; and David G.Myszka.“BIACORE J:a new platform for routine biomolecular interaction analysis.”Journal of Molecular Recognition 14.4 (2001):223-228, each of which is incorporated herein by reference in its entirety. The affinity of binding is determined by the term k a (binding rate constant of antibody from the antibody / Fc receptor complex), k d (dissociation constant), and K DAlternatively, for example, the binding signal of an SPR sensogram can be directly compared to the response signal of a reference in terms of resonance signal height and dissociation behavior.

[0127] The "CH2 domain" (also called the "Cγ2" domain) of the human IgG Fc region typically extends 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 is thought that these carbohydrates may provide a substitute for domain-domain pairing and help stabilize the CH2 domain (Burton, Molec. Immunol. 22 (1985) 161-206). In one embodiment, Figures 8, 9, and 11 show the CH domains of IgG1, IgG2, and IgG4, respectively.

[0128] 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, and 11 show the CH domains of IgG1, IgG2, and IgG4, respectively.

[0129] "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, hepatocellular carcinoma, 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.

[0130] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also 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 a different designation is intended, it will be clear from the context.

[0131] The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as 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, pp. 259-294 (each of which is incorporated herein by reference in its entirety). The heavy-chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

[0132] For example, as used herein, "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include 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 and fragments thereof, e.g., nucleases; antibiotics; toxins, e.g., 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 described herein.

[0133] "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 activates a series of enzymatic reactions that ultimately form holes in the target cell membrane. Antigen-antibody complexes (e.g., on antibody-coated target cells) bind to and activate complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation can also cause the deposition of complement components on the target cell surface, which promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0134] A "disease" 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 diseases or illnesses, including pathological conditions that predispose a mammal to the disease in question. In one embodiment, the disease is cancer.

[0135] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies, for example, depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0136] As used herein, a "reduced effector function" can refer to a reduction in a particular effector function, e.g., ADCC or CDC, of ​​at least 20% compared to a control (e.g., a polypeptide having a wild-type Fc region), and a "significantly reduced effector function" as used herein can refer to a reduction in a particular effector function, e.g., ADCC or CDC, of ​​at least 50% compared to a control.

[0137] 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 a desired therapeutic or prophylactic result.

[0138] "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, for example, comprising at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known 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).

[0139] 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, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, compared to a native-sequence Fc region or the Fc region of a parent polypeptide. In one embodiment, the 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. The variant Fc region herein can have at least about 80% homology, at least about 90% homology, at least about 95% homology, at least about 96% homology, at least about 97% homology, at least about 98% homology, or at least about 99% homology to a native-sequence Fc region and / or the Fc region of a parent polypeptide.

[0140] As used herein, "Fc variant" refers to a polypeptide containing modifications in the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that comprise them. Thus, for example, P329G is an Fc variant in which proline is substituted with glycine at position 329 compared 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 described herein, the numbering is according to the EU index. The EU index, or 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. Patent 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 fully incorporated by reference).

[0141] "Fc region-containing polypeptide" refers to a polypeptide, such as an antibody or immunoadhesin (see discussion herein), that comprises an Fc region.

[0142] For example, "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native-sequence human FcR. Yet 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 (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), 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 inhibitory motif (ITIM) in its cytoplasmic domain (see review by 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 herein by the term "FcR." This term also includes the neonatal receptor, FcRn, 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).

[0143] For example, an "IgG Fc ligand" can be a molecule, e.g., a polypeptide, derived 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 (FcRH), 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 may 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, derived from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0144] 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, fully incorporated by reference), as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγRs may 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 any unidentified mouse FcγR or FcγR isoform or allotype.

[0145] "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 may be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. 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 noted herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain with beta-2-microglobulin.

[0146] For example, a "wild-type or parent polypeptide" can be an unmodified polypeptide that is subsequently modified to generate a variant. A wild-type polypeptide can be a naturally occurring polypeptide, or a mutant 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 generate a variant, and a "wild-type antibody" refers to an unmodified antibody that is modified to generate a variant antibody. Note that "wild-type antibody" includes known, commercially available, recombinantly produced antibodies as described herein.

[0147] A "crystallizable fragment (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.

[0148] "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.

[0149] "Full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain that includes an Fc region as defined herein.

[0150] A "functional Fc region" possesses the "effector functions" 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 receptors; BCRs); and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using various assays such as those disclosed herein.

[0151] 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 S--S bonds in the same positions.

[0152] The "lower hinge region" of an Fc region corresponds, for example, to the stretch from the residues adjacent to the C-terminus to the hinge region, ie, residues 233 to 239 of the Fc region.

[0153] "Homology" refers to the percentage of residues in amino acid sequence variants that are identical, e.g., after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for alignment are well known in the art. One such computer program is "Align2," written by Genentech, Inc., which was filed with user documentation in the U.S. Copyright Office, Washington, DC 20559, on December 10, 1991.

[0154] 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," and 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.

[0155] A "human antibody" is one having 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 coding sequences. Human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0156] "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 natural sources, such as blood or PBMCs, as described herein.

[0157] 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 chains are derived from a particular source or species, while the remainder of the heavy and / or light chains are derived from a different source or species.

[0158] As used herein, a "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"). Typically, 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 from the "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 called 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.

[0159] An "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., other than the form or setting in which it may be found in nature) unless otherwise specified. However, immune complexes can be administered to a mammal, for example, to assess clearance of the immune complex in the mammal.

[0160] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0161] An "individual" or "subject" can be a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and 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 human.

[0162] The term "subject" or "patient" can refer to any organism to which an embodiment of the present invention can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects to which the compounds of the present disclosure can be administered are mammals, particularly primates, especially humans. For veterinary applications, various subjects are suitable, such as livestock such as cows, 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, various mammals are suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs. The term "living subject" refers to the above-mentioned subjects or another living organism. The term "living subject" refers to a whole subject or organism, and not just a portion (e.g., liver or other organ) removed from a living subject. [Example]

[0163] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate representative methods 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 other methods may be utilized to achieve similar results.

[0164] Example 1 - ADCC Assay ADCC assays were performed using Promega's reporter system. Pools of CHO-GITR cells were sorted to obtain cell populations with >99% purity of GITR cells. Cells were plated 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 plates were incubated for 6 hours at 37°C and 5% CO2. After incubation, Bio-Glo luciferase assay reagent was added, and luminescent signals were detected using a BMG PolarStart multilabel plate reader. The data show that, as expected, only the IgG1 wild-type monomer and hexameric constructs exhibited significant ADCC activity. It is also interesting to note that hexamerization appears to reduce the magnitude of ADCC in wild-type IgG1. The negative control IgG did not exhibit specific ADCC activity.

[0165] Other embodiments While the present invention has been described in conjunction with a detailed description thereof, the foregoing description is intended to be illustrative and 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 appended claims.

[0166] 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 described herein which are considered to be within the scope of this invention and covered by the appended claims.

[0167] Sequence information SEQUENCE LISTING <110> DANA-FARBER CANCER INSTITUTE, INC. <120> COMPOSITIONS AND METHODS FOR AUGMENTING ANTIBODY MEDIATED RECEPTOR SIGNALING <150> US 62 / 455,245 <151> 2017-02-06 <160> 77 <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> (228)..(228) <223> Lys, Gln, Arg, or Tyr <220> <221> MOD_RES <222> (313)..(313) <223> Gly, Ser, Phe, or Thr <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 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 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> (224)..(224) <223> Lys, Gln, Arg, or Tyr <220> <221> MOD_RES <222> (309)..(309) <223> Gly, Ser, Phe, or Thr <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 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 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> (225)..(225) <223> Lys, Gln, Arg, or Tyr <220> <221> MOD_RES <222> (310)..(310) <223> Gly, Ser, Phe, or Thr <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 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 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 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290,295,300 How Will Met His Xaa Ala Leu His Asn His Tr 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 tgagctggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtgta gtggtgtag 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 caacatcgga agtaatcctg taaactggta cctccaccgc 120 ccaggaacgg cccccaaact cctcatctat aatagcaatc agtggccctc aggggtccct 180 gaccgatttt ctggctccag gtctggcacc tcagcctccc tggccatcag tgggctccag 240 tctgaggatg aggctgatta ttactgtgca gcatgggatg 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 Ala 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 Has Thr With Asn Cys Lys Ser Ser Gln Ser With Leu Tyr Ser 20 25 30 Serving Gln Asn Lys Tyr Asn Only Trp Tyr 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> Homo sapiens <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 cagcaacacc aaggtggaca agaaa 285 <210> 14 <211> 48 <212> DNA <213> Homo sapiens <400> 14 gcagagccca aatcttgtga caaaactcac acatgcccac cgtgccca 48 <210> 15 <211> 330 <212> DNA <213> Homo sapiens <400> 15 gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120 180 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> Homo sapiens <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> Homo sapiens <400> 17 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> 18 <211> 97 <212> PRT <213> Homo sapiens <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 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 <210> 19 <211> 16 <212> PRT <213> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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> Homo sapiens <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 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> 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 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> 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 acagcctca 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 cccctcaag gcgggagtgg agaccaccac accctccaaa 180 caaagcaca acagtacgc ggccagcagc tatctgagcc tgacgcctga gcagtggaag 240 tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300 gccctacag 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 Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro 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 acctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttga 324 <210> 44 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 44 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> 45 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 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> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 46 Gly Phe Thr Phe Ser Ser His Ala 1 5 <210> 47 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Ala <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 49 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 49 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> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Ala Lys Ile Gly Thr Ala Asp Ala Phe Asp Ile 1 5 10 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 51 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 52 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 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> 53 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Val Pro Asn Ile Gly Ser Asn Pro 1 5 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Val Asn Trp Tyr Leu His Arg Pro Gly Thr Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 55 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 55 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> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Ala Ala Trp Asp Asp Ser Leu Asp Gly Leu Val 1 5 10 <210> 57 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 58 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 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> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Gly Tyr Thr Phe Ala Ser Ala Trp 1 5 <210> 60 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Met His Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Trp <210> 61 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 61 Ile Asn Pro Gly Asn Val Asn Thr 1 5 <210> 62 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 62 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> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Ser Thr Tyr Tyr Arg Pro Leu Asp Tyr 1 5 <210> 64 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 65 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 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> 66 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Gln Ser Ile Leu Tyr Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 67 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 67 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 68 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Trp Ala Ser Thr Arg Glu 1 5 <210> 69 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 69 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> 70 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 His Gln Tyr Met Ser Ser Tyr Thr 1 5 <210> 71 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 71 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 72 <211> 1005 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(993) <400> 72 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> 73 <211> 331 <212> PRT <213> Homo sapiens <400> 73 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> 74 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> CDS <222> (1)..(993) <400> 74 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> 75 <211> 331 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 75 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> 76 <211> 984 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> CDS <222> (1)..(981) <400> 76 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> 77 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 77 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

Claims

1. 1. An engineered polypeptide comprising an IgG1 Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions at residue positions 234, 235, 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 replaced by alanine (A); the amino acid at residue position 235 according to the EU index of Kabat is replaced by alanine (A); a substitution of glutamic acid (E) at residue position 345 according to the EU index of Kabat by lysine (K), glutamine (Q), arginine (R), or tyrosine (Y); a substitution of glutamic acid (E) at residue position 430 according to the EU index of Kabat by glycine (G), serine (S), phenylalanine (F), or threonine (T); the engineered polypeptide comprises a recombinant anti-GITR antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences GFTFSSHA, ISGSGGST, and AKIGTADAFDI, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences VPNIGSNP, NSN, and AAWDDSLDGLV, respectively; The engineered polypeptide.

2. 2. The polypeptide of claim 1, wherein the amino acid substitutions include L234A, L235A, E345K, and E430G, and the amino acid residues are numbered according to the EU index of Kabat.

3. 3. The polypeptide of claim 1 or 2, which exhibits reduced affinity for one or more human Fc receptors compared to a polypeptide comprising a wild-type IgG Fc region.

4. The polypeptide of claim 3, further exhibiting increased receptor clustering compared to a polypeptide comprising a wild-type IgG Fc region.

5. The polypeptide of any one of claims 1 to 4, wherein the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

6. The polypeptide of any one of claims 1 to 5, conjugated to a drug, a toxin, a radiolabel, or a combination thereof.

7. 7. The polypeptide of any one of claims 1 to 6, comprising a variable region amino acid sequence disclosed in Table 1B and a variant Fc region amino acid sequence disclosed in Table 5B (SEQ ID NOs: 18, 19, 22, 29, and 30).

8. A pharmaceutical composition for treating a tumor in a subject, comprising a polypeptide according to any one of claims 1 to 7.

9. A pharmaceutical composition for enhancing intracellular signal transduction in a cell, comprising a polypeptide described in any one of claims 1 to 7.

10. A pharmaceutical composition for inducing cellular receptor clustering, comprising a polypeptide described in any one of claims 1 to 7.

11. The pharmaceutical composition according to claim 8 , wherein the tumor is a solid tumor or a liquid tumor.

Images