CD70-specific antibody and use of the same

Abstract

To provide an antibody (e.g., monospecific or bispecific) which has improved effectiveness and safety profile, is suitable for used in a human patient, and treats cancer in which CD70 is expressed which is particularly mRCC.SOLUTION: The present invention provides an antibody specifically biding to CD70 (Cluster of Differentiation 70). The present invention provides a bispecific antibody specifically binding to an antigen (e.g., CD3) different from CD70. The present invention relates to nucleic acid encoding antibody, and a method for acquiring the antibody (monospecific and bispecific). The present invention further relates to a treatment method regarding use of the antibodies for treating a CD70-mediated pathological condition including cancer.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 641,873, filed Mar. 12, 2018, and U.S. Provisional Patent Application No. 62 / 625,019, filed Feb. 1, 2018, each of which is hereby incorporated by reference in its entirety.

[0002] Reference to the Sequence Listing This application was electronically filed via EFS-Web and includes a Sequence Listing that was electronically submitted in.txt format. The.txt file contains a Sequence Listing entitled “ALGN-015_02WO_SL.txt” created on Jan. 3, 2019, having a size of 234,861 bytes. The Sequence Listing contained in this.txt file is part of this specification and is hereby incorporated by reference in its entirety.

[0003] The present invention relates to antibodies, such as full-length antibodies or antigen-binding fragments thereof, that specifically bind to Cluster of Differentiation 70 (CD70). Further, the present invention relates to hetero-multimeric antibodies (e.g., bispecific antibodies) that include a CD70 antibody on one arm. Compositions containing the CD70 antibodies, methods of making and purifying the antibodies, and their use in diagnosis and treatment are also provided.

Background Art

[0004] Renal cell carcinoma (RCC) is a cancer that originates from the renal cortex and accounts for approximately 90% of kidney cancers. Histologically, RCC can be classified into several subtypes. Among them, clear cell renal cell carcinoma (ccRCC) is the most common and is responsible for the most deaths. Every year, more than 320,000 cases of RCC are reported worldwide, and approximately 140,000 people die. The incidence of RCC has been steadily increasing over the past decade and accounts for 2-3% of all adult malignancies. Patients with early-stage localized tumors can choose surgical resection. However, if the localized disease metastasizes hematogenously early, metastases occur. Sites of early metastasis include the lungs, lymph nodes, liver, bone, and brain, and less commonly, the adrenal gland and the contralateral kidney. Patients with progressive disease have a high mortality rate, with a median 5-year survival rate of 53% for stage III disease and only 8% for metastatic disease. Current first-line treatment options for progressive disease include small molecule tyrosine kinase inhibitors (TKIs) such as sunitinib and pazopanib that target vascular endothelial growth factor (VEGF) receptors, monoclonal antibodies such as bevacizumab that target VEGF, temsirolimus, an inhibitor of mammalian target of Rapamycin (mTOR), and high-dose IL-2. These VEGF-targeted therapies have improved overall survival, but disease recurrence occurs due to long-term drug resistance, and treatment options for progressive disease remain unmet needs (see, e.g., Zarrabi, K. et al., Journal of Hematology and Oncology, 10:38 (2017)).

[0005] Cluster of Differentiation 70 (CD70, also known as CD27LG or TNFSF7) is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for CD27, a TNF superfamily receptor. Transient interaction between CD27 and CD70 provides co-stimulation of T cells, which is complementary to stimulation by CD28. CD70 is expressed in blood cancers such as non-Hodgkin lymphoma and Hodgkin disease, as well as solid tumors such as glioblastoma and renal cell carcinoma, and its expression on ccRCC is nearly uniform (see, e.g., Grewal I., et al., Expert Opinion on Therapeutic Targets, 12(3):341-351 (2008)).

[0006] CD70 bispecific antibodies in the form of a bispecific approach that associates with T cells have been developed in recent years. However, many bispecific formats have limitations such as low molecular weight and short half-life, which require continuous infusion. Therefore, there remains a need for antibodies (e.g., monospecific or bispecific) that have improved efficacy and safety profiles and are suitable for use in human patients to treat cancers in which CD70 is expressed, particularly mRCC. SUMMARY OF THE INVENTION

[0007] The invention disclosed herein is directed to antibodies (e.g., monospecific or bispecific antibodies) that specifically bind to Cluster of Differentiation 70 (CD70). In some embodiments, the CD70 antibodies described herein in full-length bispecific format have a long half-life, minimized Fc interactions, and minimized non-specific cytokine release through interaction with immune cells in vivo.

[0008] Thus, in one aspect, the present invention provides an isolated antibody that specifically binds to CD70, the antibody comprising, (a)(i) a VH complementarity determining region 1 (CDR1) comprising the sequence set forth in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376, 380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405, 406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460;(ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456, 461, or 462; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, or 463, the heavy chain variable (VH) region; and / or (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527;(ii) a VL CDR2 comprising the sequences shown in 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequences shown in SEQ ID NOs: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526, or 529, comprising a variable light (VL) region.;

[0009] In another aspect, there is provided an isolated antibody that specifically binds to CD70, the antibody comprising a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, or 331, and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330. In some embodiments, the VH region described herein includes variants having one or more conservative amino acid substitutions in residues other than within the CDRs, and / or the VL region described herein includes variants having one or more amino acid substitutions in amino acids other than within the CDRs. For example, in some embodiments, the VH region or VL region may include the above-described amino acid sequence, or variants thereof having 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer conservative substitutions in residues other than within the CDRs.

[0010] In some embodiments, there is provided an isolated antibody that specifically binds to CD70, the antibody comprising a VH region comprising the sequence shown in SEQ ID NO: 18, and / or a VL region comprising the sequence shown in SEQ ID NO: 17.

[0011] In some embodiments, there is provided an antibody that specifically binds to CD70 and competes with the isolated antibodies presented herein that specifically bind to CD70.

[0012] In another aspect, a bispecific antibody is provided, where in this case the bispecific antibody is a full-length antibody and includes a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen (e.g., CD70), and a second antibody variable domain of the bispecific antibody that can recruit the activity of human immune effector cells by specifically binding to an effector antigen (e.g., Cluster of differentiation 3 (CD3)) that is localized on human immune effector cells. In some aspects, the first antibody variable domain is a heavy chain variable (VH) region that includes VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, or 331, and / or VL CDR1, VL CDR2, and VL of the VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, .....It includes a light chain variable (VL) region containing CDR3. In some embodiments, the first antibody variable domain is (a)(i) the VH complementarity determining region 1 (CDR1) comprising the sequence shown in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376, 380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405, 406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460. Complementarity determining region(i); (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456, 461, or 462; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, or 463, a heavy chain variable (VH) region; and / or (b) (i) a VL comprising the sequence shown in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527a light chain variable (VL) region comprising a VL CDR1; (ii) a VL CDR2 comprising the sequence shown in 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526, or 529.

[0013] In some embodiments, the second antibody variable domain comprises a VH region and / or a VL region specific for CD3. For example, the second antibody variable domain comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 266, and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 265. In some embodiments, the second antibody variable domain comprises (a) (i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 267, 268 or 269; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 270 or 271; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 272, and / or (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 273; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 274; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 275.

[0014] In some embodiments, the antibodies described herein include a constant region. In some embodiments, the antibodies described herein are antibodies of the subclasses human IgG1, IgG2 or IgG2Δa, IgG3, or IgG4. In some embodiments, the antibodies described herein include a glycosylated constant region. In some embodiments, the antibodies described herein include a constant region with reduced binding affinity for one or more human Fc gamma receptors.

[0015] In some embodiments, both the first and second antibody variable domains of the bispecific antibody contain amino acid modifications at positions 223, 225, and 228 (e.g., (C223E or C223R), (E225R), and (P228E or P228R)) in the hinge region of human IgG2 (SEQ ID NO: 279), as well as at position 409 or 368 in the CH3 region (e.g., K409R or L368E (EU numbering scheme)).

[0016] In some embodiments, both the first and second antibody variable domains of the bispecific antibody contain an amino acid modification (e.g., D265A) at position 265 of human IgG2.

[0017] In some embodiments, both the first and second antibody variable domains of the bispecific antibody contain an amino acid modification at one or more of positions 265 (e.g., D265A), 330 (e.g., A330S), and 331 (e.g., P331S) of human IgG2. In some embodiments, both the first and second antibody variable domains of the bispecific antibody contain an amino acid modification at each of positions 265 (e.g., D265A), 330 (e.g., A330S), and 331 (e.g., P331S) of human IgG2.

[0018] In other embodiments, the present invention provides a pharmaceutical composition containing any of the antibodies described herein.

[0019] The present invention further provides a cell line recombinantly producing any of the antibodies described herein.

[0020] The present invention further provides a nucleic acid encoding any of the antibodies described herein. The present invention further provides a nucleic acid encoding the heavy chain variable region and / or the light chain variable region of any of the antibodies described herein.

[0021] The present invention further provides a host cell containing the nucleic acid or vector provided herein. Further provided is a method for producing an antibody provided herein (e.g., monospecific or bispecific), the method comprising culturing the host cell provided herein under conditions that result in the production of the antibody, and isolating the antibody from the host cell or culture.

[0022] The present invention further provides a kit comprising an effective amount of any of the antibodies or antibody conjugates described herein.

[0023] Further provided is an antibody or bispecific antibody provided herein for use as a medicament.

[0024] The present invention further provides a method for treating a subject in need thereof, the method comprising providing an isolated antibody or bispecific antibody described herein, and administering the antibody to the subject.

[0025] Further provided is a method for treating a condition associated with malignant cells expressing CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an antibody described herein. In some embodiments, the condition is cancer. In some embodiments, the cancer is a CD70-related cancer (e.g., any cancer associated with CD70 expression) selected from the group consisting of renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin lymphoma (NHL), Hodgkin disease (HD), Waldenström's macroglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

[0026] In another aspect, the present invention provides a method for inhibiting tumor growth or progression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an isolated antibody or bispecific antibody described herein.

[0027] In another aspect, the present invention provides a method for inhibiting metastasis of malignant cells that express CD70 in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an isolated antibody or bispecific antibody described herein.

[0028] In another aspect, the present invention provides a method for inducing tumor regression in a subject having malignant cells that express CD70, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an isolated antibody or bispecific antibody described herein. **DETAILED DESCRIPTION OF THE INVENTION**

[0029] The present invention disclosed herein provides antibodies (e.g., monospecific or bispecific) that specifically bind to CD70 (e.g., human CD70). The present invention further provides polynucleotides encoding these antibodies, compositions comprising these antibodies, and methods of making and using these antibodies. The present invention further provides methods of treating conditions associated with CD70-mediated pathologies in a subject, such as cancer. In particular, the inventors of the present invention have found that the full-length bispecific form of the CD70 antibodies described herein have a long half-life, minimized Fc interactions, and minimized non-specific cytokine release mediated by interaction with immune cells in vivo.

[0030] General Techniques The practice of the present invention, unless otherwise indicated, employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, virology, methods and procedures for the production and manipulation of monoclonal antibodies, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993 - 1998) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995), etc. are fully described in the literature.

[0031] Definition An "antibody" is an immunoglobulin molecule capable of specific binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. located in the variable region of the immunoglobulin molecule via at least one antigen recognition site. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab’, F(ab’)2, Fv), single-chain (ScFv) antibodies and domain antibodies (including shark antibodies and camelid antibodies), as well as fusion proteins containing antibodies, and any other modified structure of an immunoglobulin molecule comprising an antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and the antibody does not have to be of any particular class. Immunoglobulins can be assigned to different classes according to the amino acid sequence of the constant region of their heavy chains. There are five main classes of immunoglobulins. IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional structures of various classes of immunoglobulins are known.

[0032] As used herein, the terms "antigen-binding fragment" or "antigen-binding portion" of an antibody refer to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., CD70). The antigen-binding function of an antibody can be performed by fragments of the intact antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody include Fab, Fab’, F(ab’)2, Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of the VL and VH domains of a single arm of the antibody, single-domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989), and isolated complementarity-determining regions (CDRs).

[0033] An antibody or polypeptide that "binds preferentially to" or "binds specifically to" (used interchangeably herein) a target (e.g., CD70 protein) is a term well understood in the art, and methods for determining such specific or preferential binding are also known in the art. A molecule exhibits "specific binding" or "preferential binding" when it reacts or associates with a particular cell or substance more frequently, earlier, for a longer period and / or with a higher affinity than with another cell or substance. An antibody "binds specifically to" or "binds preferentially to" a target when it binds with a higher affinity, avidity, earlier, and / or for a longer period than to other substances. For example, an antibody that binds specifically or preferentially to a CD70 epitope is an antibody that binds to that epitope with a higher affinity, avidity, earlier, and / or for a longer period than it binds to other CD70 epitopes or non-CD70 epitopes. By reading this definition, it will be understood that, for example, an antibody (or portion or epitope) that binds specifically or preferentially to a first target may or may not bind specifically or preferentially to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although exclusive binding may be included). Although not necessarily, generally, reference to binding means preferential binding.

[0034] The "variable region" of an antibody refers to the variable region of the antibody light chain, or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two methods for determining CDRs: (1) a method based on sequence diversity among different species (i.e., Sequences of Proteins of Immunological Interest by Kabat et al., (5th ed., 1991, National Institutes of Health, Bethesda MD)), and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, CDR may refer to a CDR defined by either method, or a CDR defined by a combination of both methods.

[0035] The "CDR" of a variable domain is an amino acid residue within a variable region identified according to the definitions of Kabat, Chothia, the accumulation of both Kabat and Chothia, the AbM definition, the contact definition, and / or the conformation definition, or any method of CDR determination known in the art. Antibody CDRs may be defined as the hypervariable regions first defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs may be defined as the structural loop structures first reported by Chothia et al. See, for example, Chothia et al., Nature 342:877-883, 1989. Another method for CDR identification is the "AbM definition". This method takes an intermediate position between the Kabat and Chothia methods and is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®). Alternatively, the "contact definition" of CDRs based on observed antigen contacts may also be mentioned, and this method is described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. Another method is referred to herein as the "conformation definition" of CDRs, and the positions of the CDRs may be defined as residues that contribute enthalpically to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008. Still other definitions of CDR boundaries may not strictly follow one of the methods described above but may overlap with at least a part of the Kabat CDRs. Nevertheless, in view of predictions or experimental results that certain residues or groups of residues, or even entire CDRs, do not have much effect on antigen binding, they may be too short or too long. As used herein, CDR may refer to CDRs defined by any method known in the art, including combinations of methods.The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing multiple CDRs, the CDRs may be defined according to any of Kabat, Chothia, extended, AbM, contact, and / or conformation definitions.

[0036] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies; i.e., the individual antibodies comprising the group are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Further, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous group of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or may be made by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies may also be isolated from phage libraries made using the techniques described in, for example, McCafferty et al., Nature 348:552-554, 1990.

[0037] As used herein, a "humanized" antibody refers to a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding portion sequences of an antibody, etc.) that minimally contains sequences derived from non-human immunoglobulins, in the form of a non-human (e.g., mouse) antibody. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody), such as a mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some examples, residues in the Fv framework region (FR) of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody may contain residues that are not present in the recipient antibody or in the imported CDR or framework sequences, but are included for further improvement or optimization of antibody performance. Generally, a humanized antibody contains substantially all of at least one, typically two variable domains, in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the FRs are FR regions of a human immunoglobulin consensus sequence. Further, a humanized antibody typically optimally contains at least a portion of the immunoglobulin constant region or constant domain (Fc) of a human immunoglobulin. Antibodies having an Fc region modified as described in WO99 / 58572 are preferred. Another form of a humanized antibody has one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) modified relative to the original antibody, and these are also referred to as one or more CDRs "derived" from one or more CDRs of the original antibody.

[0038] As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or a human antibody known to those of skill in the art, or an antibody produced using any of the techniques for making the human antibodies disclosed herein. This definition of a human antibody includes antibodies that contain at least one human heavy chain polypeptide or at least one human light chain polypeptide. One example of such is an antibody that contains a mouse light chain and a human heavy chain polypeptide. Human antibodies can be generated using a variety of techniques known in the art. In one embodiment, a human antibody is selected from a phage library that expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be made by immunization of animals in which the human immunoglobulin locus has been transgenically introduced in place of the endogenous locus, for example, by mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This method is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies may be prepared by immortalizing human B lymphocytes that produce antibodies directed against a target antigen (such B lymphocytes may be obtained from an individual, or from single cell cloning of cDNA, or may be immunized in vitro). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1):86-95, 1991; and U.S. Patent No. 5,750,373.

[0039] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species. For example, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody, etc.

[0040] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein and refer to amino acid chains of any length. For example, the chain may be relatively short (e.g., 10 - 100 amino acids) or long. The chain may be linear or branched, may contain modified amino acids, and / or non - amino acids may be interspersed. Further, the term includes naturally modified amino acid chains, or amino acids modified by the following interventions: for example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as binding to a labeling component. This definition further includes polypeptides containing, for example, one or more amino acid analogs (including, for example, non - natural amino acids) and other modifications known in the art. A polypeptide is understood to be able to occur as a single chain or associated chains.

[0041] A "monovalent antibody" contains one antigen - binding site per molecule (e.g., IgG or Fab). In some examples, a monovalent antibody may have multiple antigen - binding sites, but the binding sites are derived from different antigens.

[0042] A "monospecific antibody" contains two identical antigen - binding sites per molecule (e.g., IgG), such that the two binding sites bind to the same epitope on the antigen. Thus, monospecific antibodies compete with each other for binding to one antigen molecule. Many naturally occurring antibodies are monospecific. In some examples, a monospecific antibody can also be a monovalent antibody (e.g., Fab).

[0043] A "bivalent antibody" contains two antigen-binding sites per molecule (e.g., IgG). In some examples, the two binding sites have the same antigen specificity. However, bivalent antibodies can also be bispecific.

[0044] "Bispecific" or "bifunctional" refers to a hybrid antibody that has two different antigen-binding sites. The two antigen-binding sites of a bispecific antibody bind to two different epitopes, which may be present on the same or different protein targets.

[0045] A "bifunctional" antibody has the same antigen-binding site (i.e., the same amino acid sequence) in two arms, but each binding site can recognize two different antigens.

[0046] A "heteromultimer", "heteromultimeric complex", or "heteromultimeric polypeptide" is a molecule that contains at least a first polypeptide and a second polypeptide, and the second polypeptide differs from the first polypeptide by at least one amino acid residue in the amino acid sequence. A heteromultimer includes a "heterodimer" formed by the first polypeptide and the second polypeptide, or can form a higher-order tertiary structure in which there are multiple polypeptides in addition to the first and second polypeptides.

[0047] A "heterodimer", "heterodimeric protein", "heterodimeric complex", or "heteromultimeric polypeptide" is a molecule that contains a first polypeptide and a second polypeptide, and the second polypeptide differs from the first polypeptide by at least one amino acid residue in the amino acid sequence.

[0048] The terms "hinge region", "hinge sequence", and variations thereof, as used herein, include the meanings known in the art, for example, as exemplified in Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods (1997), 209:193-202.

[0049] As used herein, the terms "immunoglobulin-like hinge region", "immunoglobulin-like hinge sequence", and variations thereof refer to the hinge region and hinge sequence of an immunoglobulin-like molecule or an antibody-like molecule (e.g., an immunoadhesin). In some embodiments, the immunoglobulin-like hinge region may be derived from or induced from any IgG1, IgG2, IgG3, or IgG4 subtype, or may be derived from or induced from IgA, IgE, IgD, or IgM, including chimeric forms thereof such as a chimeric IgG1 / 2 hinge region.

[0050] The term "immune effector cell" or "effector cell", as used herein, refers to a cell within the natural repertoire of cells in the human immune system that is activated and can affect the viability of a target cell. The viability of a target cell includes the ability of the cell to survive, proliferate, and / or interact with other cells.

[0051] The antibodies of the present invention can be made using techniques known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or combinations of such techniques, or other techniques readily knowable in the art (see, for example, Jayasena, S.D., Clin. Chem., 45:1628-50, 1999 and Fellouse, F.A., et al, J. Mol. Biol., 373(4):924-40, 2007).

[0052] As is known in the art, as used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a chain of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA polymerase or RNA polymerase. Polynucleotides may include modified nucleotides such as, for example, methylated nucleotides and their analogs. Where present, modifications to the nucleotide structure may be made before or after assembly of the chain. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after multimerization, for example, by conjugation to a label component. Other types of modifications include, for example, substitution of one or more "caps" of natural nucleotides with analogs, modifications between nucleotides using, for example, uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), those containing pendant moieties of proteins (e.g., nuclease, toxin, antibody, signal peptide, poly-L-lysine, etc.), those using intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metal, radioactive metal, boron, metal oxide, etc.), those containing alkylating agents, those using modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. Further, any of the hydroxyl groups normally present on the sugar may be substituted, for example, with a phosphonate group, a phosphate group, etc., protected with standard protecting groups, or activated to prepare for additional bonding to additional nucleotides, or conjugated to a solid support. The 5' end OH and 3' end OH may be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls may be derivatized with standard protecting groups.The polynucleotide may further contain analog types of ribose sugar or deoxyribose sugar that are generally known in the art, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha or beta-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and nucleoside analogs without a base such as methyl riboside. One or more phosphodiester bonds may be replaced by alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR', CO or CH2 (formacetal), where each R or R' is independently H, or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all bonds in the polynucleotide need to be the same. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0053] As is known in the art, the "constant region" of an antibody refers to the constant region of the antibody light chain, or the constant region of the antibody heavy chain, either alone or in combination.

[0054] As used herein, "substantially pure" refers to a substance that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

[0055] "Host cell" may refer to a recipient for a vector for the integration of a polynucleotide insert, or may include an individual cell or cell culture that is the recipient. Host cells include the progeny of a single host cell, which may not always be identical to the original parent cell (either in morphology or in the complement of genomic DNA) due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with the polynucleotides of the present invention.

[0056] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226, or the amino acid residue at position Pro230, to its carboxyl terminus. The numbering of residues in the Fc region is that of the EU index in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally contains two constant regions, CH2 and CH3.

[0057] When used in the context, the terms "Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Furthermore, preferred FcRs are FcRs (gamma receptors) that bind to IgG antibodies and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA (activating receptor) and FcγRIIB (inhibitory receptor), which have similar amino acid sequences but mainly differ in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. "FcR" further includes the neonatal receptor, FcRn, which contributes to the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

[0058] As used herein with respect to antibodies, the term "competes" means that a first antibody, or an antigen-binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody or an antigen-binding portion thereof such that, in the presence of the second antibody, the binding of the first antibody to its cognate epitope is detectably reduced as compared to the binding of the first antibody in the absence of the second antibody. The binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but not necessarily. That is, the first antibody may inhibit the second binding to its epitope, but inhibition of the first antibody by the second antibody to each of its epitopes is not accompanied. However, when each antibody detectably inhibits the binding of other antibodies to their cognate epitopes or ligands to the same extent, more strongly, or more weakly, the antibodies are said to "cross-compete" with each other with respect to the binding of each of their epitopes. Both competing antibodies and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (i.e., steric hindrance, conformational change, or binding to a common epitope or a part thereof, etc.), those skilled in the art will recognize, based on the teachings provided herein, that such competing antibodies and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0059] A "functional Fc region" possesses at least one effector function of a native sequence Fc region. Examples of "effector functions" include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require an Fc region that is combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays known in the art for evaluating such antibody effector functions.

[0060] "Native Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region existing in nature. "Variant Fc region" contains an amino acid sequence different from that of the native Fc region due to at least one amino acid modification, but retains at least one effector function of the native Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution, such as about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native Fc region or the Fc region of the parental polypeptide, as compared to the native Fc region or compared to the Fc region of the parental polypeptide. The variant Fc regions herein preferably have at least about 80% sequence identity with the native Fc region and / or the Fc region of the parental polypeptide, most preferably at least about 90% sequence identity with them, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity.

[0061] The term "effector function" refers to the biological activities resulting from the Fc region of an antibody. Examples of antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, complement-dependent cytotoxicity (CDC), phagocytosis, C1q binding, and downregulation of cell surface receptors (e.g., B cell receptor, BCR). See, e.g., U.S. Patent No. 6,737,056. Such effector functions generally require an Fc region combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays known in the art for evaluating such antibody effector functions. Examples of measuring effector functions are via Fcγ3 and / or C1q binding.

[0062] As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors recognize an antibody bound to a target cell and then cause lysis of the target cell. The ADCC activity of a target molecule can be evaluated using an in vitro ADCC assay, such as an assay described in, for example, U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively, or in addition, the ADCC activity of a target molecule may be evaluated in vivo in an animal model, such as that disclosed in, for example, Clynes et al., 1998, PNAS (USA), 95:652-656.

[0063] "Complement-dependent cytotoxicity" or "CDC" refers to lysis of a target in the presence of complement. The complement activation pathway begins when the first component of the complement system (C1q) binds to a molecule (e.g., an antibody) complexed with a homologous antigen. To evaluate complement activation, a CDC assay, such as an assay described in, for example, Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may be performed.

[0064] As used herein, "treatment" refers to an approach for obtaining beneficial or desirable clinical outcomes. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: a decrease (or destruction) in the proliferation of tumor cells or cancer cells, inhibition of metastasis of tumor cells, a reduction or decrease in the size of a CD70-expressing tumor, remission of a CD70-related disease (e.g., cancer), a decrease in symptoms resulting from a CD70-related disease (e.g., cancer), an improvement in the quality of life of a person suffering from a CD70-related disease (e.g., cancer), a decrease in the dosage of other drugs required to treat a CD70-related disease (e.g., cancer), a delay in the progression of a CD70-related disease (e.g., cancer), a cure of a CD70-related disease (e.g., cancer), and / or an extension of the survival of a patient having a CD70-related disease (e.g., cancer).

[0065] "Improve" means a reduction or improvement in one or more symptoms as compared to the case where no CD70 antibody (monospecific or bispecific) is administered. "Improve" also includes a shortening or decrease in the duration of the symptoms.

[0066] As used herein, an "effective dosage" or "effective amount" of an agent, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in onset of a disease, its complications, and intermediate pathological phenotypes presented during the course of the disease, including biochemical, histological, and / or behavioral symptoms. For therapeutic use, beneficial or desired results include, for example, reduction or amelioration of the incidence of one or more symptoms of a patient's various CD70-related diseases or conditions (such as multiple myeloma), reduction in the dosage of other agents required to treat the disease, enhancement of the effect of another agent, and / or clinical outcomes such as delay in the progression of CD70-related diseases. The effective dosage can be administered in one or more administrations. For the purposes of the present invention, an effective dosage of an agent, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly effect a prophylactic or therapeutic treatment. As understood in the clinical context, an effective dosage of an agent, compound, or pharmaceutical composition may or may not be achieved in combination with another agent, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if, in combination with one or more other agents, there is a possibility or achievement of a desired result.

[0067] An "individual" or "subject" is a mammal, more preferably a human. Mammals include, but are not limited to, primates, horses, dogs, cats, mice, and rats.

[0068] As used herein, "vector" means a construct that can deliver one or more genes or sequences of interest and, preferably, can express one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0069] As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. The expression control sequence can be a constitutive promoter or an inducible promoter, or a promoter such as an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0070] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, has components for retaining biological activity and is non-reactive with the immune system of the subject. Examples include, but are not limited to, phosphate buffered saline aqueous solutions, water, emulsions such as oil / water emulsions, and standard pharmaceutical carriers such as various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or saline (0.9%). Compositions containing such carriers are formulated by known conventional methods (see, e.g., Remington’s Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

[0071] The term "acyl donor glutamine-containing tag" or "glutamine tag", as used herein, refers to a polypeptide or protein containing one or more Gln residues that act as transglutaminase amine acceptors. See, for example, WO2012059882 and WO2015015448.

[0072] As used herein, "k on " or "k a " refers to the rate constant for the association of an antibody with an antigen. Specifically, the rate constants (k on / k a and k off / k d ) and the equilibrium dissociation constant are measured using whole antibody (i.e., bivalent) and monomeric CD70 protein (e.g., histidine-tagged CD70 fusion protein).

[0073] As used herein, "k off " or "k d " refers to the rate constant for the dissociation of an antibody from an antibody / antigen complex.

[0074] As used herein, "K D " refers to the equilibrium dissociation constant for the antibody-antigen interaction.

[0075] As used herein, the term "about" in reference to a value or parameter of an embodiment includes (and describes) embodiments that essentially refer to such value or parameter. For example, an expression referring to "about X" includes the expression of "X", and a numerical range includes the numbers defining the range. Generally speaking, the term "about" refers to the specified value of a variable and all values of the variable that are within the range of experimental error (e.g., within the range of the 95% confidence interval for the mean) of the specified value or within 10% of either the greater or lesser of the specified value. When the term "about" is used in the context of a period (such as years, months, weeks, days, etc.), the term "about" means that period plus or minus one amount of the next lower period (e.g., about 1 year means 11 to 13 months, about 6 months means 6 months plus or minus 1 week, about 1 week means 6 to 8 days, etc.) or within 10% of either the greater or lesser of the specified value.

[0076] <s It is understood that in any embodiment described herein using the term "comprising", other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0077] If aspects or embodiments of the present invention are described in terms of Markush groups or other alternative groups, the present invention generally includes not only the entire recited group, but also each member of the group individually, and all possible subgroups of the main group, and the main group lacking one or more of the group members. The present invention further anticipates any obvious exclusion of one or more of the claimed group members of the present invention.

[0078] All technical and scientific terms used herein shall, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, shall control. Throughout this specification and the claims, variations such as "comprise" or "comprises" are to be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group. Unless otherwise required by context, the singular terms shall include the plural and the plural terms shall include the singular.

[0079] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described herein. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0080] CD70 antibody, and method for producing the same The present invention provides an antibody that binds to CD70 [e.g., human CD70 (e.g., accession number: NP_004110 or SEQ ID NO: 235)] and is characterized by any one or more of the following features: (a) treating, preventing, or ameliorating one or more symptoms of a condition (e.g., cancer such as AML) associated with malignant cells that express CD70 in a subject; (b) inhibiting tumor growth or progression in a subject (having a malignant tumor that expresses CD70); (c) inhibiting metastasis of cancer (malignant) cells that express CD70 in a subject (having one or more malignant cells that express CD70); (d) inducing regression of a tumor that expresses CD70 (e.g., long-term regression); (e) exerting cytotoxic activity in malignant cells that express CD70; (f) blocking the interaction of CD70 with other yet-to-be-identified factors; and / or (g) killing neighboring non-CD70-expressing malignant cells or inducing a bystander effect that inhibits their proliferation.

[0081] In one aspect, an isolated antibody that specifically binds to CD70 is provided, wherein the antibody comprises, in (a)(i), a VH complementarity determining region 1 (CDR1) having a sequence set forth in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376, 380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405, 406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460;(ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456, 461, or 462; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, or 463, a heavy chain variable (VH) region; and / or (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527;(ii) a VL CDR2 comprising the sequence shown in 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526, or 529, comprising a variable light (VL) region.;

[0082] In another aspect, an isolated antibody that specifically binds to CD70 is provided, the antibody comprising a VH region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, or 331, and / or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, .................................................. 328, or 330.

[0083] In some embodiments, an antibody is provided that has any one of the partial light chain sequences listed in Table 1 and / or any one of the partial heavy chain sequences listed in Table 1. In Table 1, the underlined sequences are CDR sequences according to Kabat, and the boldface is CDR sequences according to Chothia.

[0084] Table 1

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

[0085] Also provided herein is the CDR portion (including Chothia, Kabat CDR and CDR contact regions) of the antigen-binding domain of an antibody against CD70. Determination of the CDR regions is within the skill in the art. It should be understood that in some embodiments, the CDR can be a combination of Kabat CDR and Chothia CDR (also referred to as "mixed CR" or "extended CDR"). In some embodiments, the CDR is Kabat CDR. In other embodiments, the CDR is Chothia CDR. In other words, in embodiments with multiple CDRs, the CDR can be any of Kabat, Chothia, combined CDR, or combinations thereof. Table 2 provides examples of CDR sequences provided herein.

[0086] Table 2

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

[0087] In some embodiments, the invention provides an antibody that binds to CD70 and competes with the antibodies described herein, comprising 31H1, 63B2, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08A02, P08E02, P08F08, P08G02, P12B09, P12F02, P12G07, P13F04, P15D02, P16C05, 10A1, 10E2, 11A1, 11C1, 11D1, 11E1, 12A2, 12C4, 12C5, 12D3, 12D6, 12D7, 12F5, 12H4, 8C8, 8F7, 8F8, 9D8, 9E10, 9E5, 9F4 or 9F8.

[0088] In some embodiments, the invention further provides a CDR portion of an antibody against a CD70 antibody based on the CDR contact region. The CDR contact region is the region of the antibody that confers specificity for an antigen to the antibody. Generally, the CDR contact region includes residue positions in the CDRs and a Vernier zone that is constrained to maintain the appropriate loop structure of the antibody that binds to a particular antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determination of the CDR contact region is within the skill in the art.

[0089] The binding affinity (K) of the CD70 antibodies described herein for CD70 (e.g., human CD70 (e.g., SEQ ID NO: 278)) D) may be from about 0.001 to about 5000 nM. In some embodiments, the binding affinity is approximately 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 nM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM, 400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM, 207 nM, 200 nM, 186 nM, 172 nM, 136 nM, 113 nM, 104 nM, 101 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74 nM, 54 nM, 50 nM, 45 nM, 42 nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 12 nM, 10 nM, 9 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM, or 0.001 nM. In some embodiments, the binding affinity is less than approximately 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0090] Monoclonal antibodies that are bispecific antibodies having binding specificities for at least two different antigens may be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210, 1986). Conventionally, the recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains had different specificities (Millstein and Cuello, Nature 305, 537-539, 1983). Thus, in one aspect, a bispecific antibody is provided, wherein the bispecific antibody is a full-length human antibody and comprises a first antibody variable domain of a bispecific antibody that specifically binds to a target antigen (e.g., CD70), and a second antibody variable domain of a bispecific antibody that can recruit the activity of human immune effector cells by specifically binding to an effector antigen located on the human immune effector cells.

[0091] The human immune effector cells can be any of a variety of immune effector cells known in the art. For example, the immune effector cells can be of the human lymphoid cell lineage, including but not limited to T cells (e.g., cytotoxic T cells), B cells, and natural killer (NK) cells. Further, the immune effector cells can be of the human myeloid lineage, including but not limited to monocytes, neutrophilic granulocytes, and dendritic cells. Such immune effector cells can have a cytotoxic or apoptotic effect on target cells, or can have other desirable effects when activated by the binding of the effector antigen.

[0092] An effector antigen is an antigen (e.g., a protein or polypeptide) expressed on human immune effector cells. Examples of effector antigens that can be bound by a heterodimeric protein (e.g., a heterodimeric antibody or a bispecific antibody) include, but are not limited to, human CD3 (or the CD3 (Cluster of Differentiation) complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

[0093] Target cells may be cells that are native or foreign to a human. In the case of native target cells, the cells may be transformed to become malignant cells or may be pathologically modified (e.g., native target cells infected with a virus, plasmodium, or bacteria). In the case of foreign target cells, the cells are invasive pathogens such as, for example, bacteria, plasmodium, or viruses.

[0094] A target antigen is expressed on target cells in a disease state (e.g., an inflammatory disease, a proliferative disease (e.g., cancer), an immunological disorder, a neurological disease, a neurodegenerative disease, an autoimmune disease, an infectious disease (e.g., viral infection or parasitic infection), an allergic reaction, graft-versus-host disease, or host-versus-graft disease). A target antigen is not an effector antigen. In some embodiments, the target antigen is CD70.

[0095] In some embodiments, a bispecific antibody is provided, which is a full-length antibody and includes a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen, and a second antibody variable domain of the bispecific antibody that can recruit the activity of human immune effector cells by specifically binding to an effector antigen located on human immune effector cells. The first antibody variable domain includes the VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, or 331, and / or the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330, of the variable heavy (VH) region and / or the variable light (VL) region.

[0096] In some embodiments, a bispecific antibody is provided, which is a full-length antibody and comprises a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen, and a second antibody variable domain of the bispecific antibody that can recruit the activity of human immune effector cells by specifically binding to an effector antigen located on the human immune effector cells. The first antibody variable domain comprises (a)(i) a VH complementarity determining region 1 (CDR1) comprising the sequence shown in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376, 380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405, 406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459, or 460;(ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456, 461, or 462; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, or 463, the heavy chain variable (VH) region; and / or (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, or 527;(ii) a VL CDR2 comprising the sequences shown in 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, or 528; and (iii) a VL CDR3 comprising the sequences shown in SEQ ID NOs: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526, or 529, comprising a variable light (VL) region.;

[0097] In some embodiments, the second antibody variable domain comprises a variable heavy (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 266, and / or a variable light (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 265.

[0098] In some embodiments, the second antibody variable domain contains (a) (i) a VH complementarity determining region 1 (CDR1) comprising the sequences shown in SEQ ID NOs: 267, 268 or 269; (ii) a VH CDR2 comprising the sequences shown in SEQ ID NOs: 270 or 271; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 272, a variable heavy (VH) region, and / or (b) (i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 273; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 274; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 275, a variable light (VL) region.

[0099] Table 3 shows the specific amino acid sequences and nucleic acid sequences of the second antibody variable domains specific for CD3. In Table 3, the underlined sequences are CDR sequences according to Kabat, and the boldface ones are CDR sequences according to Chothia.

[0100] Table 3

Table 3

[0101] Table 4 shows examples of the specific CDR sequences of the second antibody variable domains specific for CD3.

[0102] Table 4

Table 4

[0103] In some embodiments, the bispecific antibodies provided herein contain a CD3-specific variable domain having an anti-CD3 sequence provided in US Patent Application Publication 20160297885, which is incorporated herein by reference for all purposes.

[0104] According to one method of making a bispecific antibody, an antibody variable domain having a desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. Fusion with an immunoglobulin heavy chain constant region including at least a part of the hinge, CH2 and CH3 regions is preferred. In at least one of the fusions, it is preferred to have a first heavy chain constant region (CH1) that includes the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptides used in the construction provide an optimal yield. However, if the expression of at least two polypeptide chains in equal ratios produces a high yield, or if the ratio is not particularly important, it is also possible to insert the coding sequences of two or all three polypeptide chains into one expression vector.

[0105] In another method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure means that only half of the bispecific molecule has an immunoglobulin light chain, thereby facilitating the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This method is described in PCT Publication WO94 / 04690.

[0106] In another method, the bispecific antibody is composed of an amino acid modification in the first hinge region of one arm, and the substituted amino acid in the first hinge region has an opposite charge to the corresponding amino acid in the second hinge region of the other arm. This method is described in International Patent Application PCT / US2011 / 036419 (WO2011 / 143545).

[0107] In another method, the formation of a desired heteromultimeric or heterodimeric protein (e.g., a bispecific antibody) is enhanced by altering or engineering the interface between the first and second immunoglobulin-like Fc regions (e.g., the hinge region and / or CH3 region). In this method, the bispecific antibody may be composed of CH3 regions, where the CH3 region contains a first CH3 polypeptide and a second CH3 polypeptide, which interact with each other to form a CH3 interface, and in this case one or more amino acids within the CH3 interface destabilize the formation of the homodimer and are electrostatically unfavorable for the formation of the homodimer. This method is described in International Patent Application PCT / US2011 / 036419 (WO2011 / 143545).

[0108] In another method, the bispecific antibody may be made using a glutamine-containing peptide tag engineered against an antibody directed to an epitope (e.g., CD70) in one arm and another peptide tag (e.g., a Lys-containing peptide tag or a reactive endogenous Lys) engineered against a second antibody directed to a second epitope in another arm in the presence of transglutaminase. This method is described in International Patent Application PCT / IB2011 / 054899 (WO2012 / 059882).

[0109] In some embodiments, the heterodimeric proteins (e.g., bispecific antibodies) described herein include full-length human antibodies, in which case the first antibody variable domain of the bispecific antibody that specifically binds to a target antigen (e.g., CD70), and the second antibody variable domain of the bispecific antibody that can recruit the activity of human immune effector cells by specifically binding to an effector antigen (e.g., CD3) localized on human immune effector cells, in which case the first and second antibody variable domains of the heterodimeric protein contain amino acid modifications (e.g., (C223E or C223R), (E225E or E225R), and (P228E or P228R)) at positions 223, 225, and 228 in the hinge region of human IgG2 (SEQ ID NO: 279), and at position 409 or 368 in the CH3 region (e.g., K409R or L368E (EU numbering scheme)).

[0110] In some embodiments, the first and second antibody variable domains of the heterodimeric protein contain amino acid modifications (e.g., (D221R or D221E) and (P228R or P228E)) at positions 221 and 228 in the hinge region of human IgG1 (SEQ ID NO: 280), and at position 409 or 368 in the CH3 region (e.g., K409R or L368E (EU numbering scheme)).

[0111] In some embodiments, the first and second antibody variable domains of the heterodimeric protein contain amino acid modifications (e.g., (P228E or P228R)) at position 228 in the hinge region of human IgG4 (SEQ ID NO: 281), and at position 409 or 368 in the CH3 region (e.g., K409R or L368E (EU numbering scheme)).

[0112] Antibodies useful in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single-chain (ScFv), variants thereof, fusion proteins containing antibody moieties (e.g., domain antibodies), humanized antibodies, and any other modified constructs of immunoglobulin molecules containing the antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies).

[0113] In some embodiments, the CD70-specific antibody, or CD70 bispecific antibody (e.g., CD70-CD3) described herein is a monoclonal antibody. For example, the CD70-specific antibody is a human monoclonal antibody. In another example, the CD70 arm of the CD70-CD3 bispecific antibody is a human monoclonal antibody, and the CD3 arm of the CD70-CD3 bispecific antibody is a humanized monoclonal antibody.

[0114] In some embodiments, the antibody includes a modified constant region, such as, but not limited to, a constant region that increases the potential to induce an immune response. For example, the constant region may be modified to increase its affinity for Fc gamma receptors such as FcγRI, FcγRIIA, or FcγIII.

[0115] In some embodiments, the antibody comprises a modified constant region such as, for example, a constant region that is immunologically inert, i.e., has a reduced potential to induce an immune response. In some embodiments, the constant region is modified as described in Eur.J.Immunol., 29:2613-2624, 1999; PCT patent application PCT / GB99 / 01441; and / or UK patent application 98099518. The Fc may be human IgG1, human IgG2, human IgG3, or human IgG4. The Fc may be human IgG2 containing the mutation from A330P331 to S330S331 (IgG2Δa), and the amino acid residues of this antibody are numbered with reference to the wild-type IgG2 sequence. Eur.J.Immunol., 29:2613-2624, 1999. In some embodiments, the antibody contains a constant region of IgG4 comprising the following mutations (Armour et al., Molecular Immunology 40 585-593, 2003): from E233F234L235 to P233V234A235 (IgG4Δc). The numbers refer to the wild-type IgG4. In yet another embodiment, the Fc is P233V234A235 with deletion of G236 from human IgG4 E233F234L235 (IgG4Δb). In another embodiment, the Fc is any human IgG4 Fc containing the hinge-stabilizing mutation from S228 to P228 (IgG4, IgG4Δb or IgG4Δc) (Aalberse et al., Immunology 105, 9-19, 2002). In another embodiment, the Fc may be a non-glycosylated Fc.

[0116] In some embodiments, the constant region is non-glycosylated by mutating oligosaccharide-binding residues (e.g., Asn297) and / or adjacent residues that are part of the glycosylation recognition sequence in the constant region. In some embodiments, the constant region is enzymatically non-glycosylated for N-linked glycosylation. The constant region may be enzymatically non-glycosylated for N-linked glycosylation or may be non-glycosylated by expression in a glycosylation-deficient host cell.

[0117] In some embodiments, the constant region has a modified constant region in which Fc gamma receptor binding has been removed or reduced. For example, the Fc may be human IgG2 containing a mutation at D265, and the amino acid residues of this antibody are numbered with reference to the wild-type IgG2 sequence (SEQ ID NO: 279). Thus, in some embodiments, the constant region has a modified constant region having the sequence shown in SEQ ID NO: 282 below: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The nucleic acid encoding the sequence shown in SEQ ID NO: 282 is shown in SEQ ID NO: 283.

[0118] In some embodiments, the constant region has a modified constant region having the sequence shown in SEQ ID NO: 284 below: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The nucleic acid encoding the sequence shown in SEQ ID NO: 284 is shown in SEQ ID NO: 285.

[0119] The amino acids of the human kappa constant region are shown in SEQ ID NO: 286 below: GTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. The nucleic acid encoding the sequence of SEQ ID NO: 286 is shown in SEQ ID NO: 287.

[0120] One way to determine the binding affinity of an antibody to CD70 is by measuring the binding affinity of a bivalent antibody to monomeric CD70 protein. The affinity of a CD70 antibody can be determined by surface plasmon resonance (Biacore™ 3000™ surface plasmon resonance (SPR) system, Biacore™, INC, Piscataway, NJ) equipped with pre - immobilized anti - mouse Fc or anti - human Fc, using HBS - EP running buffer (0.01 M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20). Monomeric 8 - histidine - tagged human CD70 extracellular domain (SEQ ID NO: 530) is diluted in HBS - EP buffer to a concentration of less than 0.5 μg / mL and injected into individual chip channels using variable contact times to achieve either of two antibody density ranges, 50 - 200 response units (RU) for detailed kinetic studies or 800 - 1,000 RU for screening assays. Regeneration experiments have shown that a 25% v / v ethanol solution of 25 mM NaOH efficiently removed bound CD70 protein over 200 injections while maintaining the activity of the CD70 antibody on the chip. Typically, serial dilutions (ranging from 0.1 - 10x the estimated K D concentration) of a purified 8 - histidine - tagged CD70 sample (SEQ ID NO: 530) are injected at 100 μL / min for 1 minute and a dissociation time of up to 2 hours is allowed. The concentration of the CD70 protein is determined by absorbance at 280 nm based on the sequence - specific extinction coefficient of the 8 - histidine - tagged CD70 protein (SEQ ID NO: 530). The kinetic association rate (k on or k a ) and dissociation rate (k off or k d ) are simultaneously obtained by globally fitting the data to a 1:1 Langmuir binding model (Karlsson, R., Roos, H., Fagerstam, L., Petersson, B. (1994). Methods Enzymology 6.99 - 110) using the BIAevaluation program. The equilibrium dissociation constant (K D) The value of k off / k on is calculated as such. This protocol is suitable for use in determining the binding affinity of antibodies to any monomeric form of CD70, including human CD70, CD70 of another mammal (e.g., mouse CD70, rat CD70, or primate CD70), and another type of CD70 (e.g., glycosylated CD70). The binding affinity of the antibody is generally measured at 25°C, but can also be measured at 37°C.

[0121] The antibodies described herein may be made by any method known in the art. For the production of hybridoma cell lines, the route and schedule of immunization of the host animal generally follow established conventional techniques for antibody stimulation and production as detailed herein. General techniques for the production of human and mouse antibodies are known in the art and / or are described herein.

[0122] Any mammalian subject, including humans, or antibody-producing cells derived therefrom can be manipulated and used as a basis for the production of mammals, including humans, and hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intradermally, and / or intrasole with an amount of immunogen, including those described herein.

[0123] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature 256:495-497, 1975, or a modified version thereof by Buck, D. W., et al., In Vitro, 18:377-381, 1982. Available myeloma strains include, but are not limited to, X63-Ag8.653, and the strains from the Salk Institute, Cell Distribution Center, San Diego, California, USA, which may be used for hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol or by electrical means known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) medium, and the unfused parental cells are removed. Any of the media described herein, with or without serum supplementation, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative means for cell fusion technology, EBV-immortalized B cells may be used to produce the monoclonal antibodies of the present invention. Hybridomas are expanded and subcloned as desired, and the supernatants are analyzed for anti-immunogen activity by conventional immunoassay methods (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[0124] Hybridomas that can be used as a source of antibodies include all derivatives and progeny cells of the parental hybridoma that produce a monoclonal antibody specific for CD70 or a portion thereof.

[0125] The hybridomas that produce the antibody may be grown in vitro or in vivo using known methods. Monoclonal antibodies may be isolated from the culture medium or body fluids by conventional immunoglobulin purification methods such as, for example, ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. If unwanted activities are present, they can be removed, for example, by flowing the preparation over an adsorbent made from an immunogen attached to a solid phase and eluting or releasing the desired antibody from the immunogen. Proteins that are immunogenic in the immunized species, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, etc., are conjugated using bifunctional agents or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (binding via cysteine residues), N-hydroxysuccinimide (binding via lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR, where R and R 1 are different alkyl groups, etc., and a group of antibodies (e.g., monoclonal antibodies) is obtained by immunizing a host animal with cells expressing human CD70, human CD70 protein, or a fragment containing the target amino acid sequence that has been conjugated using a bifunctional agent or derivatizing agent.

[0126] If desired, the antibody of interest (monoclonal or polyclonal) may be sequenced and then the polynucleotide sequence cloned into an expression vector or a propagation vector. The sequence encoding the antibody of interest may be maintained within a vector in a host cell, and then the host cell may be grown and frozen for future use. The production of recombinant monoclonal antibodies in cell culture may be performed by means known in the art via cloning of antibody genes derived from B cells. See, for example, Tiller et al., J. Immunol. Methods 329, 112, 2008; U.S. Patent No. 7,314,622.

[0127] Alternatively, a polynucleotide sequence may be used in genetic manipulation to "humanize" an antibody or to improve the affinity or other properties of the antibody. For example, the constant region may be engineered to be more closely similar to a human constant region to avoid an immune response when the antibody is used in human clinical trials and therapy. It may be desirable to genetically engineer the antibody sequence to increase the affinity for CD70 and to enhance the inhibitory effect of CD70.

[0128] There are generally four steps to humanize a monoclonal antibody. The steps are: (1) determining the nucleotide and predicted amino acid sequences of the light and heavy chain variable domains of the starting antibody; (2) designing the humanized antibody, i.e., determining which antibody framework regions to use during the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

[0129] Numerous "humanized" antibody molecules containing antigen-binding sites derived from non-human immunoglobulins have been reported, including chimeric antibodies having rodent V regions or modified rodent V regions fused to human constant regions and their associated CDRs. See, e.g., Winter et al. Nature 349:293-299, 1991; Lobuglio et al. Proc. Nat. Acad. Sci. USA 86:4220-4224, 1989; Shaw et al. J Immunol. 138:4534-4538, 1987; and Brown et al. Cancer Res. 47:3577-3583, 1987. Other references describe rodent CDRs transplanted within human auxiliary framework regions (FRs) prior to fusion with appropriate human antibody constant regions. See, e.g., Riechmann et al. Nature 332:323-327, 1988; Verhoeyen et al. Science 239:1534-1536, 1988; and Jones et al. Nature 321:522-525, 1986. Another reference describes rodent CDRs assisted by recombinant genetically engineered rodent framework regions. See, e.g., European Patent Publication 0519596. These "humanized" molecules are designed to minimize unwanted immune responses to rodent anti-human antibody molecules. Such immune responses limit the duration and effectiveness of therapeutic applications of the moiety in human recipients. For example, the antibody constant region may be engineered to be immunologically inert (e.g., not induce complement lysis). See, e.g., PCT Publication PCT / GB99 / 01441; UK Patent Application 9809951.8. Other antibody humanization methods that may be utilized are described in Daugherty et al., Nucl. Acids Res. 19:2471-2476, 1991; and U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and PCT Publication WO01 / 27160.

[0130] The general principles related to the humanized antibodies considered above are also applicable for customizing antibodies for use, for example, in dogs, cats, primates, horses, and cows. Further, one or more aspects related to the humanization of the antibodies described herein may be a combination, for example, of CDR grafting, framework mutations, and CDR mutations.

[0131] In one variation, fully human antibodies may be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more favorable (e.g., fully human) or more stable immune response may be used in the production of humanized antibodies or fully human antibodies. Examples of such technologies are Xenomouse™ of Abgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ of Medarex, Inc. (Princeton, N.J.).

[0132] Alternatively, the antibody may be recombinantly produced and expressed using any method known in the art. In another method, the antibody may be recombinantly produced by phage display technology. See, for example, U.S. Pat. Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150, and Winter et al., Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, human antibodies and antibody fragments may be produced in vitro from a repertoire of immunoglobulin variable (V) domain genes from non-immunized donors using phage display technology (McCafferty et al., Nature 348:552-553, 1990). According to this technique, antibody V domain genes are cloned in-frame into either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and are presented as functional antibody fragments on the surface of the phage particles. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also selects the gene encoding the antibody that exhibits those properties. Thus, the phage mimics part of the properties of B cells. The phage display method can be carried out in various formats. For an overview, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571, 1993. Several sources of V-gene segments may be used for phage display. Clackson et al., Nature 352:624-628, 1991, isolated a diverse array of anti-oxazolone antibodies from a low-random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from non-immunized human donors is constructed, and antibodies against a diverse array of antigens (including autoantigens) can in principle be isolated according to the techniques described in Mark et al., J. Mol. Biol. 222:581-597, 1991, or Griffith et al., EMBO J. 12:725-734, 1993.In the natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the introduced changes result in high affinity, and B cells presenting high-affinity surface immunoglobulins are preferentially replicated and differentiated upon subsequent antigen challenge. This natural process can be mimicked by adopting a technique known as "chain shuffling" (Marks et al., Bio / Technol. 10:779-783, 1992). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by successively replacing the V-region genes of the heavy and light chains with natural variants (repertoires) of V-domain genes obtained from non-immunized donors. This technique enables the production of antibodies and antibody fragments with affinities in the pM to nM range. Strategies for generating a very large phage antibody repertoire (also known as "the mother-of-all libraries") are described in Waterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene shuffling can also be used to derive human antibodies from rodent antibodies. In this case, the human antibodies have an affinity and specificity similar to the starting rodent antibodies. According to this method, also called "epitope imprinting", the V-domain genes of the heavy and light chains of rodent antibodies obtained by phage display technology are replaced with a repertoire of human V-domain genes, generating rodent-human chimeras. By performing selection against the antigen, human variable regions capable of restoring a functional antigen-binding site are isolated. That is, the epitope dictates (imprints) the partner selection. When the process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT publication WO93 / 06213). Unlike the humanization of rodent antibodies by conventional CDR grafting, this technique provides fully human antibodies that do not have framework or CDR residues of rodent origin.

[0133] Antibodies may be recombinantly produced by first isolating antibodies and antibody-producing cells from a host animal, obtaining the gene sequences, and using the gene sequences to recombinantly express the antibodies in a host cell (e.g., CHO cells). Another method that may be employed is to express the antibody sequences in plants (e.g., tobacco) or transgenic milk. Methods for recombinantly expressing antibodies in plants or milk have been reported. See, for example, Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods 231:147, 1999. Methods for making derivatives of antibodies, such as humanization, making single chains, etc. are also known in the art.

[0134] Immunoassays, and flow cytometry sorting techniques such as fluorescence activated cell sorting (FACS) can also be employed to isolate antibodies specific for CD70 or for a target tumor antigen.

[0135] The antibodies described herein can bind to many different carriers. The carrier may be active and / or inactive. Examples of known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural cellulose and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier may be either soluble or insoluble for the purposes of the present invention. One of ordinary skill in the art will know of other suitable carriers for antibody binding or will be able to identify such carriers using routine experimentation. In some embodiments, the carrier contains a moiety that targets the myocardium.

[0136] DNA encoding a monoclonal antibody can be readily isolated and sequenced by using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of a monoclonal antibody). Hybridoma cells function as a preferred source of such DNA. Upon isolation, the DNA may be placed into an expression vector (e.g., an expression vector disclosed in PCT Publication WO87 / 04462). Thereafter, the vector is transfected into host cells such as, for example, E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins by other means, and synthesis of the monoclonal antibody is obtained in the recombinant host cells. See, e.g., PCT Publication WO87 / 04462. The DNA may be modified, for example, by substituting the coding sequences of the constant regions of human heavy and light chains for homologous mouse sequences. Morrison et al., Proc. Nat. Acad. Sci. 81:6851, 1984. Or it may be modified by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the coding sequence of an immunoglobulin. In this way, "chimeric" or "hybrid" antibodies having the binding specificity of the monoclonal antibodies herein are prepared.

[0137] The CD70 antibodies described herein can be identified or characterized using methods known in the art, whereby a decrease in the expression level of CD70 is detected and / or measured. In some embodiments, the CD70 antibody is identified by incubating CD70 with a candidate substance and monitoring the binding and / or any decrease in the expression level of CD70 associated with the binding. The binding assay may be performed using a purified CD70 polypeptide, or cells that naturally express the CD70 polypeptide, or cells transfected to express the CD70 polypeptide. In one embodiment, the binding assay is a competitive binding assay, in which the ability of a candidate antibody to compete with a known CD70 antibody for CD70 binding is evaluated. This assay may be performed in various formats, including an ELISA format.

[0138] After initial identification, the activity of a candidate CD70 antibody can be further confirmed and improved by a bioassay known to verify the target biological activity. Alternatively, a bioassay can be used to directly screen candidate antibodies. Some of the methods for identifying and characterizing antibodies are detailed in the Examples.

[0139] The CD70 antibody may be characterized using methods known in the art. For example, one method is to identify the epitope to which the antibody binds, or “epitope mapping”. For example, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999, many methods for mapping and characterizing the location of epitopes on proteins are known in the art, including analysis of the crystal structure of the antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays. In additional examples, epitope mapping can be used to determine the sequence to which the antibody binds. Epitope mapping is commercially available from various suppliers, such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope, i.e., contained in a single sequence of amino acids. Or it may be a conformational epitope formed by the three-dimensional interaction of amino acids, which may not necessarily be contained in a single sequence. Peptides of various lengths (e.g., at least 4-6 amino acids in length) may be isolated or synthesized (e.g., recombinantly) and used in binding assays with antibodies to CD70 or other tumor antigens. In another example, the epitope to which the CD70 antibody binds can be determined by performing a systematic screening using overlapping peptides derived from the CD70 sequence and determining binding by the CD70 antibody. According to the gene fragment expression assay, the open reading frame encoding CD70 is fragmented either randomly or by a specific gene construct, and the reactivity of the expression fragment of CD70 with the validation antibody is determined. The gene fragment may be generated, for example, by PCR and then transcribed and translated in vitro into protein in the presence of radiolabeled amino acids. Then, the binding of the antibody to radiolabeled CD70 is determined by immunoprecipitation and gel electrophoresis.An epitope may be identified by using a large library of random peptide sequences presented on the surface of phage particles (phage library). Alternatively, a defined library of overlapping peptide fragments may be screened for binding to a test antibody in a simple binding assay. In additional examples, mutagenesis of antigen-binding domains, domain swapping experiments, and alanine scan mutagenesis may be performed to identify residues necessary, sufficient, and / or essential for epitope binding. For example, domain swapping experiments may be performed using mutant CD70, in which case various fragments of the CD70 protein are replaced (swapped) with the sequence of CD70 from another species (e.g., mouse), or the sequence of a closely related but antigenically distinct protein. By evaluating the binding of an antibody to mutant CD70, the importance of a particular CD70 fragment for antibody binding can be assessed. In the case of a CD70-specific antibody (i.e., an antibody that does not bind to CD70wt (wild type) or any other protein), the epitope can be deduced from the sequence alignment of CD70 and CD70wt.

[0140] Yet another method that can be used to characterize a CD70 antibody is to use a competition assay with other antibodies known to bind to various fragments on the same antigen, i.e., CD70, to determine whether the CD70 antibody binds to the same epitope as the other antibodies. Competition assays are known to those of skill in the art.

[0141] An expression vector may be used to induce the expression of a CD70 antibody. Those of skill in the art understand the administration of expression vectors to obtain the expression of foreign proteins in vivo. See, for example, U.S. Pat. Nos. 6,436,908, 6,413,942, and 6,376,471. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and topical administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion, or directly into the coronary artery, atrium, ventricle, or pericardium.

[0142] Targeted delivery of therapeutic compositions containing an expression vector or a subgenomic polynucleotide may be used. Receptor-mediated DNA delivery methods are described, for example, in Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J.A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 263:621, 1988; Wu et al., J. Biol. Chem., 269:542, 1994; Zenke et al., Proc. Natl. Acad. Sci. USA, 87:3655, 1990; and Wu et al., J. Biol. Chem., 266:338, 1991. Therapeutic compositions containing polynucleotides are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. Concentrations in the range of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA may be used during gene therapy protocols. Therapeutic polynucleotides and polypeptides may be delivered using a gene delivery vehicle. The gene delivery vehicle may be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy, 1:51, 1994; Kimura, Human Gene Therapy, 5:845, 1994; Connelly, Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature Genetics, 6:148, 1994). Expression of such coding sequences may be induced using an endogenous mammalian promoter or a heterologous promoter. Expression of the coding sequence may be either structural or regulatory.

[0143] Viral vectors for delivery of a desired polynucleotide and for expression in a desired cell are known in the art. Examples of viral vehicles include, but are not limited to, recombinant retroviruses (e.g., PCT publications WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, WO93 / 11230, WO93 / 10218, WO91 / 02805, U.S. Pat. Nos. 5,219,740 and 4,777,127, British Pat. No. 2,200,651, and European Pat. No. 0345242), alphavirus vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67, ATCC VR-1247), Ross River virus (ATCC VR-373, ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923, ATCC VR-1250, ATCC VR 1249, ATCC VR-532)), and adeno-associated virus (AAV) vectors (e.g., PCT publications WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655). Administration of DNA conjugated to a killed adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 may also be employed.

[0144] Non-viral delivery vehicles and non-viral delivery methods may be employed, including but not limited to polycationic condensed DNA (e.g., Curiel, Hum. Gene Ther., 3:147, 1992) either alone or conjugated to inactivated adenovirus, ligand-conjugated DNA (e.g., Wu, J. Biol. Chem., 264:16985, 1989), eukaryotic cell delivery vehicle cells (e.g., U.S. Patent No. 5,814,482, PCT Publications WO95 / 07994, WO96 / 17072, WO95 / 30763, and WO97 / 42338), and nuclear charge neutralization or cell membrane fusion methods. Naked DNA may be employed. Examples of naked DNA introduction methods are described in PCT Publication WO90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Publications WO95 / 13796, WO94 / 23697, WO91 / 14445, and EP 0524968. Additional methods are described in Philip, Mol. Cell Biol., 14:2411, 1994, and Woffendin, Proc. Natl. Acad. Sci., 91:1581, 1994.

[0145] In some embodiments, the invention encompasses compositions comprising a pharmaceutical composition comprising an antibody described herein, an antibody made by the methods of the invention, or an antibody having the characteristics described herein. As used herein, a composition comprises one or more antibodies that bind CD70 and / or one or more polynucleotides comprising sequences encoding one or more of these antibodies. These compositions may further comprise suitable excipients such as pharmaceutically acceptable excipients including buffers known in the art.

[0146] The present invention further provides a method for producing any of these antibodies. The antibodies of the present invention can be produced by methods known in the art. The polypeptide can be produced by proteolysis or other degradation of the antibody, by the recombinant methods described above (i.e., as a single polypeptide or a fusion polypeptide), or by chemical synthesis. The polypeptide of the antibody, particularly a short polypeptide of about 50 amino acids or less, can be readily produced by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an antibody can be produced by an automated polypeptide synthesizer employing a solid-phase method. See U.S. Pat. Nos. 5,807,715, 4,816,567, and 6,331,415.

[0147] In another alternative method, the antibody can be produced recombinantly using procedures known in the art. In one embodiment, the polynucleotide comprises a sequence encoding the variable region of the heavy and / or light chain of antibody 31H1, 63B2, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08A02, P08E02, P08F08, P08G02, P12B09, P12F02, P12G07, P13F04, P15D02, or P16C05. The sequence encoding the antibody of interest may be maintained in a vector within a host cell, and then the host cell may be grown and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[0148] Heteroconjugate antibodies that include two covalently linked antibodies are also within the scope of the present invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and have been used in the treatment of HIV infection (PCT Publications WO91 / 00360 and WO92 / 200373, EP 03089). Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents and cross-linking methods are known in the art and are described in U.S. Pat. No. 4,676,980.

[0149] Chimeric or hybrid antibodies can be prepared in vitro using known synthetic protein chemical methods, including methods involving crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0150] In recombinant humanized antibodies, the Fcγ region may be modified to avoid interaction with Fcγ receptors and the complement and immune systems. Methods for preparing such antibodies are described in WO99 / 58572. For example, the constant region can be further engineered to resemble the human constant region more closely to avoid immune responses when the antibody is used in human clinical trials and therapy. See, for example, U.S. Pat. Nos. 5,997,867 and 5,866,692.

[0151] The present invention encompasses modifications to the antibodies and polypeptides of the present invention, including variants shown in Table 5, functionally equivalent antibodies that are not significantly affected in their properties, and variants with enhanced or decreased activity and / or affinity. For example, the amino acid sequence may be mutated such that an antibody having the desired binding affinity for CD70 is obtained. Modifications of polypeptides are routinely performed in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, polypeptides having one or more deletions or additions of amino acids that do not significantly deteriorate or alter the functional activity or that mature (enhance) the affinity of the polypeptide for its ligand, or polypeptides in which chemical analogs have been used.

[0152] Amino acid sequence insertions include amino-terminal fusions and / or carboxyl-terminal fusions of polypeptides ranging in length from 1 residue to hundreds or more residues, as well as insertions between the sequences of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue, or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include fusions of enzymes or polypeptides to the antibody N-terminus or antibody C-terminus, thereby extending the half-life of the antibody in blood circulation.

[0153] Substitution variants are those in which at least one amino acid residue in the antibody molecule has been removed and a different residue inserted in its place. The sites of greatest interest for substitution mutagenesis are the hypervariable regions, although modification of the FRs is also anticipated. Conservative substitutions are shown in Table 5 under the heading "Conservative Substitutions". Where such substitutions result in a change in biological activity, more substantial substitution changes are indicated in Table 5 as "Exemplary Substitutions", or as described in more detail below with respect to classes of amino acids, such substitutions can be introduced and the products screened. In some embodiments, the substitution variants of the antibodies provided herein have 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer conservative substitutions in the VH or VL region compared to the reference parental antibody. In some embodiments, the substitutions are not within the CDRs of the VH or VL region.

[0154] Table 5: Amino Acid Substitutions

Table 5

[0155] Substitution modifications in the biological properties of an antibody are achieved by selecting widely different substitutions in terms of their effect on (a) the structure of the polypeptide backbone in the substitution region, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Natural amino acid residues are grouped based on the following common side-chain characteristics: (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile, (2) Uncharged polar: Cys, Ser, Thr, Asn, Gln, (3) Acidic (negatively charged): Asp, Glu, (4) Basic (positively charged): Lys, Arg, (5) Residues affecting the directionality of the chain: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe, His.

[0156] Non-conservative substitutions are made by exchanging one of these classes for another.

[0157] All cysteine residues that do not participate in maintaining the proper conformation of the antibody may generally be substituted with serine, thereby improving the oxidative stability of the molecule and preventing abnormal cross-linking. Conversely, adding cysteine bonds to the antibody, especially when the antibody is an antibody fragment such as an Fv fragment, improves its stability.

[0158] Amino acid modifications may range from the alteration or modification of one or more amino acids to the complete redesign of a region such as the variable region. Altering the variable region may change the binding affinity and / or binding specificity. In some embodiments, within the CDR domain, 1 to 5 or fewer conservative amino acid substitutions are made. In other embodiments, within the CDR domain, 1 to 3 or fewer conservative amino acid substitutions are made. In yet other embodiments, the CDR domain is CDR H3 and / or CDR L3.

[0159] The modifications include glycosylated polypeptides and non-glycosylated polypeptides, as well as polypeptides having other post-translational modifications, such as polypeptides having glycosylation with different saccharides, acetylation and phosphorylation. Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, Chem. Immunol. 65:111-128, 1997; Wright and Morrison, TibTECH 15:26-32, 1997). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al., Mol. Immunol. 32:1311-1318, 1996; Wittwe and Howard, Biochem. 29:4175-4180, 1990), and can affect the intramolecular interactions between portions of the glycoprotein that affect the three-dimensional structure and the presented three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. 7:409-416, 1996). Oligosaccharides can also play a role in targeting a given glycoprotein to specific molecules based on specific recognition structures. The glycosylation of antibodies has also been reported to affect antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, CHO cells having tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of branched GlcNAc, have been reported to have improved ADCC activity (Umana et al., Mature Biotech. 17:176-180, 1999).

[0160] Antibody glycosylation is typically either N-linked or O-linked. N-linked refers to the addition of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid other than proline, are recognition sequences for the enzymatic addition of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide potentially creates a glycosylation site. O-linked glycosylation refers to the addition of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, and 5-hydroxyproline or 5-hydroxylysine may also be used.

[0161] The addition of glycosylation sites to an antibody can be readily achieved by altering the amino acid sequence to contain one or more of the above-described (for N-linked glycosylation sites) tripeptide sequences. (For O-linked glycosylation sites) modifications may be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody.

[0162] The glycosylation pattern of an antibody may be altered without changing the underlying nucleotide sequence. Glycosylation is highly dependent on the host cell used to express the antibody. Since it is rare for the cell type used to express a recombinant glycoprotein, such as an antibody potentially for therapeutic use, to be a native cell, variation in the glycosylation pattern of an antibody can be predicted (see, e.g., Hse et al., J. Biol. Chem. 272:9062-9070, 1997).

[0163] In addition to the selection of host cells, factors that affect glycosylation during recombinant production of antibodies include growth mode, medium composition, culture density, oxygenation, pH, purification scheme, and the like. Various methods have been proposed to alter the glycosylation patterns achieved in specific host organisms, including introducing or overexpressing specific enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335, 5,510,261, and 5,278,299). Glycosylation, or a specific type of glycosylation, may be enzymatically removed from glycoproteins using, for example, endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. Furthermore, recombinant host cells may be genetically engineered to lack the processing of specific types of polysaccharides. These and similar techniques are known in the art.

[0164] Other methods of modification include, but are not limited to, the use of coupling techniques known in the art, including enzymatic means, oxidative substitution, and chelation. For example, modifications for the purpose of adding labels for immunoassays may be used. Modified polypeptides may be made using procedures established in the art and screened using standard assays known in the art, some of which are described below and in the examples.

[0165] Other antibody modifications include antibodies modified as described in PCT Publication WO99 / 58572. These antibodies include an effector domain having an amino acid sequence substantially homologous to all or part of the constant region of the human immunoglobulin heavy chain, in addition to a binding domain directed to a target molecule. These antibodies can bind to target molecules without inducing significant target complement-dependent lysis or cell-mediated destruction. In some embodiments, the effector domain can specifically bind to FcRn and / or FcγRIIb. These domains typically consist of two or more human immunoglobulin heavy chain C HIt is based on a chimeric domain derived from two domains. The antibody modified in this way is particularly suitable for use in long-term antibody therapy and avoids inflammation and other adverse reactions to conventional antibody therapy.

[0166] The present invention includes affinity matured embodiments. For example, affinity matured antibodies may be made by methods known in the art (Marks et al., Bio / Technology, 10:779-783, 1992; Barbas et al., Proc Nat.Acad.Sci, USA 91:3809-3813, 1994; Schier et al., Gene, 169:147-155, 1995; Yelton et al., J.Immunol., 155:1994-2004, 1995; Jackson et al., J.Immunol., 154(7):3310-9, 1995, Hawkins et al., J.Mol.Biol., 226:889-896, 1992; and PCT publication WO2004 / 058184).

[0167] The following methods can be used to modulate the affinity of an antibody and to characterize the CDRs. One way to characterize the CDRs of an antibody and / or to vary (e.g., improve) the binding affinity of a polypeptide such as an antibody is called "library scanning mutagenesis." Generally, library scanning mutagenesis works as follows. One or more amino acid positions within the CDR are substituted with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using methods recognized in the art. This creates a small clone library (in some embodiments, one for each amino acid position being analyzed). (If two or more amino acids are substituted at each position), each has a complexity of two or more members. Generally, the library also contains clones that include the native (unsubstituted) amino acid. From each library, a small number of clones, e.g., about 20-80 clones depending on the complexity of the library, are screened for binding affinity to a target polypeptide (or other binding target), and candidates with increased binding, equivalent binding, decreased binding, or no binding are identified. Methods for determining binding affinity are known in the art. Binding affinity may be determined using the Biacore™ surface plasmon resonance assay method, which can detect differences in binding affinity of about two-fold or more. Biacore™ is particularly useful, for example, when the starting antibody already binds with a relatively high affinity, such as K of about 10 nM or less. Screening using the Biacore™ surface plasmon resonance method is described in the Examples herein. D When the starting antibody already binds with a relatively high affinity, such as K of about 10 nM or less. Screening using the Biacore™ surface plasmon resonance method is described in the Examples herein.

[0168] Binding affinity may also be determined using a Kinexa Biocensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and / or yeast display. Binding affinity may be screened using an appropriate bioassay.

[0169] In some embodiments, the position of each amino acid in the CDR is substituted with all 20 natural amino acids (one at a time in some embodiments) using mutagenesis methods recognized in the art, some of which are described herein. This creates a small clone library (one for each amino acid position analyzed in some embodiments). (If all 20 amino acids are substituted at each position), each has a complexity of 20 members.

[0170] In some embodiments, the library being screened contains substitutions at two or more positions, which may be within the same CDR or within two or more CDRs. Thus, the library may contain substitutions at two or more positions within one CDR. The library may contain substitutions at two or more positions within two or more CDRs. The library may contain substitutions at 3, 4, 5, or more positions, which are present in 2, 3, 4, 5, or 6 CDRs. The substitutions may be prepared using low-redundancy codons. See, for example, Table 2 of Balint et al., Gene 137(1):109-18, 1993.

[0171] The CDR may be CDRH3 and / or CDRL3. The CDR may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and / or CDRH3. The CDR may be a Kabat CDR, a Chothia CDR, or an extended CDR.

[0172] Candidate(s) with improved binding may be sequence analyzed, thereby identifying CDR substitution mutants that result in improved affinity (also named "improved" substitutions). Candidate(s) that bind may be sequence analyzed, thereby identifying CDR substitutions that retain binding.

[0173] Multiple rounds of screening may be performed. For example, candidates with improved binding (each containing an amino acid substitution at one or more positions of one or more CDRs) are also useful for designing a second library containing at least the original amino acid and the substituted amino acid at each improved CDR position. The preparation and screening or selection of this library are further considered below.

[0174] To the extent that the frequency of clones with improved binding, same binding, decreased binding, or no binding provides information on the importance of each amino acid position for the stability of the antibody-antigen complex, the library screening mutagenesis method also provides a means for characterizing CDRs. For example, when all 20 amino acids are changed and the CDR position retains binding, that position is identified as a position that may not be required for antigen binding. Conversely, when the CDR position retains binding with only a few substitutions, that position is identified as a position important for CDR function. Thus, the library screening mutagenesis method provides information on positions in the CDR that can be changed to many different amino acids (including all 20 amino acids), and information on positions in the CDR that cannot be changed or can be changed to only a few amino acids.

[0175] Candidates with improved affinity may be combined in a second library. The library may further contain additional substitutions at that position, depending on the desired complexity of the library or the complexity of the library that allows the use of the desired screening or selection method. If further desired, adjacent amino acid positions can also be randomized to at least two or more amino acids. Randomization of adjacent amino acids may allow additional structural adaptability in the mutant CDR, which may similarly allow or facilitate the introduction of a number of improved mutations. The library may contain substitutions at positions that did not show an improvement in affinity in the first round of screening.

[0176] The second library is screened or selected for members of the library having improved and / or altered binding affinities using any method known in the art. Such methods include screening using Biacore™ surface plasmon resonance analysis, and selection using any method known in the art for selection including phage display, yeast display, and ribosome display.

[0177] The present invention also encompasses fusion proteins containing one or more fragments or regions of the antibodies of the present invention. In one embodiment, a fusion polypeptide is provided that contains at least 10 contiguous amino acids of the variable light chain region shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47, and / or at least 10 amino acids of the variable heavy chain region shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. In other embodiments, a fusion polypeptide is provided that contains at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable light chain region, and / or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the variable heavy chain region. In another embodiment, the fusion polypeptide comprises one or more CDRs. In yet other embodiments, the fusion polypeptide comprises CDR H3 (VH CDR3) and / or CDR L3 (VL CDR3). For the purposes of the present invention, the fusion protein comprises one or more antibodies and another amino acid sequence not added in native molecules such as, for example, a heterologous sequence or a homologous sequence from another region. Examples of heterologous sequences include, but are not limited to, "tags" such as, for example, a FLAG tag or a 6His tag (SEQ ID NO: 531). Tags are known in the art.

[0178] The fusion polypeptide can be produced by methods known in the art, such as synthesis or recombination. Typically, the fusion proteins of the present invention are made by preparing and expressing the polynucleotides encoding them using the recombinant methods described herein, but may also be prepared by other means known in the art, including, for example, chemical synthesis.

[0179] The present invention also provides a composition comprising an antibody bound (e.g., linked) to a substance (e.g., biotin or avidin) that promotes coupling to a solid support. For purposes of simplicity, reference is made generally to the antibodies in any of the embodiments of the CD70 antibodies described herein with the understanding that these methods are applied to the antibodies. Binding generally refers to linking these components described herein. Linking (generally fixing these components in a proximate relationship for at least administration) can be achieved in any number of ways. For example, a direct reaction between a substance and an antibody is possible if each bears substituents capable of reacting with the other. For example, a nucleophilic group such as an amino group or a sulfhydryl group can react with a carbonyl-containing group such as an anhydride or an acid halide on the one hand, or with an alkyl group containing a sufficient leaving group (e.g., a halide) on the other.

[0180] The present invention also provides an isolated polynucleotide encoding the antibody of the present invention, as well as a vector and a host cell comprising the polynucleotide.

[0181] Accordingly, the present invention provides a polynucleotide (or a composition comprising a pharmaceutical composition) comprising a polynucleotide encoding any of the following: 31H1, 63B2, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08A02, P08E02, P08F08, P08G02, P12B09, P12F02, P12G07, P13F04, P15D02 or P16C05, or any fragment or portion thereof having the ability to bind to CD70.

[0182] In another aspect, the present invention provides a polynucleotide encoding any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides with impaired effector functions. The polynucleotide can be made and expressed by methods known in the art.

[0183] In another aspect, the present invention provides a composition (e.g., a pharmaceutical composition) comprising any of the polynucleotides of the present invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies described herein.

[0184] The administration of the expression vector and the polynucleotide composition is further described herein.

[0185] In another aspect, the present invention provides a method of making any of the polynucleotides described herein.

[0186] The present invention also encompasses any polynucleotide complementary to such an array. The polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be a DNA molecule (genomic, cDNA or synthetic) or an RNA molecule. RNA molecules include HnRNA molecules containing introns and corresponding to DNA molecules in a one-to-one manner, and mRNA molecules not containing introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the present invention, and the polynucleotides may, but need not, be linked to other molecules and / or support substances.

[0187] The polynucleotide may include a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof), or may include variants of such sequences. Polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions such that the immunoreactivity of the encoded polypeptide is not reduced as compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be evaluated as described herein. The variant preferably exhibits at least about 70% identity to the polynucleotide sequence encoding the native antibody or a portion thereof, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity.

[0188] Two polynucleotide sequences or polypeptide sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are the same when optimally aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50 contiguous positions, in which the sequences may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0189] Optimal alignment of the sequences for comparison may be performed using the Megalign program of the Lasergene suite of the bioinformatics software (DNASTAR, Inc., Madison, Wis.) using the default parameters. This program embodies several alignment schemes described in the following references. Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345 - 358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626 - 645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151 - 153; Myers, E.W. and Muller W., 1988, CABIOS 4:11 - 17; Robinson, E.D., 1971, Comb.Theor. 11:105; Santou, N., Nes, M., 1987, Mol.Biol.Evol. 4:406 - 425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc.Natl.Acad.Sci.USA 80:726 - 730.

[0190] Preferably, the "percentage of sequence identity" is determined by comparing two optimally juxtaposed sequences over a comparison window of at least 20 positions, wherein a portion of the polynucleotide sequence or polypeptide sequence in the comparison window may include additions or deletions of 20% or less, usually 5-15%, or 10-12% (i.e., gaps) as compared to the reference sequence (without addition or deletion) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues are present in both sequences, counting the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to give the percentage of sequence identity.

[0191] The variant may further or alternatively be substantially homologous to a native gene, or a part thereof, or its complement. Such polynucleotide variants have the ability to hybridize to the native DNA sequence (or complementary sequence) encoding the native antibody under moderately stringent conditions.

[0192] Suitable "moderately stringent conditions" include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C - 65°C, 5 X SSC, overnight; and then washing twice at 65°C for 20 minutes each with 2X, 0.5X, and 0.2X SSC containing 0.1% SDS each.

[0193] As used herein, "highly stringent conditions" or "high stringency conditions" refer to (1) for washing, employing low ionic strength and high temperature, e.g., at 50°C, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate, etc.; (2) during hybridization, employing a denaturing agent such as formamide, e.g., 50% (v / v) formamide containing 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, with 750 mM sodium chloride and 75 mM sodium citrate at 42°C; or (3) using 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, at 42°C, washing in 0.2x SSC (sodium chloride / sodium citrate) at 42°C, and washing in 50% formamide at 55°C, followed by high stringency washing in 0.1x SSC containing EDTA at 55°C. One of ordinary skill in the art will recognize how to adjust the temperature, ionic strength, etc. as needed to adapt to factors such as the length of the probe, etc.

[0194] One skilled in the art will recognize that due to the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides carry minimal homology to the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage frequency are specifically contemplated by the present invention. Further, alleles of genes containing the polynucleotide sequences provided herein are within the scope of the present invention. An allele is an endogenous gene that varies as a result of one or more mutations such as, for example, nucleotide deletions, additions and / or substitutions. The resulting mRNA and protein may or may not have an altered structure or function. Alleles may be identified using standard techniques such as hybridization, amplification and / or database sequence comparison.

[0195] The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Chemical polynucleotide synthesis methods are known in the art and need not be described in detail herein. One skilled in the art can generate the desired DNA sequence using the sequences provided herein and a commercially available DNA synthesizer.

[0196] As further contemplated herein, to prepare a polynucleotide using recombinant methods, a polynucleotide containing the desired sequence may be inserted into a suitable vector, which is then introduced into a suitable host cell and may be replicated and amplified. The polynucleotide can be inserted into the host cell by any means known in the art. The cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Upon introduction, the exogenous polynucleotide can be maintained intracellularly as a non-integrated vector (e.g., plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods known in the art. See, for example, Sambrook et al., 1989.

[0197] Alternatively, it is possible to regenerate a DNA sequence by PCR. PCR technology is known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkhauswer Press, Boston, 1994.

[0198] RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. For example, when the cell is replicated and the DAN is transcribed into RNA as described in Sambrook et al., 1989 above, the RNA can be isolated using methods known to those skilled in the art.

[0199] A suitable cloning vector may be constructed according to standard techniques or selected from any of a number of cloning vectors available in the art. The selected cloning vector may vary depending on the host cell intended for use, but useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry a gene for a marker that can be used to select clones containing the vector. Suitable examples include plasmids and bacteriophage viruses. For example, pUC18, pUC19, Bluescript (e.g., pBS SK+), and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These vectors, and many other cloning vectors, are available from commercial sources such as BioRad, Strategene, and Invitrogen.

[0200] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the invention. The expression vector is suggested to be replicable in the host cell, either as an episome or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenovirus, adeno-associated virus, retrovirus, cosmids, and the expression vectors disclosed in PCT Publication WO87 / 04462. Components of the vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, and appropriate transcriptional control elements (e.g., a promoter, an enhancer, and a terminator). For expression (i.e., translation), one or more translational control elements are also usually required, such as a ribosome binding site, a translation initiation site, and a stop codon.

[0201] Vectors containing the polynucleotide of interest can be introduced into host cells by any of a number of suitable means including, but not limited to, electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, microprojectile bombardment, lipofection, and infection (where the vector is an infectious entity such as, for example, vaccinia virus). The choice of means for introducing the vector or polynucleotide often depends on the characteristics of the host cell.

[0202] The present invention also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA may be used for the purpose of isolating a gene encoding a subject antibody, polypeptide, or protein. Non-limiting examples of mammalian host cells include, but are not limited to, COS cells, HeLa cells, and CHO cells. See, for example, PCT Publication WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or Bacillus subtilis) and yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Kluyveromyces lactis). The host cell expresses the cDNA at a level about 5-fold higher, more preferably 10-fold higher, and even more preferably 20-fold higher than the corresponding endogenous subject antibody or subject protein, if present, in the host cell. Screening of host cells for specific binding to CD70 is performed by immunoassay or FACS. Cells overexpressing the subject antibody or protein can be identified.

[0203] Method of using the CD70 antibody The antibodies of the present invention are useful in a variety of applications including, but not limited to, therapeutic and diagnostic procedures.

[0204] The antibodies obtained by the above method (e.g., monospecific and bispecific) can be used as pharmaceuticals. In some embodiments, such pharmaceuticals can be used for the treatment of cancer. In some embodiments, the cancer is a hematopoietic-origin cancer such as, for example, lymphoma or leukemia. In some embodiments, the cancer is renal cell carcinoma, glioblastoma, glioma such as low-grade glioma, non-Hodgkin lymphoma (NHL), Hodgkin disease (HD), Waldenström's macroglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

[0205] In some embodiments, a method of inhibiting tumor growth or progression is provided in a subject having malignant cells expressing CD70, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a CD70 antibody (e.g., a CD70-CD3 bispecific antibody) described herein. In other embodiments, a method of inhibiting metastasis of cells expressing CD70 is provided in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a CD70 antibody (e.g., a CD70-CD3 bispecific antibody) described herein. In other embodiments, a method of inducing tumor regression in malignant cells is provided in a subject, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a CD70 antibody (e.g., a CD70-CD3 bispecific antibody) described herein.

[0206] In some embodiments, the antibody according to the present invention (e.g., a CD70-CD3 bispecific antibody) can be used in the manufacture of a pharmaceutical for the treatment of cancer in a patient in need thereof.

[0207] In some embodiments, the treatment can be combined with one or more treatments for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, targeted therapy, vaccine therapy, dendritic cell therapy, gene therapy, hormone therapy, surgical resection, laser therapy, and radiation therapy.

[0208] For example, in some embodiments, the CD70 antibodies of the invention (e.g., CD70-CD3 bispecific antibodies) are administered to a patient in combination with (e.g., before, simultaneously, or after) a small molecule tyrosine kinase inhibitor (TKI) such as sunitinib and pazopanib that target vascular endothelial growth factor (VEGF) receptors, a monoclonal antibody that targets VEGF such as bevacizumab, temsirolimus which is an inhibitor of mammalian target of Rapamycin (mTOR), and treatment with high-dose IL-2. In some embodiments, the CD70 antibodies of the invention (e.g., CD70-CD3 bispecific antibodies) are administered to a patient in combination with one or more of the following: anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, or PF-06801591), anti-PD-L1 antibodies (e.g., avelumab, atezolizumab or durvalumab), anti-OX40 antibodies (e.g., PF-04518600), anti-4-1BB antibodies (e.g., PF-05082566), anti-MCSF antibodies (e.g., PD-0360324), anti-GITR antibodies, and / or anti-TIGIT antibodies.

[0209] Administration of the antibodies (e.g., monospecific or bispecific) according to the invention may be carried out by any convenient method including aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intracranially, intra-articularly, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the antibody composition of the invention is preferably administered by intravenous injection.

[0210] In some embodiments, administration of the antibody (e.g., monospecific or bispecific) may include administration of, for example, about 0.01 to about 20 mg per kg of body weight, including all integer values of mg per kg within that range. In some embodiments, administration of the antibody may include administration of about 0.1 to 10 mg per kg of body weight, including all integer values of mg per kg within that range. The antibody may be administered in one or more doses. In some embodiments, the effective amount of the antibody may be administered as a single dose. In some embodiments, the effective amount of the antibody may be administered as multiple doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the patient. While individual needs vary, determination of the optimal range of the effective amount of a given antibody (e.g., monospecific or bispecific) for a particular disease or condition is within the skill of the art. The effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administered depends on the age, health and weight of the recipient, the type of co-therapy if any, the frequency of treatment, and the nature of the desired effect. In some embodiments, the effective amount of the heteromultimeric antibody or composition comprising the antibody is administered parenterally. In some embodiments, the administration may be intravenous administration. In some embodiments, the administration may be performed directly by intratumoral injection.

[0211] In some embodiments, the anti-CD70 antibodies provided herein may be used for diagnostic purposes, and in such assays, CD70 protein in a sample (e.g., immunohistochemical assay) or in a patient is identified.

[0212] Composition In one aspect, there is provided a pharmaceutical composition comprising the antibody (monospecific or bispecific) of the present invention or a portion thereof in a pharmaceutically acceptable carrier. In certain embodiments, the polypeptide of the present invention may be in a neutral form (including zwitterionic), or may exist as a positively charged or negatively charged species. In some embodiments, the polypeptide may complex with a counterion to form a "pharmaceutically acceptable salt", which are referred to as complexes comprising one or more polypeptides and one or more counterions, where the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

[0213] The antibody (e.g., monospecific or bispecific) or a portion thereof may be administered alone, or in combination with one or more other polypeptides of the present invention, or in combination with one or more other agents (or as any combination thereof). Thus, the pharmaceutical compositions, methods and uses of the present invention also encompass embodiments of combination (co-administration) with other active agents as detailed below.

[0214] As used herein, with respect to an antibody of the invention and one or more other therapeutic agents, the terms "co-administered", "co-administering", and "administered in combination" are intended to mean, refer to, and include the following: (i) simultaneous administration of the combination of the antibody and therapeutic agent disclosed herein to a patient in need of treatment, where the components are formulated together in a single dosage form and the components are released to the patient substantially simultaneously; (ii) substantially simultaneous administration of the combination of the antibody and therapeutic agent disclosed herein to a patient in need of treatment, where the components are formulated separately in distinct dosage forms and are taken by the patient substantially simultaneously, and at that time the components are released to the patient substantially simultaneously; (iii) sequential administration of the combination of the antibody and therapeutic agent disclosed herein to a patient in need of treatment, where the components are formulated separately in distinct dosage forms and are taken by the patient sequentially with a significant interval between each administration, and at that time the components are released to the patient at substantially different times; and (iv) sequential administration of the combination of the antibody and therapeutic agent disclosed herein to a patient in need of treatment, where the components are formulated together in a single dosage form that releases the components in a controlled manner, and are released to the patient at the same time and / or at different times, simultaneously, sequentially, and / or repetitively, and in this case each portion may be administered by the same route or different routes.

[0215] Generally, the antibodies (monospecific or bispecific) or portions thereof disclosed herein are suitable for administration as formulations associated with one or more pharmaceutically acceptable excipients. As used herein, the term "excipient" is used to describe any component other than the compounds of the invention. The choice of excipient depends largely on factors such as, for example, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyalcohols such as methanol, sorbitol, or sodium chloride. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers that enhance the shelf life or effectiveness of the antibody.

[0216] The pharmaceutical compositions of the invention, and methods for their preparation, will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably manufactured under GMP conditions.

[0217] The pharmaceutical composition of the present invention may be prepared, packaged, or sold in large quantities as a single unit dose or as multiple single unit doses. As used herein, "unit dose" refers to an individual quantity of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject, or a convenient fraction of such a dose, such as half or one-third of such a dose. Any method of administration of a peptide, protein, or antibody acceptable in the art may be suitably employed for the heterodimeric proteins and portions thereof disclosed herein.

[0218] The pharmaceutical composition of the present invention is typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by the formation of a physical hole in the tissue of a subject and any route of administration of the pharmaceutical composition through such a hole in the tissue, and thus generally results in direct administration into the bloodstream, muscle, or internal organ. Thus, but not limited to, parenteral administration includes administration of the pharmaceutical composition by injection of the composition, administration of the pharmaceutical composition by application of the composition through a surgical incision, administration of the pharmaceutical composition by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is expected to include, but is not limited to, injection or infusion subcutaneously, intraperitoneally, intramuscularly, intrasternal, intravenously, intraarterially, intrathecally, intraventricularly, intraurethrally, intracranially, into a bursa, and renal dialysis infusion methods. Preferred embodiments include the intravenous route and the subcutaneous route.

[0219] Formulations of pharmaceutical compositions suitable for parenteral administration typically generally contain the active ingredient in combination with a pharmaceutically acceptable carrier such as, for example, sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form in, for example, ampoules or multi-dose containers containing a preservative. Parenteral formulations include, but are not limited to, suspensions, solutions, emulsions, pastes, etc. in oily vehicles or aqueous vehicles. Such formulations may further contain one or more additional ingredients including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a parenteral formulation, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution using a suitable vehicle (e.g., sterile pyrogen-free water), and then the reconstituted composition is administered parenterally. Parenteral formulations also include aqueous solutions that may contain excipients such as, for example, salts, carbohydrates, and buffers (preferably up to a pH of 3 to 9), but for some applications may be more suitably formulated as a sterile non-aqueous solution or as a dry form for use in combination with a suitable vehicle such as, for example, sterile pyrogen-free water. Examples of forms of parenteral administration include, for example, solutions or suspensions in sterile aqueous solutions such as aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered if desired. Other useful parenteral formulations include formulations containing the active ingredient in microcrystalline form or liposome preparations. Parenteral formulations may be formulated to be immediate release and / or modified release. Modified release formulations include controlled release formulations, delayed release formulations, sustained release formulations, pulsatile release formulations, targeted release formulations, and programmed release formulations. For example, in one example, a sterile injectable solution can be prepared by combining, as necessary, one or a combination of the ingredients listed above with the necessary amount of a heterodimeric protein such as, for example, a bispecific antibody in a suitable solvent and then filter sterilizing.Generally, the dispersion is prepared by combining the active compound with a sterile vehicle containing a basic dispersion medium and other ingredients required from the substances listed above. In the case of sterile powders for the preparation of sterile injection solutions, the preferred methods of preparation are the vacuum drying method and the freeze-drying method, by which powders of the active ingredient and any additional desired ingredients are obtained from its previously sterile-filtered solution. By using coatings such as lecithin, for example, the required particle size in the case of the dispersion can be maintained, and by using surfactants, the appropriate fluidity of the solution can be maintained. Sustained absorption of the injectable composition can be brought about, for example, by containing in the composition agents that delay absorption such as monostearates and gelatin.

[0220] The dosage regimen may be adjusted to provide the optimal desired response. For example, a single bolus may be administered, the dosage may be administered in divided doses over time, or the dosage may be relatively decreased or increased as indicated by the requirements of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the parenteral composition in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unitary forms of dosage for the patient / subject to be treated, each unit being associated with the required pharmaceutical carrier and containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect. The specifications for the unit dosage forms of the present invention are generally determined by, and directly dependent on, the inherent characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the individual's therapeutic susceptibility, as well as the limitations inherent in the art of compound formulation of the active compound.

[0221] Therefore, one of ordinary skill in the art will recognize that, based on the disclosure provided herein, dosages and dosing regimens will be adjusted according to methods known in the art of treatment. That is, the maximum tolerated dose can be readily established, and the effective amount that provides a detectable therapeutic benefit to the patient can also be determined, along with the time requirements for administration of each agent that provides a detectable therapeutic benefit to the patient. Accordingly, specific dosages and dosing regimens are exemplified herein, but these exemplifications do not limit the dosages and dosing regimens that can be provided to patients in the practice of the present invention.

[0222] It should be noted that the values of the dosages may vary with the type and severity of the condition being alleviated and may include single or multiple administrations. For any particular subject, the specific dosing regimen should be adjusted over time according to the individual needs and the professional judgment of the person implementing or supervising the administration of the composition, and it is further understood that the dosage ranges described herein are merely exemplary and do not limit the scope or practice of the claimed composition. Further, dosing regimens using the compositions of the present invention can be based on a variety of factors, including the type of disease, the age, weight, gender, medical condition, severity of the condition, route of administration, and the specific antibody employed. Accordingly, dosing regimens can vary widely but can be routinely determined using standard methods. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or experimental values. Therefore, the present invention encompasses dose escalation within the same patient determined by one of ordinary skill in the art. It is understood that the determination of appropriate dosages and regimens is known in the relevant art and will be encompassed by one of ordinary skill in the art in view of the teachings disclosed herein.

[0223] Generally, with respect to the administration of the antibodies (monospecific or bispecific) disclosed herein, candidate doses can be administered daily, weekly, every other week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every ten weeks, every twelve weeks, or more than once every twelve weeks. For repeated administration over several days, depending on the condition, treatment is continued until a desirable suppression of symptoms occurs or a sufficient therapeutic level is achieved, for example, until cancer-related symptoms are reduced. The progress of this treatment is readily monitored by conventional techniques and assays. The dosing regimen (including the anti-FLT monospecific or bispecific antibody used) can vary over time.

[0224] In some embodiments, the candidate dose is administered daily at a dose in the approximate range of from 1 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors described above. For example, a daily dose of about 0.01 mg / kg, about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, and about 25 mg / kg may be used.

[0225] In some embodiments, the candidate dose is administered weekly at a dose in the approximate range of from 1 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors described above. For example, a weekly dose of about 0.01 mg / kg, about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, and about 30 mg / kg may be used.

[0226] In some embodiments, the candidate dosage is administered every other week at a dosage in the approximate range of 1 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors described above. For example, an every-other-week dosage of about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, and about 30 mg / kg may be used.

[0227] In some embodiments, the candidate dosage is administered every three weeks at a dosage in the approximate range of 1 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors described above. For example, an every-three-week dosage of about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, and about 50 mg / kg may be used.

[0228] In some embodiments, the candidate dosage is administered monthly or every four weeks at a dosage in the approximate range of 1 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg to 30 mg / kg to 100 mg / kg or more, depending on the factors described above. For example, a monthly dosage of about 0.1 mg / kg, about 0.3 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, and about 50 mg / kg may be used.

[0229] In other embodiments, the candidate dosage is administered daily at a dosage in the range of about 0.01 mg to about 1200 mg or more, depending on the factors described above. For example, a daily dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, or about 1200 mg may be used.

[0230] In other embodiments, the candidate dosage is administered weekly at a dosage in the range of about 0.01 mg to about 2000 mg or more, depending on the factors described above. For example, a weekly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg may be used.

[0231] In other embodiments, the candidate dosage is administered every two weeks at a dosage in the range of about 0.01 mg to about 2000 mg or more, depending on the factors described above. For example, a bi-weekly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg may be used.

[0232] In other embodiments, the candidate dosage is administered every three weeks at a dosage in the range of about 0.01 mg to about 2500 mg or more, depending on the factors described above. For example, a dosage administered every three weeks of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, or about 2500 mg may be used.

[0233] In other embodiments, the candidate dosage is administered every four weeks or monthly at a dosage in the range of about 0.01 mg to about 3000 mg or more, depending on the factors described above. For example, a monthly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or about 3000 mg may be used.

[0234] Kit The present invention also provides a kit for use in the present method. The kit of the present invention includes one or more containers, which contain the antibodies described herein (e.g., monospecific or bispecific), and instructions regarding use according to any of the methods described herein. Generally, these instructions include descriptions regarding the administration of the antibody protein for the above-described therapeutic treatment.

[0235] Instructions regarding the use of the antibodies described herein (e.g., monospecific or bispecific) generally include information regarding the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dosage, a bulk package (e.g., a multiple dosage package), or a sub-unit dosage. Instructions supplied in the kits of the invention are often written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions executed on a magnetic storage disk or an optical storage disk) are also acceptable.

[0236] The kits of the invention are in a suitable packaging material. Suitable packaging materials include, but are not limited to, vials, bottles, flasks, flexible packaging materials (e.g., sealed Mylar bags or plastic bags), etc. Further, packaging materials for use in combination with certain devices, such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices such as mini-pumps, for example, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one of the active agents in the composition is a bispecific antibody. The container may further contain a second pharmaceutically active agent.

[0237] The kit may optionally provide additional components, such as, for example, buffers and explanatory information. Usually, the kit includes a container and a label or package insert on or associated with the container.

[0238] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims.

Example

[0239] Example 1: Determination of the kinetics of human CD70 and the interaction of CD70 antibodies at 37°C and affinity The kinetics and affinity of the anti-CD70 antibodies disclosed herein can be measured on a Biacore T200 surface plasmon resonance biosensor (GE Lifesciences, Piscataway, NJ).

[0240] Example 2: T cell-mediated killing of RCC cell lines using CD70-CD3 bispecific IgG in vitro Human anti-CD70 antibody and human anti-CD3 (h2B4-VH-hnps VL-TK(H2B4)) antibody were expressed as human IgG2dA_D265A engineered with EEEE on one arm and RRRR on the other arm for bispecific exchange at positions 223, 225, and 228 (e.g., (C223E or C223R), (E225E or E225R), and (P228E or P228R)) in the hinge region of human IgG2 (SEQ ID NO: 279), and at position 409 or 368 (e.g., K409R or L368E (EU numbering scheme)) in the CH3 region. The CD70 / CD3 bispecific antibody also has a mutation from D to A at position 265 (EU numbering scheme).

[0241] CD3+ T cells derived from human PBMCs were negatively selected using the Pan T Cell Isolation Kit, human (Miltenyi, San Diego, CA). Target-expressing (786-O) cells and CD3+ T cells were seeded onto clear U-bottom plates at 20,000 cells and 100,000 cells / well, respectively. Cells were treated with serially diluted bispecific antibodies 8-fold. Depletion of RCC cells was determined by flow cytometry analysis 24 hours after treatment. Cell depletion was measured relative to control-treated cells. EC50 was calculated by Prism software.

[0242] Example 3: CD70-CD3 bispecific IgG induces tumor regression in a subcutaneous RCC xenograft model NOD scid gamma (NSG) mice are subcutaneously implanted with 786-O tumors. When the tumor volume reaches 200 mm 3 20 million expanded T cells are intraperitoneally administered to each mouse. Two days later, anti-CD70 bispecific antibody is intravenously administered via tail vein injection at 300, 100, or 30 μg / mL to determine the optimal dosage of the bispecific antibody.

[0243] Materials and methods NOD scid gamma (NSG) mice are prepared by shaving and subcutaneously implanted with tumors on the right flank. 786-O tumor cells known to express CD70 are expanded in RPMI supplemented with 10% FBS. On day 0, 786-O cells are resuspended at the required concentration in serum-free RPMI and 5 million cells are injected per animal. The tumor cells are subcutaneously injected in 100 μL of serum-free RPMI mixed with 100 μL of Matrigel (Corning) per animal. Immediately after tumor implantation, the baseline body weight on day 0 is recorded for all animals. Tumors are measured twice a week starting on day 9 using Digimatic Calipers (Mitutoyo), and body weight is recorded. On day 14, when the tumor reaches 200 mm 3 (standard error 8.39), 40 tumor-bearing mice are randomized into 4 groups of 10 mice each. T cells are lysed, expanded, and then resuspended at the required concentration in serum-free RPMI, and 20 million T cells are injected per animal. The T cells are intraperitoneally injected in 200 μL of serum-free RPMI per animal. Two days later, bispecific antibody is intravenously administered via the tail vein at 300, 100, or 30 μg / mL per animal. Tumors are measured twice a week and body weight is recorded until the non-treated group reaches the test endpoint (tumor volume of 1500 mm 3 ).

[0244] The tumor volume (mean and error SEM) is plotted on GraphPad Prism, and statistics are calculated using repeated measures results with one-way analysis of variance.

[0245] The disclosed teachings have been described with reference to various uses, methods, kits and compositions, but it will be understood that various changes and modifications can be made without departing from the teachings herein and the inventions claimed below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. Although the present teachings have been described from the perspective of these exemplary embodiments, those skilled in the art will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

[0246] All references cited herein, including references such as patents, patent applications, papers, textbooks, and references cited therein, are hereby incorporated by reference in their entirety to the extent that they are not already so incorporated. If one or more of the incorporated documents and similar materials are different from or conflict with the present application, the present application controls, including but not limited to defined terms, term usage, described techniques, or the like.

[0247] The foregoing description and examples detail certain embodiments of the invention and describe the best mode contemplated by the inventors. However, it will be understood that the invention may be practiced in many ways, and the invention should be construed in accordance with the appended claims and any equivalents thereof, however detailed the foregoing may be in text.

Claims

1. An isolated antibody that specifically binds to Cluster of Differentiation 70 (CD70), wherein the antibody comprises: a) (i) A VH complementarity-determining region 1 (CDR1) comprising the sequence shown in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376, 380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405, 406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459 or 460; (ii) a VH comprising the sequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456, 461 or 462a heavy chain variable (VH) region comprising: i) a CDR2; and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and / or b) (i) a VL CDR1 comprising a sequence shown in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524 or 527; (ii) a VL CDR2 comprising a sequence shown in SEQ ID NO: 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525 or 528; and (iii) a VL CDR3 comprising a sequence shown in SEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529, and a light chain variable (VL) region comprising the same, an isolated antibody.

2. An isolated antibody that specifically binds to Cluster of Differentiation 70 (CD70), wherein the antibody comprises: a) a VH region comprising VH CDR1, VH CDR2 and VH CDR3 of the VH sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331, and / or b) a VL region comprising VL CDR1, VL CDR2 and VL CDR3 of the VL sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328 or 330, an isolated antibody comprising the same. **Claim 3** An isolated antibody that specifically binds to CD70 and competes with the antibody according to claim 1. **Claim 4** A bispecific antibody, wherein the bispecific antibody is a full-length antibody, comprises a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen, and a second antibody variable domain of the bispecific antibody that can recruit the activity of the human immune effector cell by specifically binding to an effector antigen located on the human immune effector cell, wherein the first antibody variable domain comprises a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, or 331, and / or a light chain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, or 330, a bispecific antibody. [

5. ] A bispecific antibody, wherein the bispecific antibody is a full-length antibody, comprises a first antibody variable domain of the bispecific antibody that specifically binds to a target antigen, and a second antibody variable domain of the bispecific antibody that can recruit the activity of the human immune effector cell by specifically binding to an effector antigen located on the human immune effector cell, wherein the first antibody variable domain is a) (i) A VH complementarity-determining region 1 (CDR1) comprising the sequence shown in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376, 380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405, 406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459 or 460; (ii) A VH comprising the sequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456, 461 or 462a heavy chain variable (VH) region comprising: i) a CDR2; and iii) a VH CDR3 comprising a sequence shown in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; and / or b) (i) A VL CDR1 comprising the sequence shown in SEQ ID NO: 93, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524 or 527; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525 or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526 or 529, a bispecific antibody comprising a variable light (VL) region. **Claim 6** The bispecific antibody according to claim 5, wherein the second antibody variable domain specifically binds to the effector antigen CD3. **Claim 7** The second antibody variable domain is a) (i) A VH complementarity determining region 1 (CDR1) comprising the sequence shown in SEQ ID NO: 267, 268, 269; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 270 or 271; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 272, a variable heavy (VH) region: and / or b) (i) A VL CDR1 comprising the sequence shown in SEQ ID NO: 273; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 274; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 275, a bispecific antibody according to claim 6, comprising a variable light (VL) region.

8. The bispecific antibody according to claim 4, wherein both the first and second antibody variable domains of the heterodimeric protein contain amino acid modifications at positions 223, 225 and 228 in the hinge region of human IgG2 (SEQ ID NO: 279), and at position 409 or 368 in the CH3 region (EU numbering scheme).

9. The bispecific antibody according to claim 8, further comprising amino acid modifications at one or more of positions 265, 330 and 331 of the human IgG2.

10. A nucleic acid encoding the antibody according to any one of claims 1 to 9.

11. A vector comprising the nucleic acid according to claim 10.

12. A host cell comprising the nucleic acid according to claim 10.

13. The antibody according to any one of claims 1 to 9 for use as a medicament.

14. The antibody according to claim 13, wherein the medicament is for use in the treatment of a CD70-related cancer selected from the group consisting of renal cell carcinoma, glioblastoma, such as glioma including low-grade glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), Waldenström's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

15. A method of treating a subject in need thereof, comprising: a) providing an antibody according to any one of claims 1 to 9; and b) administering the antibody to the subject.

16. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 9.

17. A method of treating a condition associated with malignant cells expressing CD70 in a subject, comprising administering to a subject in need thereof an effective amount of an antibody according to any one of claims 1 to 9, or a pharmaceutical composition according to claim 16.

18. The method according to claim 17, wherein the condition is cancer.

19. The method according to claim 18, wherein the cancer is a CD70-related cancer selected from the group consisting of renal cell carcinoma, glioblastoma, such as glioma including low-grade glioma, non-Hodgkin lymphoma (NHL), Hodgkin's disease (HD), Waldenström's hypergammaglobulinemia, acute myeloid leukemia, multiple myeloma, diffuse large B-cell lymphoma, follicular lymphoma, or non-small cell lung cancer.

20. A method for inhibiting tumor growth or progression in a subject having malignant cells that express CD70, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to claim 16 for the subject.

21. A method for inhibiting metastasis of malignant cells that express CD70 in a subject, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to claim 16 for the subject.

22. A method for inducing tumor regression in a subject having malignant cells that express CD70, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to claim 16 for the subject.

23. A method for producing an antibody, the method comprising culturing the host cell according to claim 12 under conditions that result in production of the antibody, and isolating the antibody from the host cell or culture.

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