Binding molecules for bcma and cd3

0001background BCMA (B cell maturation antigen, TNFRSF17, CD269) is a transmembrane protein belonging to the TNF receptor superfamily. BCMA was first reported as an integral membrane protein of the Golgi apparatus of human mature B lymphocytes, i.e., an intracellular protein (Gras et al., (1995) International Immunol 7 (7): 1093-1105). Suggested that BCMA may play an important role in B cell development and homeostasis. The findings of Gras et al. May also be associated with the fact that the BCMA protein described by Gras et al. Is a fusion protein between BCMA and IL-2 by chromosomal translocation. However, at the same time, BCMA is a ligand for its ligand, BAFF (B cell activating factor), which is also called TALL-1 or TNFSF13B, and probably from an essential interaction with APRIL (proliferation-inducing ligand) of B cells. It has been identified as an essential B cell marker for development and homeostasis (Schliemann et al., (2001) Science 293 (5537): 2111-2114).

0002Expression of BCMA is confined to B cell lines, predominantly present in plasma cells and precursor cells, to some extent in memory B cells, but substantially absent in peripheral and naive B cells. BCMA is also expressed in multiple myeloma (MM) cells. Together with its family members, the transmembrane activator / cyclophilin ligand interacting factor (TACI) and the TNF family receptor B cell activating factor (BAFF-R), BCMA is associated with humoral immunity, B cell development and It regulates various aspects of homeostasis. BCMA expression is found slightly later in B cell differentiation and contributes to long-term survival of plasmablasts and plasma cells in the bone marrow. Specific deletion of the BCMA gene in mice does not affect the production of mature B cells, the quality and scale of the humoral immune response, the formation of germinal centers and the production of short-lived plasma cells. However, such mice significantly reduced the number of long-lived plasma cells in the bone marrow, indicating that BCMA is important for their survival (O'Connor et al., 2004).

0003As evidenced by this finding, BCMA also supports the growth and survival of multiple myeloma (MM) cells. Novak et al. Found that MM cell lines and newly isolated MM cells express BCMA and TACI proteins on their cell surface and that BAFF-R protein expression on their cell surface varies. Found (Novak et al., (2004) Blood 103 (2): 689-694).

0004Multiple myeloma (MM) is the second most common hematological malignancies, accounting for 2% of all cancer deaths. MM is a heterogenous disease, mainly by chromosomal translocations, especially by t (11; 14), t (4; 14), t (8; 14), del (13), del (17). Caused (Drach et al. (1998) Blood 92 (3): 802-809; Gertz et al., (2005) Blood 106 (8): 2837-2840; Facon et al., (2001) Blood 97 (6) ): 1566-1571). Patients with MM may experience a variety of disease-related symptoms due to the psychosocial burden of diagnosing bone marrow infiltration, bone destruction, renal failure, immunodeficiency and cancer. As of 2006, MM had a 5-year relative survival rate of approximately 34%, emphasizing that MM is a difficult disease to treat and there is currently no curative option.

0005Breakthrough new therapies such as chemotherapy and stem cell transplantation approaches have become available and improved survival, but these often have unwanted side effects that make MM still difficult to treat (M). Lee et al., (2004) J Natl Compr Canc Netw 8 (4): 379-383). To date, the two most frequently used treatment options for patients with multiple myeloma are steroids, thalidomide, lenalidomide, bortezomib or a combination of various cytotoxic agents, and autologous stem cell transplantation in young patients. Is a high-dose chemotherapy concept using. Most transplants are autologous, that is, they use the patient's own cells. Although such transplants are not curative, they have been shown to prolong lifespan in selected patients. They can be given as initial treatment for newly diagnosed patients or at the time of recurrence. Occasionally, two or more transplants may be recommended for selected patients to better control the disease.

0006Chemotherapeutic agents used to treat this disease are cyclophosphamide, doxorubicin, vincristine and melphalan, and immunomodulators such as thalidomide (Thalomid®), lenalidomide (Revlimid®), Combination therapy with bortezomib (Velcade®) and corticosteroids (eg, dexamethasone) for the treatment of myeloma in both newly diagnosed patients and patients with advanced disease who have failed chemotherapy or transplantation Appeared as an important option.

0007The treatments currently used are usually not curative. Stem cell transplantation may not be an option for many patients due to old age, the presence of other serious illnesses, or other physical constraints. Chemotherapy controls multiple myeloma only partially and rarely leads to complete remission. Therefore, there is an urgent need for new and innovative treatments.

0008Bellucci et al. (Blood, 2005; 105 (10)) identified BCMA-specific antibodies in patients with multiple myeloma after undergoing donor lymphocyte infusion (DLI). The sera of these patients could mediate BCMA-specific cell lysis by ADCC and CDC, which was detected only in patients with an antitumor response (4/9) and not in non-responding patients. (0/6). The authors speculate that induction of BCMA-specific antibodies contributes to the elimination of myeloma cells and long-term remission of patients.

0009Ryan et al. (Mol. Cancer Ther. 2007; 6 (11)) produced antagonistic BCMA-specific antibodies that prevent activation of NF-κB associated with strong survival-promoting signaling pathways in normal and malignant B cells. I reported that I did. In addition, the antibody showed strong antibody-dependent cellular cytotoxicity (ADCC) against multiple myeloma cell lines in vitro, significantly enhanced by Fc engineering.

0010Other approaches to combating hematological tumors or autoimmune disorders include BAFF and APRIL, the ligands of the TNF ligand superfamily, and their receptors TACI, BAFF-R and BCMA activated by BAFF and / or APRIL. It focuses on the interaction between them. For example, Zymogenetics, Inc. produced atacicept (TACI-Ig), which neutralizes both of these ligands and prevents receptor activation by fusing the Fc domain of human immunoglobulin to TACI. Atacicept is currently in clinical trials for the treatment of systemic lupus erythematosus (SLE, phase III), multiple sclerosis (MS, phase II) and rheumatoid arthritis (RA, phase II), as well as B-cell malignant chronic lymphocytic lymph. It is under Phase I clinical trials for the treatment of lupus erythematosus (CLL), non-Hodgkin's lymphoma (NHL) and MM. In preclinical studies, atacicept was used in vitro (Moreaux et al, Blood, 2004, 103) and in vivo (Yaccoby et al, Leukemia, 2008, 22, 22). In 406-13), the growth and survival of primary MM cells and MM cell lines were reduced, demonstrating the association between TACI ligand and MM cells. Since most MM cells and derived cell lines express BCMA and TACI, both receptors are thought to contribute to ligand-mediated growth and survival. These data suggest that antagonistic effects on both BCMA and TACI may be beneficial in the treatment of plasmacytosis. In addition, BCMA-specific antibodies that cross-react with TACI have also been described (WO 02/066516).

0011Human Genome Sciences and GlaxoSmithKline have developed an antibody that targets BAFF called belimumab. Belimumab blocks soluble BAFF from binding to its receptors on B cells, BAFF-R, BCMA and TACI. Belimumab does not bind directly to B cells, but by binding to BAFF, belimumab inhibits the survival of B cells, including autoreactive B cells, and reduces the differentiation of B cells into immunoglobulin-producing plasma cells. ..

0012Although BCMA; BAFF-R and TACI, the B-cell receptors belonging to the TNF receptor superfamily and their ligands BAFF and APRIL, are used in the treatment of cancer and / or autoimmune disorders, such It is still desired that additional options for the treatment of various medical conditions become available.

0013Therefore, in the present specification, the means and methods for solving this problem are at least bispecific binding having one binding domain for cytotoxic cells, that is, cytotoxic T cells, and a second binding domain for BCMA. Provided in molecular form.<u style="single">[Invention 1001]</u><u style="single"> A binding molecule that is at least bispecific and contains the first and second binding domains.</u><u style="single">(a) The first binding domain can bind to epitope cluster 3 and epitope cluster 4 of BCMA, and</u><u style="single">(b) The second binding domain can bind to the T cell CD3 receptor complex,</u><u style="single">Here, the epitope cluster 3 of BCMA corresponds to the amino acid residues 24 to 41 of the sequence shown in SEQ ID NO: 1002, and the epitope cluster 4 of BCMA corresponds to the amino acid residues 42 to 42 of the sequence shown in SEQ ID NO: 1002. Binding molecule corresponding to 54.</u><u style="single">[Invention 1002]</u><u style="single"> The binding molecule of the invention 1001 in which the first binding domain cannot bind to the chimeric extracellular domain of BCMA shown in SEQ ID NO: 1015.</u><u style="single">[Invention 1003]</u><u style="single"> A binding molecule of the invention 1001 or 1002, wherein the first binding domain can further bind to macaque BCMA.</u><u style="single">[Invention 1004]</u><u style="single"> A binding molecule of any of the inventions, wherein the second binding domain is capable of binding to CD3 epsilon.</u><u style="single">[Invention 1005]</u><u style="single"> The binding molecule of any of the inventions, wherein the second binding domain is capable of binding to human CD3 and macaque CD3.</u><u style="single">[Invention 1006]</u><u style="single"> The binding molecule of any of the inventions, wherein the first and / or second binding domain is derived from an antibody.</u><u style="single">[Invention 1007]</u><u style="single"> (scFv)</u>_{<u style="single">2</u>}<u style="single">, (Single domain mAb)</u>_{<u style="single">2</u>}<u style="single">, ScFv-A binding molecule of the invention 1006 selected from the group consisting of single domain mAbs, diabodies and oligomers thereof.</u><u style="single">[Invention 1008]</u><u style="single"> The first binding domain is</u><u style="single">(a) CDR-H1 shown in SEQ ID NO: 231, CDR-H2 shown in SEQ ID NO: 232, CDR-H3 shown in SEQ ID NO: 233, CDR-L1 shown in SEQ ID NO: 234. , SEQ ID NO: 235, CDR-L2 and SEQ ID NO: 236, CDR-L3;</u><u style="single">(b) CDR-H1 shown in SEQ ID NO: 241, CDR-H2 shown in SEQ ID NO: 242, CDR-H3 shown in SEQ ID NO: 243, and CDR-L1 shown in SEQ ID NO: 244. , SEQ ID NO: 245, CDR-L2 and SEQ ID NO: 246, CDR-L3;</u><u style="single">(c) CDR-H1 shown in SEQ ID NO: 251, CDR-H2 shown in SEQ ID NO: 252, CDR-H3 shown in SEQ ID NO: 253, CDR-L1 shown in SEQ ID NO: 254. , SEQ ID NO: 255, CDR-L2 and SEQ ID NO: 256, CDR-L3;</u><u style="single">(d) CDR-H1 shown in SEQ ID NO: 261, CDR-H2 shown in SEQ ID NO: 262, CDR-H3 shown in SEQ ID NO: 263, and CDR-L1 shown in SEQ ID NO: 264. , SEQ ID NO: 265, CDR-L2 and SEQ ID NO: 266, CDR-L3;</u><u style="single">(e) CDR-H1 shown in SEQ ID NO: 271, CDR-H2 shown in SEQ ID NO: 272, CDR-H3 shown in SEQ ID NO: 273, CDR-L1 shown in SEQ ID NO: 274. , SEQ ID NO: 275, CDR-L2 and SEQ ID NO: 276, CDR-L3;</u><u style="single">(f) CDR-H1 shown in SEQ ID NO: 281, CDR-H2 shown in SEQ ID NO: 282, CDR-H3 shown in SEQ ID NO: 283, CDR-L1 shown in SEQ ID NO: 284. , SEQ ID NO: 285, CDR-L2 and SEQ ID NO: 286, CDR-L3;</u><u style="single">(g) CDR-H1 shown in SEQ ID NO: 291, CDR-H2 shown in SEQ ID NO: 292, CDR-H3 shown in SEQ ID NO: 293, CDR-L1 shown in SEQ ID NO: 294. , SEQ ID NO: 295, CDR-L2 and SEQ ID NO: 296, CDR-L3;</u><u style="single">(h) CDR-H1 shown in SEQ ID NO: 301, CDR-H2 shown in SEQ ID NO: 302, CDR-H3 shown in SEQ ID NO: 303, and CDR-L1 shown in SEQ ID NO: 304. , SEQ ID NO: 305, CDR-L2 and SEQ ID NO: 306, CDR-L3;</u><u style="single">(i) CDR-H1 shown in SEQ ID NO: 391, CDR-H2 shown in SEQ ID NO: 392, CDR-H3 shown in SEQ ID NO: 393, CDR-L1 shown in SEQ ID NO: 394. , SEQ ID NO: 395, CDR-L2 and SEQ ID NO: 396, CDR-L3;</u><u style="single">(k) CDR-H1 shown in SEQ ID NO: 401, CDR-H2 shown in SEQ ID NO: 402, CDR-H3 shown in SEQ ID NO: 403, CDR-L1 shown in SEQ ID NO: 404. , SEQ ID NO: 405, CDR-L2 and SEQ ID NO: 406, CDR-L3;</u><u style="single">(l) CDR-H1 shown in SEQ ID NO: 411, CDR-H2 shown in SEQ ID NO: 412, CDR-H3 shown in SEQ ID NO: 413, CDR-L1 shown in SEQ ID NO: 414. , SEQ ID NO: 415, CDR-L2 and SEQ ID NO: 416, CDR-L3;</u><u style="single">(m) CDR-H1 shown in SEQ ID NO: 421, CDR-H2 shown in SEQ ID NO: 422, CDR-H3 shown in SEQ ID NO: 423, CDR-L1 shown in SEQ ID NO: 424. , SEQ ID NO: 425, CDR-L2 and SEQ ID NO: 426, CDR-L3;</u><u style="single">(n) CDR-H1 shown in SEQ ID NO: 431, CDR-H2 shown in SEQ ID NO: 432, CDR-H3 shown in SEQ ID NO: 433, CDR-L1 shown in SEQ ID NO: 434. , SEQ ID NO: 435, CDR-L2 and SEQ ID NO: 436, CDR-L3;</u><u style="single">(o) CDR-H1 shown in SEQ ID NO: 441, CDR-H2 shown in SEQ ID NO: 442, CDR-H3 shown in SEQ ID NO: 443, CDR-L1 shown in SEQ ID NO: 444. , SEQ ID NO: 445, CDR-L2 and SEQ ID NO: 446, CDR-L3;</u><u style="single">(p) CDR-H1 shown in SEQ ID NO: 451, CDR-H2 shown in SEQ ID NO: 452, CDR-H3 shown in SEQ ID NO: 453, CDR-L1 shown in SEQ ID NO: 454. , SEQ ID NO: 455, CDR-L2 and SEQ ID NO: 456, CDR-L3;</u><u style="single">(q) CDR-H1 shown in SEQ ID NO: 461, CDR-H2 shown in SEQ ID NO: 462, CDR-H3 shown in SEQ ID NO: 463, CDR-L1 shown in SEQ ID NO: 464. , SEQ ID NO: 465, CDR-L2 and SEQ ID NO: 466, CDR-L3;</u><u style="single">(r) CDR-H1 shown in SEQ ID NO: 471, CDR-H2 shown in SEQ ID NO: 472, CDR-H3 shown in SEQ ID NO: 473, CDR-L1 shown in SEQ ID NO: 474. , SEQ ID NO: 475, CDR-L2 and SEQ ID NO: 476, CDR-L3;</u><u style="single">(s) CDR-H1 shown in SEQ ID NO: 481, CDR-H2 shown in SEQ ID NO: 482, CDR-H3 shown in SEQ ID NO: 483, CDR-L1 shown in SEQ ID NO: 484. , SEQ ID NO: 485, CDR-L2 and SEQ ID NO: 486, CDR-L3;</u><u style="single">(t) CDR-H1 shown in SEQ ID NO: 491, CDR-H2 shown in SEQ ID NO: 492, CDR-H3 shown in SEQ ID NO: 493, CDR-L1 shown in SEQ ID NO: 494. , SEQ ID NO: 495, CDR-L2 and SEQ ID NO: 496; and</u><u style="single">(u) CDR-H1 shown in SEQ ID NO: 501, CDR-H2 shown in SEQ ID NO: 502, CDR-H3 shown in SEQ ID NO: 503, CDR-L1 shown in SEQ ID NO: 504. , SEQ ID NO: 505, CDR-L2 and SEQ ID NO: 506, CDR-L3</u><u style="single">A binding molecule of any of the present invention comprising a VH region containing CDR-H1, CDR-H2 and CDR-H3 and a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of: ..</u><u style="single">[Invention 1009]</u><u style="single"> The first binding domain is SEQ ID NO: 237, SEQ ID NO: 247, SEQ ID NO: 257, SEQ ID NO: 267, SEQ ID NO: 277, SEQ ID NO: 287, SEQ ID NO: 297, SEQ ID NO: 307, SEQ ID NO: 397, SEQ ID NO: 407, SEQ ID NO: 417, SEQ ID NO: 427, SEQ ID NO: 437, SEQ ID NO: 447, SEQ ID NO: 457, SEQ ID NO : 467, SEQ ID NO: 477, SEQ ID NO: 487, SEQ ID NO: 497 and SEQ ID NO: 507. Binding molecule.</u><u style="single">[Invention 1010]</u><u style="single"> The first binding domain is SEQ ID NO: 238, SEQ ID NO: 248, SEQ ID NO: 258, SEQ ID NO: 268, SEQ ID NO: 278, SEQ ID NO: 288, SEQ ID NO: 298, SEQ ID NO: 308, SEQ ID NO: 398, SEQ ID NO: 408, SEQ ID NO: 418, SEQ ID NO: 428, SEQ ID NO: 438, SEQ ID NO: 448, SEQ ID NO: 458, SEQ ID NO : 468, SEQ ID NO: 478, SEQ ID NO: 488, SEQ ID NO: 498 and SEQ ID NO: 508. Binding molecule.</u><u style="single">[Invention 1011]</u><u style="single"> The first binding domain is</u><u style="single">(a) VH region shown by SEQ ID NO: 237 and VL region shown by SEQ ID NO: 238;</u><u style="single">(b) VH region shown by SEQ ID NO: 247 and VL region shown by SEQ ID NO: 248;</u><u style="single">(c) VH region shown by SEQ ID NO: 257 and VL region shown by SEQ ID NO: 258;</u><u style="single">(d) VH region shown by SEQ ID NO: 267 and VL region shown by SEQ ID NO: 268;</u><u style="single">(e) VH region shown by SEQ ID NO: 277 and VL region shown by SEQ ID NO: 278;</u><u style="single">(f) VH region shown by SEQ ID NO: 287 and VL region shown by SEQ ID NO: 288;</u><u style="single">(g) VH region shown by SEQ ID NO: 297 and VL region shown by SEQ ID NO: 298;</u><u style="single">(h) VH region shown by SEQ ID NO: 307 and VL region shown by SEQ ID NO: 308;</u><u style="single">(i) VH region shown by SEQ ID NO: 397 and VL region shown by SEQ ID NO: 398;</u><u style="single">(k) VH region shown by SEQ ID NO: 407 and VL region shown by SEQ ID NO: 408;</u><u style="single">(l) VH region shown by SEQ ID NO: 417 and VL region shown by SEQ ID NO: 418;</u><u style="single">(m) VH region shown by SEQ ID NO: 427 and VL region shown by SEQ ID NO: 428;</u><u style="single">(n) VH region shown by SEQ ID NO: 437 and VL region shown by SEQ ID NO: 438;</u><u style="single">(o) VH region shown by SEQ ID NO: 447 and VL region shown by SEQ ID NO: 448;</u><u style="single">(p) VH region shown by SEQ ID NO: 457 and VL region shown by SEQ ID NO: 458;</u><u style="single">(q) VH region shown by SEQ ID NO: 467 and VL region shown by SEQ ID NO: 468;</u><u style="single">(r) VH region shown by SEQ ID NO: 477 and VL region shown by SEQ ID NO: 478;</u><u style="single">(s) VH region shown by SEQ ID NO: 487 and VL region shown by SEQ ID NO: 488;</u><u style="single">(t) VH region shown in SEQ ID NO: 497 and VL region shown in SEQ ID NO: 498; and</u><u style="single">(u) VH region shown by SEQ ID NO: 507 and VL region shown by SEQ ID NO: 508</u><u style="single">A binding molecule of any of the present invention comprising a VH region and a VL region selected from the group consisting of.</u><u style="single">[Invention 1012]</u><u style="single"> The first binding domain is SEQ ID NO: 239, SEQ ID NO: 249, SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279, SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419, SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449, SEQ ID NO: 459, SEQ ID NO A binding molecule of the invention 1010 comprising an amino acid sequence selected from the group consisting of: 469, SEQ ID NO: 479, SEQ ID NO: 489, SEQ ID NO: 499 and SEQ ID NO: 509.</u><u style="single">[Invention 1013]</u><u style="single"> A binding molecule of any of 1001 to 1007 of the present invention having the amino acid sequence shown in SEQ ID NO: 300 or SEQ ID NO: 500.</u><u style="single">[Invention 1014]</u><u style="single"> A nucleic acid sequence encoding any of the binding molecules of the present invention 1001 to 1013.</u><u style="single">[Invention 1015]</u><u style="single"> A vector comprising the nucleic acid sequence of the present invention 1014.</u><u style="single">[Invention 1016]</u><u style="single"> Host cells transformed or transfected with the nucleic acid sequence of the present invention 1014 or the vector of the present invention 1015.</u><u style="single">[Invention 1017]</u><u style="single"> The present invention includes a step of culturing the host cell of the present invention 1016 under conditions that enable expression of any of the binding molecules of the present invention 1001 to 1013, and a step of recovering the produced binding molecule from the culture. The process for producing any of the binding molecules 1001 to 1013.</u><u style="single">[Invention 1018]</u><u style="single"> A pharmaceutical composition comprising a binding molecule of any of 1001 to 1013 of the present invention or a binding molecule produced by the process of 1017 of the present invention.</u><u style="single">[Invention 1019]</u><u style="single"> A binding molecule of any of 1001-1013 of the present invention for use in the prevention, treatment or remission of a disease selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases. Or a binding molecule produced by the process of the present invention 1017.</u><u style="single">[Invention 1020]</u><u style="single"> A method for the treatment or amelioration of a disease selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases, the present invention of the present invention 1001 ~ A method comprising the step of administering any of the binding molecules of 1013 or the binding molecules produced by the process of the present invention 1017.</u><u style="single">[Invention 1021]</u><u style="single"> Plasma cell disorders include multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, bone The method of the invention 1020 selected from the group consisting of sclerosing myeloma, heavy chain disease, unclear monoclonal gamma globulinemia, and smoldering multiple myeloma.</u><u style="single">[Invention 1022]</u><u style="single"> The method of the present invention 1020, wherein the autoimmune disease is systemic lupus erythematosus.</u><u style="single">[Invention 1023]</u><u style="single"> A kit comprising a binding molecule of any of the inventions 1001 to 1013, a nucleic acid molecule of the invention 1014, a vector of the invention 1015, and / or a host cell of the invention 1016.</u><u style="single">[1024 of the present invention]</u><u style="single"> Use of epitope cluster 3 and epitope cluster 4 of BCMA, preferably for the production of binding molecules capable of binding BCMA, wherein the epitope cluster 3 of BCMA is the sequence shown in SEQ ID NO: 1002. Corresponds to amino acid residues 24-41 of, and epitope cluster 4 of BCMA corresponds to amino acid residues 42-54 of the sequence shown in SEQ ID NO: 1002, used.</u><u style="single">[Invention 1025]</u><u style="single"> An antibody capable of binding BCMA, preferably a method of producing a binding molecule,</u><u style="single">(a) In the step of immunizing an animal with a polypeptide containing epitope cluster 3 and epitope cluster 4 of BCMA, epitope cluster 3 of BCMA corresponds to amino acid residues 24-41 of the sequence shown in SEQ ID NO: 1002. The process, in which the epitope cluster 4 of BCMA corresponds to amino acid residues 42-54 of the sequence shown in SEQ ID NO: 1002.</u><u style="single">(b) The step of obtaining the antibody, and</u><u style="single">(c) Optionally, the step of converting the antibody into a binding molecule capable of binding to human BCMA and preferably the T cell CD3 receptor complex.</u><u style="single">Including methods.</u>

0014It should be noted that as used herein, the singular form ("one", "is" and "that") contains multiple references unless the context clearly indicates otherwise. Must be. Thus, for example, "one reagent" includes one or more such different reagents, and references to "the method" can be modified or replaced by those skilled in the art described herein. Includes references to equivalent steps and methods known in.

0015Unless indicated otherwise, the term "at least" that precedes a series of elements should be understood to refer to any element in this series. One of ordinary skill in the art can recognize or confirm many equivalents of the specific embodiments of the invention described herein without the use of more conventional experiments. Such equivalents are intended to be included in the present invention.

0016The terms "and (and) / or (or)" are connected by "and (and)", "or (or)" and "by this term, wherever they are used herein. Includes the meaning of "all or any other combination of elements".

0017As used herein, the term "about" or "approximately" is within ± 20%, preferably within ± 15%, more preferably within ± 10%, and most preferably within ± 5 of the values or ranges indicated. Means within%.

0018Throughout the present specification and the appended claims, unless the context requires otherwise, the word "contains" and derivatives such as "contains" are the integer values or steps or arrangements referred to. It should be understood to include a group of numbers or processes, but imply that it does not exclude any other integer value or process or group of integer values or processes. As used herein, the term "comprising" is "containing" or "including," or as used herein, "having." It may be replaced.

0019As used herein, "consisting of" excludes any element, process or component not specified in the elements of the claimed invention. As used herein, "becomes essential" does not exclude a substance or process that does not substantially affect the basic and novel features of the claimed invention.

0020In each of the examples herein, the terms "contains," "consisting essentially of," and "consisting of" can all be replaced with any of the other two terms.

0021Overview<u style="single">Epitope clusters 1, 2, 3, 4, 5, 6, 7</u>Is included in the extracellular domain of BCMA. "BCMA extracellular domain" or "BCMA ECD" represents a form of BCMA that is essentially free of the transmembrane and cytoplasmic domains of BCMA. It will be appreciated by those skilled in the art that the transmembrane domain identified in the BCMA polypeptide of the invention will be identified according to standards commonly used in the art to identify the type of hydrophobic domain. Let's do it. The exact boundaries of the transmembrane domain can vary, but it is reasonable to assume that they are about 5 amino acids or less from the end of any of the domains specifically mentioned herein. Preferred BCMA ECDs are shown in SEQ ID NO: 1007. Preferred mouse ECDs are shown in SEQ ID NO: 1008. Epitope cluster 1 corresponds to amino acids 1-7 of the human BCMA extracellular domain (SEQ ID NO: 1007), and epitope cluster 2 corresponds to amino acids 8-21 of the human BCMA extracellular domain (SEQ ID NO: 1007). Epitope cluster 3 is the extracellular domain of human BCMA (SEQ ID NO: Corresponding to amino acids 24-41 of 1007), Epitope cluster 4 corresponds to amino acids 42-54 of the human BCMA extracellular domain (SEQ ID NO: 1007), and epitope cluster 5 corresponds to the human BCMA extracellular domain (SEQ ID NO: 1007). Corresponding to amino acid 22 of NO: 1007), epitope cluster 6 corresponds to amino acid 25 of the human BCMA extracellular domain (SEQ ID NO: 1007), and epitope cluster 7 corresponds to the human BCMA extracellular domain (SEQ ID NO: 1007). : Corresponds to amino acid 39 of 1007). It is also envisioned that epitope clusters 5-7 are single amino acid substitutions.

0022The T cell CD3 receptor complex is a protein complex, composed of four distinct strands. In mammals, this complex comprises a CD3γ chain, a CD3δ chain and two CD3ε (epsilon) chains. These chains associate with molecules known as the T cell receptor (TCR) and ζ chains to generate activation signals in T lymphocytes. Redirect lysis of target cells through T cell recruitment by bispecific molecules results in the formation of cytolytic synapses and the delivery of perforins and granzymes. The T cells involved are capable of continuous target cell lysis and are unaffected by immune avoidance mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation; see, eg, WO 2007/042261. thing.

0023In the context of the present disclosure, the term "binding molecule" refers to any molecule that can (specifically) bind to, interact with, or recognize the target molecules BCMA and CD3. According to the present invention, the binding molecule is preferably a polypeptide. Such polypeptides can include protein and non-protein moieties (eg, chemical linkers or chemical cross-linking agents, such as glutaraldehyde). The binding molecule provides, so to speak, a scaffold of the binding domains so that one or more binding domains can bind / interact with the target molecules BCMA and CD3. For example, such scaffolds include protein A, especially its Z domain (affibody), ImmE7 (immune protein), BPTI / APPI (Kunitz domain), Ras binding protein AF-6 (PDZ domain), caribbean toxin (scorpion). Can be provided by Toxic), CTLA-4, Min-23 (Notton), Lipocalin (Anticarin), Neocardinostatin, Fibronectin Domain, Ankyrin Consensus Repeat Domain or Thioredoxin (Skerra, Curr. Opin. Biotechnol. 18, 295-304 (2005); Hosse et al., Protein Sci. 15, 14-27 (2006); Nicaise et al., Protein Sci. 13, 1882-1891 (2004); Nygren and Uhlen , Curr. Opin. Struc. Biol. 7, 463-469 (1997)). A preferred binding molecule is an antibody. The binding molecule of the present invention is also expected to have additional functions in addition to its function of binding to the target molecules BCMA and CD3. In this form, the binding molecule targets plasma cells through binding to BCMA, mediates activation of cytotoxic T cells through binding to CD3, and through further function, eg, recruitment of effector cells such as NK cells. Fully functional Fc constant domain-mediated antibody-dependent cytotoxicity, labeling (such as fluorescence), therapeutic agents such as toxins or radionuclear species, and / or means for prolonging serum half-life, etc., trifunctional or multifunctional It is a sex-binding molecule.

0024As used herein, the term "bispecific" means that the binding molecule comprises at least the first and second binding domains, the first binding domain can bind to one antigen or target, and the second. Indicates that the binding domain can bind to another antigen or target. The "binding molecule" of the present invention also includes a multispecific binding molecule, eg, a trispecific binding molecule containing three binding domains. Binding molecules are produced (or by phage display or library screening methods, as well as by transplantation of CDR sequences from existing (monoclonal) antibodies into scaffolds, eg, scaffolds disclosed herein. (Available by) is expected.

0025The term "binding domain", in the context of the present invention, characterizes a domain capable of specifically binding / interacting with a given target epitope or given target site on the target molecules BCMA and CD3. Binding domains include binding domain donors such as antibody, protein A, ImmE7 (immune protein), BPTI / APPI (Kunitz domain), Ras binding protein AF-6 (PDZ domain), caribdotoxin (scorpion poison), CTLA-4. , Min-23 (Notton), Lipocalin (Anti-Carlin), Neocardinostatin, Fibronectin Domain, Ankyrin Consensus Repeated Domain or Thioredoxin (Skerra, Curr. Opin. Biotechnol. 18, 295-304 (2005)) Hosse et al., Protein Sci. 15, 14-27 (2006); Nicaise et al., Protein Sci. 13, 1882-1891 (2004); Nygren and Uhlen, Curr. Opin. Struc. Biol. 7, 463-469 (1997)). The preferred binding domain is obtained from the antibody. The binding domain of the present invention is expected to include at least a portion of any of the above binding domains required for binding / interaction to a given target epitope or given target site on the target molecule BCMA and CD3. There is. The binding domains of the above binding domain donors are bound by a portion of these donors responsible for binding to their respective targets, i.e., by the donor losing its binding capacity when that portion is removed from the binding domain donor. It is supposed to be characterized. "Loss" means that its binding capacity is reduced by at least 50% compared to a binding donor. Methods of mapping these binding sites are well known in the art and therefore it is a standard of skill in the art to position / map the binding sites of binding domain donors and thereby "obtain" the binding domain from each binding domain donor. Within the scope of knowledge.

0026The term "epitope" refers to a site on an antigen to which a binding domain, eg, an antibody or immunoglobulin or a derivative or fragment of an antibody or immunoglobulin, specifically binds. "Epitope" is antigenic, so the term epitope may also refer herein to "antigenic structure" or "antigenicity determinant". Therefore, the binding domain is the "antigen interaction site". It is understood that this binding / interaction also defines "specific recognition". In one example, the binding domain that binds / interacts (specifically) with a given target epitope or given target site on the target molecules BCMA and CD3 is an antibody or immunoglobulin, and the binding domain is an antibody or immunoglobulin. VH and / or VL region. An "epitope" can be formed by both continuous amino acids or discontinuous amino acids that are adjacent by the three-dimensional folding of a protein.

0027A "linear epitope" is an epitope that contains an epitope in which the primary sequence of an amino acid is recognized. Linear epitopes typically contain at least 3 or at least 4, and more generally at least 5 or at least 6 or at least 7, such as about 8 to about 10 amino acids within a unique sequence.

0028A "three-dimensional epitope" is an epitope in which the primary sequence of an amino acid containing the epitope is not the only defining element of the recognized epitope with respect to a linear epitope (for example, the primary sequence of an amino acid is not necessarily recognized by the binding domain. It is not an epitope). Typically, conformational epitopes contain more amino acids than linear epitopes. With respect to the recognition of conformational epitopes, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or a fragment thereof (in the present invention, an antigen for one of the binding domains is contained within the BCMA protein. ing). For example, when a protein molecule is folded to form a three-dimensional structure, the specific amino acid and / or polypeptide backbones that form the conformational epitope are in close proximity, which allows the antibody to recognize that epitope. .. Methods for determining the conformation of an epitope include, but are not limited to, x-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and site-specific spin-label and electron paramagnetic resonance (EPR) spectroscopy. .. In addition, the provided examples describe additional methods for testing whether a given binding domain binds to one or more epitope clusters of a given protein, particularly BCMA.

0029As used herein, the term "epitope cluster" refers to the entire epitope present in a given contiguous sequence of antigen stretches. Epitope clusters can contain one, two or more epitopes. Epitope clusters defined in the extracellular domain of BCMA in relation to the present invention are described above and are shown in FIG.

0030The terms "binding (can)", "specifically recognize", "directed" and "react" are according to the invention and the binding domain is one or more of the epitopes, preferably at least one. It means that it can specifically interact with two, more preferably at least three, and most preferably at least four amino acids.

0031As used herein, the terms "specifically interact" or "specifically bind" indicate the affinity that the binding domain can sense for a particular protein or antigen, and usually. It means that it does not show significant reactivity to proteins or antigens other than BCMA or CD3. "Perceptible affinity" is about 10^-6 Includes M (KD) or stronger affinity binding. Preferably, the binding has a binding affinity of about 10^-12~10^-8 M, 10^-12~10^-9 M, 10^-12~10^-10 M, 10^-11~10^-8 M, preferably about 10^-11~10^-9 It is considered specific if it is M. Whether a binding domain reacts or binds specifically to a target is determined, in particular, by comparing the reaction of that binding domain to a target protein or antigen with the reaction of that binding domain to a protein or antigen other than BCMA or CD3. It can be easily tested. Preferably, the binding domain of the invention essentially does not or cannot bind to a protein or antigen other than BCMA or CD3 (ie, the first binding domain cannot bind to a protein other than BCMA and the second binding. The domain cannot bind to proteins other than CD3). Specific binding is believed to be brought about by specific motifs within the binding domain and amino acid sequence of the antigen. Therefore, binding is achieved as a result of their primary, secondary and / or tertiary structures and as a result of secondary modifications of these structures. The specific interaction between the antigen interaction site and its specific antigen can result in mere binding of the site to the antigen. Further, the specific interaction between the antigen interaction site and the specific antigen thereof can initiate the signal alternative or additionally, for example, by inducing a change in the three-dimensional structure of the antigen, oligomerization of the antigen, or the like.

0032The terms "essentially non-binding" or "non-binding" mean that the binding domain of the invention does not bind to any protein or antigen other than BCMA or CD3, i.e. 100% binding to each of BCMA or CD3. Reactivity of greater than 30%, preferably greater than 20%, more preferably greater than 10%, particularly preferably greater than 9%, 8%, 7%, 6% or greater than 5% with respect to proteins or antigens other than BCMA or CD3. Means not to show.

0033"Proteins" (including fragments, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) are linked to each other through covalent peptide bonds (thus forming a chain of amino acids). Contains one or more amino acids (to form). As used herein, the term "polypeptide" refers to a group of molecules consisting of more than 30 amino acids. Polypeptides can further form multimers, such as dimers, trimers and higher order oligomers, i.e., can consist of two or more polypeptide molecules. The polypeptide molecules that form such dimers, trimers, etc. can be identical or non-identical. As a result, the corresponding higher order structures of such multimers are referred to as homo or heterodimers, homo or heterotrimers, and the like. An example of a heteromultimer is an antibody molecule in its natural form consisting of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins that have been modified by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. The "polypeptide" herein can also be chemically modified, eg, pegged. Such modifications are well known in the art.

0034The term "amino acid" or "amino acid residue" typically refers to an amino acid having a definition known in the art, such as alanin (Ala or A); arginine (Arg or R); aspartic acid (Asn). Or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He) Or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or) Represents an amino acid selected from the group consisting of T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic or rare amino acids may also be used if desired. In general, amino acids are non-polar side chains (eg Ala, Cys, He, Leu, Met, Phe, Pro, Val); loaded side chains (eg Asp, Glu); positively charged side chains (eg Arg, His, Lys); or can be grouped in that they have uncharged polar side chains (eg, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp and Tyr).

0035The definition of the term "antibody" includes aspects such as monoclonal, chimeric, single chain, humanized and human antibody. In addition to full-length antibodies, this definition also includes antibody derivatives and antibody fragments, especially such as Fab fragments. Antibody fragments or derivatives are further F (ab')₂, Fv, scFv fragment or single domain antibody, eg, domain antibody or Nanobody, containing the only variable domain that can be VHH, VH or VL that specifically binds to an antigen or epitope independently of other V regions or domains. Includes single variable domain antibodies or immunoglobulins; eg, Harlow and Lane (1988) and (1999), loc. Cit .; Kontermann and Dubel, Antibody Engineering, Springer, 2nd ed. 2010 and Little, Recombinant See Antibodies for Immunotherapy, Cambridge University Press 2009. The term also includes diabodies or Dual-Affinity Re-Targeting (DART) antibodies. In addition, (bispecific) single chain diabody, tandem diabody (Tandab), the following structure: (VH-VL-CH3)₂, (ScFv-CH3)₂Or (scFv-CH3-scFv)₂The "minibody", "Fc DART" and "IgG DART" exemplified by, multibody, eg triabodies, are envisioned. An immunoglobulin single variable domain comprises not only an isolated antibody single variable domain polypeptide, but also a larger polypeptide containing one or more monomers of the antibody single variable domain polypeptide sequence. Various procedures are known in the art and can be used for the production of such antibodies and / or fragments. Thus, (antibody) derivatives can be produced by peptide mimetics. In addition, techniques described for the production of single chain antibodies (particularly US Pat. No. 4,946,778, Kontermann and Dubel (2010), loc. Cit. And Little (2009), loc. (See cit.) Can be adapted to produce single chain antibodies specific for the polypeptide of choice. Transgenic animals can also be used to express humanized antibodies specific for the polypeptides and fusion proteins of the invention. For the preparation of monoclonal antibodies, any technique can be used to provide the antibody produced by continuous cell line culture. Examples of such techniques include hybridoma technology (Kohler and Milstein Nature 256 (1975), 495-497), trioma technology, human B cell hybridoma technology (Kozbor, Immunology Today 4 (1983), 72) and human monoclonal antibodies. EBV hybridoma technology to be manufactured (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96) is included. Surface plasmon resonance used in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to target polypeptides, such as the epitope of CD3 epsilon (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In the context of the present invention, the term "antibody" refers to an antibody construct that can be expressed in a host as described below, eg, an antibody construct that can be transfected and / or transduced, especially through a viral or plasmid vector. Is also expected to include. In addition, the term "antibody" as used herein also relates to a derivative or variant of an antibody described herein that exhibits the same specificity as the antibody described. Examples of "antibody variants" are humanized variants of non-human antibodies, "affinity maturation" antibodies (eg, Hawkins et al., J. Mol. Biol. See 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)) and antibody variants with altered effector function (eg, US Pat. No. 5,648,260, Kontermann and Dubel). (2010), loc. Cit. And Little (2009), see loc. Cit.). One exemplary method of making an antibody involves screening a protein expression library, such as a phage or ribosome display library. Phage displays are described, for example, in US Pat. No. 5,223,409 of Ladner et al.; Smith (1985) Science 228: 1315-1317; Clackson et al. (1991) Nature, 352: 624-628. In addition to the use of display libraries, the antigen of interest can be used to immunize non-human animals such as rodents such as mice, hamsters or rats. In one embodiment, the non-human animal comprises at least a portion of the human immunoglobulin gene. For example, a mouse strain lacking the ability to produce mouse antibodies can be modified using a large fragment of the human Ig locus. Hybridoma techniques can be used to produce and select antigen-specific monoclonal antibodies derived from genes with the desired specificity. For example, XENOMOUSE , Green et al. (1994) Nature Genetics 7: See 13-21, US 2003-0070185, WO96 / 34096 and WO96 / 33735. Antibodies or fragments thereof can also be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II; these peptides (as defined in WO 98/52976 and WO 00/34317). It is a potential T cell epitope. A computer modeling approach called "peptide threading" can be applied for the detection of potential T cell epitopes, as described in WO 98/52976 and WO 00/34317, in addition to human MHC. A database of class II binding peptides can be searched for motifs present in the VH and VL sequences. These motifs are 18 major MHC class II It binds to any of the DR allotypes and thus constitutes a potential T cell epitope. Detected potential T cell epitopes can be removed by substituting a small number of amino acid residues within the variable domain or preferably by a single amino acid substitution. Conservative substitutions are typically made. In many cases, but not all, common amino acids can be used for positions in the human germline antibody sequence. Human germline sequences are, for example, Tomlinson, et al. (1992) J. Mol. Biol. 227: 776-798; Cook, GP et al. (1995) Immunol. Today Vol. 16 (5): 237-242. ; And Tomlinson et al. (1995) EMBO J. 14:14: 4628-4638. The V BASE directory is (Tomlinson, LA. Et al. Provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by the MRC Center for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example for framework regions and CDRs. Consensus human framework areas, such as those described in US Pat. No. 6,300,064, can also be used. VH and VL<u style="single">versus</u>Jointly form one antigen binding site. The CH domain closest to VH is called CH1. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chain. The VH and VL domains consist of four relatively conserved sequence regions (FR1, FR2, FR3 and FR4) called framework regions, which are the three hypervariable sequence regions (complementarity determining regions, CDRs). Form a scaffold. The CDR contains most of the residues responsible for the specific interaction between the antibody and the antigen. CDRs are referred to as CDR1, CDR2 and CDR3. Therefore, the CDR elements of the heavy chain are referred to as H1, H2 and H3, and the CDR elements of the light chain are referred to as L1, L2 and L3.

0036The term "variable" refers to the variability in those sequences and refers to a portion of the immunoglobulin domain (ie, the "variable domain") that is involved in determining the specificity and binding affinity of individual antibodies. .. The variability is not uniformly distributed throughout the variable domain of the antibody; it is concentrated in the partial domains of each variable region of the heavy and light chains. These subdomains are called "hypervariable" regions or "complementarity determining regions" (CDRs). The more conserved (non-super variable) part of the variable domain is called the "framework" area (FRM). Each of the naturally occurring heavy and light chain variable domains contains four FRM regions, most of which have a β-sheet arrangement, which form loop connections and, in some cases, β-sheets. It is connected by three hypervariable regions that form part of the structure. The hypervariable regions of each strand are held together in close proximity by FRM and, together with the hypervariable regions of the other strands, contribute to the formation of antigen-binding sites (Kabat et al., Loc. See cit.). The constant domain is not directly involved in antigen binding, but exhibits various effector functions such as antibody-dependent cell-mediated cytotoxicity and complement activation.

0037The term "hypervariable regions" (also known as "complementarity determining regions" or CDRs), as used herein, are immunoglobulins that form the antigen binding site and are the primary determinants of antigen specificity. Represents an amino acid residue of an antibody (usually three or four short regions showing extremely high sequence variability) within the V region domain of. There are at least two methods for identifying CDR residues: (1) an approach based on interspecific sequence variability (ie, Kabat et al., Loc. Cit.); And (2) crystals of the antigen-antibody complex. A study-based approach (Chothia C. et al., J. Mol. Biol. 196: 901-917 (1987)). However, they can be combined to define hybrid CDRs to the extent that two residue identification techniques define overlapping regions that are not the same region. However, in general, CDR residues are preferably identified according to the so-called Kabat (numbering) system.

0038The terms "antigen-binding domain," "antigen-binding fragment," and "antibody-binding region," as used herein, refer to a portion of an antibody molecule that contains an amino acid that is responsible for the specific binding between an antibody and an antigen. .. The portion of the antigen that is specifically recognized and bound by the antibody is referred to herein as an "epitope". As mentioned above, the antigen binding domain can typically include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH); but it does not have to. For example, an Fd fragment has two VH regions and often retains some antigen-binding function of an intact antigen-binding domain. Examples of antigen-binding fragments of antibodies are (1) Fab fragments, which are monovalent fragments with VL, VH, CL and CH1 domains; (2) two Fab fragments linked by disulfide bridges in the hinge regions. F (ab') 2 fragment, which has a divalent fragment; (3) Fd fragment, which has two VH and CH1 domains; (4) Fv fragment, which has the VL and VH domains of one arm of the antibody. (5) dAb fragments with VH domains (Ward et al., (1989) Nature 341: 544-546); (6) isolated complementarity determining regions (CDRs), and (7) single-chain Fv ( It contains scFv), the latter being preferred (eg, from the scFV library). The two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they are connected by a synthetic linker using a recombination method, and the VL and VH regions are paired to form a monovalent molecule. One protein chain (known as a single chain Fv (scFv); eg, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: It can be generated as (see 5879-5883). These antibody fragments are obtained using prior art known to those of skill in the art, and the fragments are evaluated for function in the same manner as intact antibodies.

0039As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the potential for the individual antibodies that make up that population to be present in trace amounts. Identical except for spontaneous mutations and / or post-translational modifications (eg, isomerization, amidation). Monoclonal antibodies are highly specific and are directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is relative to a single determinant on the antigen. It is aimed. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma cultures that are free of other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody that it is obtained from a substantially homogeneous population of antibodies and should be construed as requiring the antibody to be produced by any particular method. is not it. For example, the monoclonal antibodies used according to the present invention are Kohler et al., Nature 256: 495 It can be made by the hybridoma method first described by (1975) or by the recombinant DNA method (see, eg, US Pat. No. 4,816,567). "Monoclonal antibodies" are also described in, for example, Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Can be isolated from the phage antibody library using. The monoclonal antibodies of the invention are specifically identical or homologous to the corresponding sequences of antibodies derived from or belonging to a particular antibody class or subclass of a heavy chain and / or part of the light chain. , A "chimeric" antibody (immunoglobulin), in which the rest of the chain is identical or homologous to the corresponding sequence of an antibody of another species or belonging to another antibody class or subclass, and the desired biology. Fragments of such antibodies are included, as long as they are active (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81; 6851-6855 (1984)). Chimeric antibodies of interest herein include "primate" antibodies that include variable domain antigen binding sequences and human constant region sequences from non-human primates (eg, Old World monkeys, apes, etc.). Monoclonal antibodies can be obtained from non-human animals, followed by modified, eg, humanized, deimmunized, chimeric, recombinant DNA techniques known in the art. Can be manufactured using. Various approaches for making chimeric antibodies have been described. For example, Morrison et al., Proc. Natl. Acad. ScL USA 81: 6851, 1985; Takeda et al., Nature 314: 452, 1985, Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; See EP0171496; EP0173494, GB2177096 by Tanaguchi et al. The "humanized" form of a non-human (eg, mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (eg, eg) of mostly human sequences, including minimal sequences from non-human immunoglobulins. Fv, Fab, Fab', F (ab') 2 or other antigen-binding partial sequence of an antibody). Humanized antibodies are often human immunoglobulins (recipient antibodies), but non-human species in which residues in the hypervariable region (or CDR) of the recipient have the desired specificity, affinity and competence. (Donator antibody), eg, one that has been replaced with a residue in the hypervariable region of a mouse, rat or rabbit. In some examples, residues in the Fv framework region (FR) of human immunoglobulin are replaced with corresponding non-human residues. In addition, the "humanized antibody" used herein may also contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize the performance of the antibody. Humanized antibodies may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2 : See 593-596 (1992). Humanized antibodies can also be produced, for example, using transgenic mice that express human heavy and light chain genes but not endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR transplantation method that can be used to prepare the humanized antibodies described herein (US Pat. No. 5,225,539). All of the CDRs of a particular human antibody can be replaced by at least a portion of the non-human CDRs, or only some of the CDRs can be replaced by non-human CDRs. It is only necessary to replace the number of CDRs required to bind the humanized antibody to a given antigen. Humanized antibodies or fragments thereof can be generated, for example, by replacing the sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from the human Fv variable domain. Illustrative methods for producing humanized antibodies or fragments thereof are by Morrison (1985) Science 229: 1202-1207; by Oi et al. (1986) BioTechniques 4: 214; and US5,585,089; US5,693,761. Provided by US5,693,762; US5,859,205; and US6,407,213. These methods involve the isolation, manipulation and expression of nucleic acid sequences encoding all or part of an immunoglobulin Fv variable domain from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas that produce antibodies against predetermined targets, as described above, and from other sources. Recombinant DNA encoding a humanized antibody molecule can then be cloned into a suitable expression vector. Humanized antibodies can be optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and / or reversion mutations. Such modified immunoglobulin molecules can be made by any of a variety of techniques known in the art (eg, Teng et al., Proc. Natl. Acad. Sci. USA, 80: 7308). -7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982), and can be made according to the teachings of EP 239 400.

0040The term "human antibody" substantially relates to human germline immunoglobulin sequences known in the art, including those described, for example, by Kabat et al. (See Kabat et al. (1991) loc. Cit.). Includes antibodies with corresponding variable and constant regions. The human antibodies of the invention can be used, for example, in CDR, especially in CDR3, by amino acid residues not encoded by the human germline immunoglobulin sequence (eg, by random or site-directed mutagenesis in vitro or by somatic mutations in vivo. Mutations to be introduced) can be included. Human antibodies may have at least one, two, three, four, five or more positions replaced by amino acid residues not encoded by the human germline immunoglobulin sequence.

0041As used herein, an "in vitro generated antibody" is a variable region (eg, at least one CDR) in which all or part of a non-immune cell selection (eg, in vitro phage display, protein chip or candidate sequence). Represents an antibody produced by any other method) that can be tested for their antigen-binding ability. Therefore, the term preferably excludes sequences produced by the rearrangement of genes within immune cells.

0042A "bispecific" or "bifunctional" antibody or immunoglobulin is an artificial hybrid antibody or immunoglobulin having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab'fragments. For example, Songsivilai &amp; Lachmann, Clin. Exp. Immunol. 79: See 315-321 (1990). Many methods known to those of skill in the art are available for obtaining an antibody or antigen-binding fragment thereof. For example, antibodies can be produced using recombinant DNA (US Pat. No. 4,816,567). Monoclonal antibodies can also be produced by the production of hybridomas according to known methods (see, eg, Kohler and Milstein (1975) Nature, 256: 495-499). Hybridomas formed in this manner are then standard methods for identifying one or more hybridomas that produce antibodies that specifically bind to the antigen of interest, such as an enzyme-linked immunosorbent assay (ELISA). And screened using surface plasmon resonance (BIACORE ) analysis. Any form of the antigen of interest, such as recombinant antigens, naturally occurring forms, any variants or fragments thereof and their antigenic peptides, can be used as immunogens.

0043The term "CDR" and its plural form represent complementarity determining regions (CDRs), three of which constitute the binding properties of light chain variable regions (CDRL1, CDRL2 and CDRL3), and three of which are heavy chain variable regions. Configure binding properties (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of antibody molecules and are disrupted by amino acid sequences containing scaffold or framework regions. The exact CDR boundaries and lengths depend on the classification and numbering system. CDRs can therefore be represented by the definition of Kabat, Chothia, contact or any other boundary, including the numbering schemes described herein. Even if the boundaries are different, each of these systems overlaps to some extent with respect to the parts that make up the so-called "hypervariable regions" within the variable array. Therefore, the definitions of CDRs according to these schemes can differ in terms of length and boundary areas with adjacent framework areas. For example, Kabat, Chothia and / or MacCallum (Kabat et al., Loc. Cit .; Chothia et al., J. See Mol. Biol, 1987, 196: 901; and MacCallum et al., J. Mol. Biol, 1996, 262: 732). However, numbering according to the so-called Kabat system is preferable. Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the conformation of the main chain adopted by the antigen binding (CDR) loop. Many structural studies have found that five of the six antigen-binding loops have a limited repertoire of available conformations. Each canonical structure can be characterized by the helix angle of the polypeptide backbone. Thus, the corresponding loops between antibodies can have very similar three-dimensional structures, despite the high degree of amino acid sequence variability in most of those loops (each in its entirety here by reference). Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al., Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996, 263: 800). In addition, there is a link between the loop structure adopted and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues present at key positions within the loop and within the conserved framework (ie, outside the loop). Therefore, assignments to specific canonical classes can be made based on the presence of these key amino acid residues. The term "canonical structure" is also used, for example, in the catalog by Kabat (Kabat et al., Loc. It may include consideration for the linear sequence of the antibody, as described in cit.). Kabat's numbering scheme is a widely adopted standard for numbering amino acid residues in antibody variable domains in a common manner, as described elsewhere herein. This is the preferred scheme applied to the invention. Further structural considerations can also be used to determine the canonical structure of the antibody. For example, differences that are not fully reflected by Kabat's numbering can be explained by the numbering system of Chothia et al. And / or revealed by other techniques such as crystallography and 2D or 3D computer modeling. Thus, a given antibody sequence can be classified into a canonical class in which a particularly suitable chassis sequence can be found (eg, based on the desire to include various canonical structures in the library). Kabat numbering of the amino acid sequence of the antibody and Chothia et al., Loc. Consideration of the structures described by cit. And their implications for interpreting the canonical aspects of antibody structure are described in the literature. CDR3 is typically the largest source of molecular diversity at the antibody binding site. For example, H3 can be as short as two amino acid residues or larger than 26 amino acids. The subunit and three-dimensional structures of various classes of immunoglobulins are well known in the art. When reviewing the antibody structure, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. See Harlow et al., 1988. Those skilled in the art will appreciate that each subunit structure, eg, CH, VH, CL, VL, CDR, FR structure, is an active fragment, eg, a portion of a VH, VL or CDR subunit that binds to an antigen, i.e. an antigen binding fragment, or eg, You will understand that it contains, for example, a portion of the CH subunit that binds to and / or activates the Fc receptor and / or complement. CDRs typically represent Kabat CDRs as described in the Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another standard that characterizes the antigen binding site is to refer to the hypervariable loop described by Chothia. For example, Chothia, et al. (1987; J. Mol. Biol. 227; 799-817); and Tomlinson et al. (1995) EMBO J. 14: See 4628-4638. Yet another standard is the AbM definition used by Oxford Molecular's AbM antibody modeling software. For example, see Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed .: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg) for an overview. The embodiments described based on the Kabat CDR can optionally be implemented using the relationships similarly described based on the Chothia hypervariable loop or the AbM-defined loop. The sequences of antibody genes after assembly and somatic mutations are highly diversified, with 10 of these diversified genes.¹⁰Estimated to encode different antibody molecules (Immunoglobulin Genes, 2)^nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). The immune system thus provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived in whole or in part from at least one sequence encoding at least one immunoglobulin. These sequences can be generated by rearrangement of heavy chain V, D and J segments and light chain V and J segments in vivo. Alternatively, these sequences can be generated from cells in response to what causes the rearrangement, such as in vitro stimulation. Alternatively, some or all of these sequences may be obtained by DNA splicing, nucleotide synthesis, mutagenesis and other methods, see, eg, US Pat. No. 5,565,332. The repertoire may contain only one sequence, or may contain multiple sequences, including sequences from a multigene collection.

0044The term "framework region" refers to a well-known portion of an antibody variable region that resides between more variegated (ie, hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1-4 (FR1, FR2, FR3 and FR4) and have six CDRs (three heavy chains and three light chains) in three-dimensional space. To provide a scaffold for forming an antigen-binding surface.

0045The binding molecule of the present invention is preferably an "isolated" binding molecule. As used to describe a binding molecule disclosed herein, "isolated" means a binding molecule that has been identified, separated and / or recovered from the components of its production environment. Preferably, the isolated binding molecule is free of all other components from its production environment. Contaminating components of the production environment, such as those resulting from recombinant transfected cells, are substances that would typically interfere with the diagnostic or therapeutic use of the polypeptide, including enzymes, hormones and other proteinaceous or non-proteinaceous substances. May contain proteinaceous solutes. In a preferred embodiment, the binding molecule is (1) to the extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by use of a spin-cup sequencing device, or (2) Coomassie blue or preferably silver. Purify until homogenous by SDS-PAGE under non-reducing or reducing conditions using staining. However, isolated antibodies are usually prepared by at least one purification step.

0046Amino acid sequence modifications of the binding molecules described herein are taken into account. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the binding molecule are prepared by introducing appropriate nucleotide changes into the nucleic acid of the binding molecule or by peptide synthesis.

0047Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the binding molecule. Any combination of deletions, insertions and substitutions is made to reach the final construct, as long as the final construct retains the desired properties. Amino acid changes can also change the post-translational process of the binding molecule, eg, change the number or position of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids can be substituted in the CDRs and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids can be substituted in the framework region (FR). The substitution is preferably a conservative substitution as described herein. Additional or alternative, 1, 2, 3, 4, 5 or 6 amino acids can be inserted or deleted in each of the CDRs (depending on their length, of course), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids are inserted or deleted in each of the FRs. obtain.

0048A useful method for identifying specific residues or regions of binding molecules that are preferred positions for mutagenesis is "alanine scan mutagenesis" as described by Cunningham and Wells in Science, 244: 1081-1085 (1989). It is called. In this method, residues or target residues within the binding molecule are identified (eg, charged residues such as arg, asp, his, lys and glu) and affect the interaction of the amino acid with the epitope. So, it is replaced with a neutral or ponted amino acid (most preferably alanine or polyalanine).

0049Amino acid positions that are functionally sensitive to this substitution are then carefully selected by introducing additional or other variants at or for the substitution site. Therefore, the site of introduction of the amino acid sequence variant is predetermined, but the nature of the mutation itself does not need to be determined in advance. For example, to analyze the function of mutations at a given site, an ala scan or random mutagenesis is performed at the target codon or region, and variants of the expressed binding molecule are screened for the desired activity.

0050Preferably, the amino acid sequence insertion is amino and / / in the length range of the polypeptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to 100 residues or more. Or it includes carboxyl-terminal fusion as well as intra-sequence insertion of single or multiple amino acid residues. Insertion variants of the binding molecule include fusion of the N- or C-terminus of the antibody to the enzyme or fusion to a polypeptide that increases the serum half-life of the antibody.

0051Another type of variant is the amino acid substitution variant. These variants are preferably those in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the binding molecule have been replaced with different residues. Sites of greatest interest for substitution mutagenesis include the CDRs of heavy and / or light chains, especially hypervariable regions, but changes in FR in heavy and / or light chains are also taken into account.

0052For example, if the CDR sequence contains 6 amino acids, it is assumed that one, two or three of these amino acids will be replaced. Similarly, if the CDR sequence contains 15 amino acids, it is assumed that 1, 2, 3, 4, 5 or 6 of these amino acids will be replaced.

0053In general, if an amino acid is substituted in one or more or all of the heavy and / or light chain CDRs, the resulting "substituted" sequence is at least 60% identical to the "original" CDR sequence. It is preferably 65%, even more preferably 70%, particularly preferably 75%, and even more particularly preferably 80%. This means that how much it is identical to the "replaced" sequence depends on the length of the CDR. For example, a CDR having 5 amino acids is preferably at least 80% identical to the substituted amino acid sequence when at least one amino acid is substituted. Thus, the CDRs of binding molecules can have different degrees of identity to their substituted sequences, eg, CDRL1 can have 80% and CDRL3 can have 90%.

0054A preferred substitution (or substitution) is a conservative substitution. However, as long as the binding molecule retains the ability to bind BCMA through the first binding domain and to CD3 epsilon through the second binding domain and / or its CDRs be identical to the sequences so substituted. Any having (at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, even more particularly preferably 80% identical to the "original" CDR sequence). Substitutions (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table 1 below) are envisioned.

0055Conservative substitutions are shown under the heading "Favorable substitutions" in Table 1. If such substitutions alter biological activity, more substantive changes are introduced, such as those listed as "exemplary substitutions" in Table 1 or further described below with respect to the amino acid class. The product can be screened for the desired properties.

0056(Table 1) Amino acid substitution<img id="000002" he="137" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0057Substantial modifications of the biological properties of the binding molecule of the invention include (a) the structure of the polypeptide skeleton at the substitution region, eg, a sheet or spiral conformation, (b) the charge or hydrophobicity of the molecule at the target site, or c) Their effect on the maintenance of side chain bulk, is achieved by choosing substitutions that are significantly different. Naturally occurring residues are grouped based on common side chain properties: (1) hydrophobicity: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic; cys, ser, thr; (3) Acidity: asp, glu; (4) Basicity: asn, gin, his, lys, arg; (5) Residues affecting chain orientation: gly, pro; and (6) Aromatic : trp, tyr, phe.

0058Non-conservative replacement would involve the exchange of one member of one of these classes with another. Any cysteine residue that is not involved in maintaining the proper conformation of the bound molecule can usually be replaced with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine binding can be added to an antibody to improve its stability, especially if the antibody is an antibody fragment, eg, an Fv fragment.

0059A particularly preferred type of substitution variant is one that involves the substitution of one or more hypervariable region residues of the parent antibody (eg, humanized or human antibody). Usually, the resulting variants selected for further development will have improved biological properties compared to the parent antibody from which they originated. A convenient method for producing such substitution variants involves affinity maturation using a phage display. Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus produced are presented in monovalent form from the filamentous phage particles as a fusion with the Gene III product of M13 packaged within each particle. Phage-displayed variants are then described for their biological activity (eg, binding affinity) as disclosed herein to identify hypervariable region site candidates for modification. Alanine scan mutagenesis can be performed to be screened and identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or in addition, it may be useful to analyze the crystal structure of the antigen-antibody complex to identify the binding domain and the point of contact between, for example, human BCMA. Such contact and flanking residues are candidates for substitution according to the techniques detailed herein. After such variants have been generated, a panel of variants is subjected to the screenings described herein, and antibodies exhibiting superior properties in one or more related assays are selected for further development. Can be done.

0060Other modifications of the binding molecule are also taken into account herein. For example, the binding molecule can be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyalkylene or copolymers of polyethylene glycol and polypropylene glycol. The binding molecule is also attached to a colloidal drug delivery system (eg, a liposome) into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (eg, hydroxymethyl cellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively). , Albumin microspheres, microemulsions, nanoparticles and nanocapsules), or can be captured in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

0061The binding molecules disclosed herein can also be formulated as immunoliposomes. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and / or surfactants that are useful for drug delivery to mammals. Liposomal components are usually arranged in a bilayer format similar to the arrangement of lipids in biological membranes. Liposomes containing antibodies include, for example, Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); Prepared by methods known in the art as described in US Pat. Nos. 4,485,045 and 4,544,545; and WO 97/38731 published October 23, 1997. .. Liposomes with enhanced circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol and PEG derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of defined pore size to obtain liposomes of the desired diameter. Fab'fragments of the antibodies of the invention can be conjugated to liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) through a disulfide exchange reaction. Optionally, a chemotherapeutic agent is included within the liposome. See Gabizon et al. J. National Cancer Inst. 81 (19) 1484 (1989).

0062When using recombinant techniques, the binding molecule can be produced intracellularly in the periplasmic cleft or secreted directly into the medium. When the binding molecule is produced intracellularly, the first step is to remove the granular debris of either the host cell or the lysed fragment, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe the procedure for isolating antibodies secreted in the periplasmic cleft of E. coli.

0063The bound molecular composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique.

0064In a further aspect, the invention relates to a nucleic acid sequence encoding a binding molecule of the invention. The term "nucleic acid" is well known to those of skill in the art and includes DNA (eg, cDNA) and RNA (eg, mRNA). Nucleic acids can be double-stranded and single-stranded, linear and cyclic. The nucleic acid molecule is preferably contained in a vector, preferably contained within the host cell. The host cell can express the binding molecule, for example, after transformation or transfection with the nucleic acid sequences of the invention. For that purpose, the nucleic acid molecule is functionally linked to the control sequence.

0065Vectors are nucleic acid molecules used as vehicles to carry (foreign) genetic material into cells. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids and artificial chromosomes. In general, engineering vectors include an origin of replication, a multicloning site and a selectable marker. The vector itself is generally a nucleotide sequence, usually a DNA sequence, that contains an insert (transgene) and a larger sequence that acts as the "skeleton" of the vector. Recent vectors may include transgene inserts and skeletons plus additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. A vector called an expression vector (expression construct) is particularly intended for expressing a transgene in a target cell, and generally has a control sequence, for example, a promoter sequence that promotes the expression of the transgene. Input of a vector into a target cell is usually called "transformation" in the case of bacterial cells and "transfection" in the case of eukaryotic cells, but input of a viral vector is also called "transduction".

0066As used herein, the term "host cell" is intended to refer to a cell into which a nucleic acid encoding a binding molecule of the invention is introduced by transformation, transfection, or the like. It should be understood that such terms refer not only to a particular cell of interest, but also to the progeny or potential progeny of such cells. Such progeny may not actually be identical to their parent cells, as certain modifications can occur in subsequent generations, either due to mutations or environmental influences, but even so. When used in writing, it falls within the scope of this term.

0067As used herein, the term "expression" includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion of any step involved in the production of the binding molecule of the invention. including.

0068The term "regulatory sequence" refers to the DNA sequence required to express a functionally linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

0069A nucleic acid is "functionally linked" if it is placed under a functional relationship with another nucleic acid sequence. For example, a DNA for a presequence or secretory leader is functionally linked to a DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or An enhancer is functionally linked to a coding sequence if it affects transcription of that sequence; or if a ribosome binding site is functionally linked to a coding sequence, it translates. This is the case when it is arranged to promote. In general, "functionally linked" means that the linked DNA sequences are contiguous and, in the case of a secretory leader, contiguous and within the leading phase. However, enhancers do not have to be continuous. Coupling is achieved by ligation at a convenient restriction site. In the absence of such sites, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

0070The terms "host cell", "target cell" or "recipient cell" can be or have already become recipients for the integration of vectors or exogenous nucleic acid molecules, polynucleotides and / or proteins. It is intended to include any individual cell or cell culture that is present. It is also intended to contain the progeny of one cell, which progeny is not necessarily with the original parent cell (in terms of morphological or genomic or total DNA complementarity) by natural, accidental or deliberate mutations. It may not be exactly the same. Cells can be prokaryotic cells or eukaryotic cells, including but not limited to bacterial cells, yeast cells, animal cells and mammalian cells such as mice, rats, mackerel or humans.

0071Suitable host cells include prokaryotes and eukaryotic host cells including yeast, fungal, insect and mammalian cells.

0072The binding molecule of the present invention can be produced in bacteria. After expression, the binding molecules of the invention, preferably binding molecules, can be isolated from E. coli cell paste in solution fractions and purified, for example, through affinity chromatography and / or size exclusion. Final purification can be performed, for example, in the same manner as the process for purifying antibodies expressed in CHO cells.

0073In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the binding molecules of the invention. Saccharomyces cerevisiae or baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful in the present invention, such as fission yeast (Schizosaccharomyces pombe), Kluyveromyces host, eg K. lactis (K. .lactis), K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. Walchii (K. waltii) (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerans and K. marxianus (K. marxianus; yarrowia (EP 402 226); Pichia pastoris (EP 183 070); Candida; Trichoderma reesia (EP 244 234); Neurospora crassa); Schwanniomyces, eg Schwanniomyces occidentalis; and filamentous fungi, such as neurospora, Penicillium, Tolypocladium and Aspergillus hosts, eg A. A. nidulans and A. niger.

0074Host cells suitable for expressing the glycosylation-binding molecule of the present invention, preferably an antibody-derived binding molecule, are obtained from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants as well as Spodoptera frugiperda (hairworm), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (Drosophila melanogaster) Corresponding acceptable insect host cells from hosts such as Aedes albopictus have been identified. Various viral strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Spirogyra NPV, are publicly available, and such viruses are available. According to the present invention, it can be used as a virus herein, especially for transfection of Spirogyra cells.

0075Plant cell cultures of cotton, corn, potatoes, soybeans, peanuts, petunias, tomatoes, Arabidopsis thaliana and tobacco can also be used as hosts. Cloning and expression vectors useful for protein production in plant cell cultures are known to those of skill in the art. For example, Hitatt et al., Nature (1989) 342: 76-78, Owen et al. (1992) Bio / Technology 10: 790-794, Artsaenko et al. (1995) The Plant J 8: 745-750 and Fecker. See et al. (1996) Plant Mol Biol 32: 979-986.

0076However, there is the greatest interest in vertebrate cells, and breeding of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human fetal kidney line (293 or subcloned for growth in suspension culture 293). Cells, Graham et al., J. Gen Virol. 36: 59 (1977)); Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells /-DHFR (CHO, Urlaub et al., Proc. Natl) Acad. Sci. USA 77: 4216 (1980)); Mammalian cell line (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); Human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (HELA, ATCC CCL 2) MDCK, ATCC CCL 34); Buffalo Lat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, 1413 8065); Mouse breast cancer (MMT 060562, ATCC CCL5) 1); TRI cells (Mather et al., Annals N. Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma strain (Hep G2).

0077When using recombinant techniques, the binding molecules of the invention can be intracellularly produced in the periplasmic cleft or secreted directly into the medium. When the binding molecule is produced intracellularly, the first step is to remove the granular debris of either the host cell or the lysed fragment, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes the procedure for isolating antibodies secreted into the periplasmic cleft of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When the antibody is secreted into the medium, usually first, the supernatant of such an expression system is centrifuged using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

0078The binding molecule of the present invention prepared from a host cell can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, and affinity chromatography is the preferred purification technique.

0079The matrix to which the affinity ligand is added is most often agarose, but other matrices are also available. A mechanically stable matrix, such as glass or poly (styrenedivinyl) benzene with defined pores, provides faster flow rates and shorter processing times than can be achieved with agarose. Bakerbond ABX Mrresin (JT Baker, Phillipsburg, NJ) is useful for purification when the binding molecule of the present invention contains the CH3 domain. Other protein purification techniques such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin SEPHAROSE chromatography, anion or cation exchange resin (eg polyaspartic acid column) chromatography Imaging, chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation are also available depending on the antibody recovered.

0080In another aspect, the present invention comprises the steps of culturing the host cells as defined herein under conditions that allow the expression of the binding molecule and the step of recovering the produced binding molecule from the culture. A process for producing the binding molecule of the invention is provided.

0081The term "culture" refers to in vitro maintenance, differentiation, growth, proliferation and / or reproduction of cells under appropriate conditions in the medium.

0082In an alternative embodiment, a composition comprising the binding molecule of the present invention or prepared according to the process of the present invention is provided. Preferably, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a composition administered to a patient, preferably a human patient. Certain preferred pharmaceutical compositions of the invention include binding molecules of the invention. Preferably, the pharmaceutical composition comprises suitable formulations of carriers, stabilizers and / or excipients. In a preferred embodiment, the pharmaceutical composition comprises a composition for parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or direct injection into tissue. It is specifically envisioned that the composition be administered to the patient via infusion or injection. Administration of the appropriate composition can be performed by different methods, eg, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In particular, the present invention provides uninterrupted administration of suitable compositions. As a non-limiting example, uninterrupted or continuous administration can be performed by a small pump system worn on the patient that measures the influx of therapeutic agent into the patient's body. Pharmaceutical compositions containing the binding molecules of the invention can be administered by using this pump system. Such pump systems are generally known in the art and generally require regular replacement of cartridges containing the infused therapeutic agent. Replacing the cartridge in such a pump system can result in a temporary interruption of the influx of therapeutic agent into the patient's body that is otherwise uninterrupted. Even in such cases, the dosing phase before cartridge replacement and the dosing phase after cartridge replacement remain "uninterrupted" in such therapeutic agents, in both the sense of pharmaceutical means and the methods of the invention. Will be considered to constitute administration.

0083Continuous or uninterrupted administration of these binding molecules of the invention is venous by a fluid delivery device or small pump system, including a fluid delivery mechanism for pumping fluid from the reservoir and a working mechanism for activating the delivery mechanism. It can be administered intraorally or subcutaneously. A pump system for subcutaneous administration may include a needle or cannula to penetrate the patient's skin and deliver the appropriate composition into the patient's body. The pump system may be fixed or attached directly to the patient's skin so that the pump system is in direct contact with the patient's skin, regardless of veins, arteries or blood vessels. The pump system can be attached to the patient's skin for 24 hours to several days. The pump system can be of small size with a small amount of reservoir. As a non-limiting example, the volume of the reservoir for a suitable pharmaceutical composition to be administered can be between 0.1 and 50 ml. Continuous administration can be transdermal administration with patches that are attached to the skin and replaced at regular intervals. Those skilled in the art are familiar with patch systems for drug delivery suitable for this purpose. For transdermal administration, replacement of the first used patch places a new second patch, for example, on the skin surface immediately next to the first used patch and just before removal of the first used patch. Note that it is particularly suitable for uninterrupted administration because of the benefits that can be achieved at the same time. There is no problem of flow interruption or power loss.

0084The compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include solutions such as phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, liposomes and the like. .. Compositions containing such carriers can be formulated by well-known conventional methods. Formulations may include carbohydrates, buffer solutions, amino acids and / or surfactants. Carbohydrates can be non-reducing sugars, preferably trehalose, sucrose, octasulfhate, sorbitol or xylitol. In general, as used herein, a "pharmaceutically acceptable carrier" is any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retardants that are compatible with drug administration. Means. The use of such vehicles and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosage and concentration used and are additional buffers; preservatives; co-solvents; antioxidants including ascorbic acid and methionine. Chelating agents, such as EDTA; metal complexes (eg, Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polysaccharide alcohols; amino acids, such as alanine, glycerin, asparagine. , 2-Phenylalanine and threonins; sugars or sugar alcohols such as trehalose, sucrose, octasulfide, sorbitol or xylitol stachiose, mannose, sorbose, xylose, ribose, myoinisitose, galactose, lactitol, ribitol, myoinicitol, ga Lactitol, glycerol, cyclitol (eg, inositol), polyethylene glycol; sulfur-containing reducing agents such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha] -monothioglycerol and sodium thiosulfate; Includes low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone. Such formulations can be used for continuous administration, which can be intravenous or subcutaneous administration with and / or without a pump system. Amino acids can be charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and / or histidine. Surfactants can be detergent, preferably those with a molecular weight of&gt; 1.2 KD, and / or polyethers, preferably those with a molecular weight of&gt; 3 KD. Non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples of preferred polyethers are PGE 3000, PEG 3350, PEG 4000 or PEG 5000. The buffer system used in the present invention can have a preferred pH of 5-9 and may include citrates, succinates, phosphates, histidine and acetates. It can be that of 3KD. Non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples of preferred polyethers are PGE 3000, PEG 3350, PEG 4000 or PEG 5000. The buffer system used in the present invention can have a preferred pH of 5-9 and may include citrates, succinates, phosphates, histidine and acetates. It can be that of 3KD. Non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples of preferred polyethers are PGE 3000, PEG 3350, PEG 4000 or PEG 5000. The buffer system used in the present invention can have a preferred pH of 5-9 and may include citrates, succinates, phosphates, histidine and acetates.

0085Suitable compositions of the invention can be determined, for example, by dose-increasing studies in which increasing doses of the polypeptides of the invention exhibiting the interspecific specificities described herein are administered to non-chimpanzee primates, such as macaques. Can be administered to the subject at a dose. As mentioned above, the binding molecules of the invention that exhibit the interspecificity described herein have the advantage that they can be used in preclinical studies in non-chimpanzee primates and in the same form as drugs in humans. These compositions can also be administered in combination with other proteinaceous and non-proteinaceous drugs. These drugs may be administered simultaneously using a composition comprising the polypeptide of the invention as defined herein, or separately administered at defined time intervals and doses before or after administration of the polypeptide. obtain. The dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for a single patient is the size of the patient, the surface area of the body, the age, the individual compounds to be administered, the gender, the time and route of administration, the overall health condition and the simultaneous administration. It depends on many factors, including other drugs. Preparations for parenteral administration include sterilized aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffering media. Parenteral vehicles include sodium chloride solution, ringer dextrose, dextrose and sodium chloride, lactated ringer or fixed oil. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (eg, those based on Ringer dextrose) and the like. Preservatives and other additives such as antibacterial substances, antioxidants, chelating agents, inert gases and the like may also be present. In addition, the compositions of the invention may include, for example, a proteinaceous carrier, preferably of human origin, such as serum albumin or immunoglobulin. It is assumed that the compositions of the invention may include additional biologically active agents in addition to the polypeptides of the invention as defined herein, depending on the intended use of the composition. Has been done. Such drugs include drugs that act on the gastrointestinal system, drugs that act as cytostatics, drugs that prevent hyperurikemia, drugs that inhibit the immune response (eg, corticosteroids), inflammatory responses. It can be a drug that regulates, a drug that acts on the circulatory system and / or a drug known in the art, such as a cytokine. It is also envisioned that the binding molecules of the invention will be applied in co-therapy, ie in combination with another anti-cancer drug.

0086The biological activity of the pharmaceutical compositions defined herein is described, for example, in WO 99/54440 or Schlereth et al. (Cancer Immunol. Immunother. 20 It can be determined by a cytotoxicity assay as described by (2005), 1-12). As used herein, "efficacy" or "in vivo efficacy" refers to a response to treatment with a pharmaceutical composition of the invention, eg, using standardized NCI response criteria. The success or in vivo efficacy of treatment with the pharmaceutical compositions of the present invention determines the effectiveness of the composition for its intended purpose, i.e. its desired effect, i.e. depletion of pathological cells, eg, tumor cells. Represents the ability of the composition to bring. In vivo efficacy can be monitored by standard methods established in each disease, including but not limited to white blood cell count, differentiation, fluorescence activated cell selection, bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods may be used. In addition, computed tomography, X-rays, and nuclear magnetic resonance tomography (eg, for response assessment based on National Cancer Institute criteria [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999 Apr; 17 (4): 1244]), Positron-releasing tomography, leukocyte count, differentiation, fluorescence-activated cell selection, bone marrow aspiration, lymph node biopsy / Histology, and various lymphoma-specific clinical chemical parameters (eg, lactated dehydrogenase) and other established standard methods can be used.

0087Another major challenge in the development of drugs, eg, the pharmaceutical compositions of the present invention, is to adjust the pharmacokinetic properties to be predictable. For this purpose, a pharmacokinetic profile of a candidate drug, i.e. a profile of pharmacokinetic parameters that affect the ability of a particular drug to treat a given condition, can be established. Pharmacokinetic parameters of a drug that affect the ability of the drug to treat a particular disease include, but are not limited to, half-life, volume of distribution, hepatic first-pass metabolism and blood serum binding. The efficacy of a given drug can be influenced by each of the above parameters. "Half-life" means the time at which 50% of a administered drug is eliminated through biological processes such as metabolism, excretion, etc.

0088"Liver first pass metabolism" means the property of a drug to be metabolized when it first comes into contact with the liver, i.e. during the liver's first pass.

0089"Volume of distribution" refers to the degree of drug retention and distribution within these compartments of the body, such as intracellular and extracellular spaces, tissues and organs, and the like.

0090"Blood serum binding" means the property that a drug interacts with and binds to a blood serum protein, such as albumin, to reduce or lose the biological activity of the drug.

0091Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, rate of absorption, more onset and / or Cmax for a given amount of drug administered. "Bioavailability" means the amount of drug in the blood compartment. "Ragtime" means the time delay between the administration of a drug and its detection and measurable stage in blood or plasma.

0092"Tmax" is the time after which the maximum blood concentration of the drug is achieved, and "Cmax" is the maximum blood concentration obtained by a given drug. The time to reach the blood or tissue concentration of the drug required for its biological effect is affected by all parameters. The pharmacokinetic parameters of bispecific single-chain antibodies exhibiting interspecific specificity that can be determined in preclinical animal studies in non-chimpanzee primates as described above include, for example, Schlereth et al. (Cancer Immunol. Immunother. 20 (2005)). , 1-12) is also shown in the publication.

0093As used herein, the term "toxicity" refers to the toxic effects of a drug as seen in an adverse event or serious adverse event. These side events may represent an overall lack of tolerability of the drug and / or a local lack of tolerability after administration. Toxicity can also include teratogenic or carcinogenic effects caused by the drug.

0094As used herein, the terms "safety," "in vivo safety," or "tolerability" are serious adverse events immediately after dosing (locally tolerated) and during longer drug application periods. Is defined as the administration of drugs that do not induce. "Safety," "in vivo safety," or "tolerability" can be assessed, for example, at regular intervals during treatment and follow-up. Measurements include clinical evaluation, such as screening for organ signs, and laboratory abnormalities. Clinical evaluations can be performed and deviations from normal findings according to NCI-CTC and / or MedDRA standards can be recorded / encoded. Organ signs, for example, Common Terminology Criteria for adverse events Criteria such as allergy / immunology, blood / bone marrow, cardiac arrhythmia, coagulation, etc. may be included as shown in v3.0 (CTCAE). Test parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profile and urinalysis and other body fluid tests such as serum, plasma, lymph or spinal fluid, liquor and the like. Therefore, safety is determined, for example, by physical examination, imaging techniques (ie, ultrasound, x-ray, CT scan, magnetic resonance imaging (MRI), other measurements using specialized equipment (ie, electrocardiogram), vital signs. It can be assessed by measuring test parameters and recording adverse events. For example, in use and methods according to the invention, adverse events in non-chimpanzee primates can be tested by histopathology and / or histochemical methods.

0095An "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially suppress the disease and its complications in patients already suffering from the disease. The amount effective for this application will depend on the severity of the infection and the overall condition of the subject's own immune system. The term "patient" includes human and other mammalian subjects undergoing either prophylactic or therapeutic treatment.

0096As used herein, the term "effective / non-toxic dose" refers to pathological cell depletion, tumor removal, tumor shrinkage or disease with or without significant toxic effects. Represents an acceptable dose of the binding molecule of the invention, high enough to provide stabilization of. Such effective and non-toxic doses can be determined, for example, by dose-increasing studies described in the art, and it should be less than or equal to the dose that induces serious adverse side events (dose limiting toxicity). , DLT).

0097The above terms also refer to, for example, the Preclinical safety evaluation of biotechnology derived pharmaceuticals S6; ICH Harmonized Tripartite Guideline; ICH Steering Committee meeting on July 16, 1997.

0098The appropriate dosage or therapeutically effective amount of the binding molecule of the present invention depends on the condition being treated, the severity of the condition, previous treatment and the patient's medical history and response to the therapeutic agent. Appropriate doses may be adjusted according to the discretion of the attending physician so that they can be administered to the patient in a single or series of doses. The pharmaceutical composition may be administered as a single treatment or in combination with additional treatments, such as anti-cancer treatments, if required.

0099The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, i.e., subcutaneous, intramuscular, intravenous, intra-articular and / or intrasynovial sac. Parenteral administration can be by bolus injection or continuous infusion.

0100When the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with a suitable liquid prior to administration. The lyophilized material is reconstituted with, for example, bacteriostatic water for injection (BWFI), physiological saline, phosphate buffered saline (PBS) or the same formulation as the protein was added prior to lyophilization. obtain.

0101Preferably, the binding molecule of the invention or produced by the process of the invention is used for the prevention, treatment or remission of a disease selected from proliferative, neoplastic or immunological disorders.

0102An alternative aspect of the invention is a method for the prevention, treatment or amelioration of a disease selected from proliferative, neoplastic or immunological disorders, the present invention or for patients in need thereof. Provided is a method comprising the step of administering a binding molecule produced by the process of the present invention.

0103The formulations described herein are useful as pharmaceutical compositions that treat, ameliorate and / or prevent the pathological medical conditions described herein in patients in need thereof. The term "treatment" refers to both therapeutic treatment and preventive or deterrent measures. Treatment is a disease / disorder, disease for the purpose of curing, repairing, alleviating, releasing, correcting, relieving, ameliorating, improving or affecting the disease, symptoms of the disease or predisposition to the disease. Includes application or administration of a formulation to the body, isolated tissue or cells of a patient with a symptom or disease / predisposition to the disorder.

0104"Needing treatment" includes those who already have a disability and those who want to prevent the disability. The term "disease" is any condition that would benefit from treatment with the protein formulations described herein. This includes chronic and acute disorders or diseases, including pathological conditions that make mammals more susceptible to the disease in question. Non-limiting examples of diseases / disorders treated by the present invention include proliferative diseases, neoplastic diseases or immunological disorders.

0105Preferably, the binding molecules of the invention are used for the prevention, treatment or remission of B cell disorders that correlate with (over) expression of BCMA, such as plasma cell disorders and / or autoimmune diseases. Autoimmune diseases are, for example, systemic lupus erythematosus or rheumatoid arthritis.

0106Cytotoxicity mediated by BCMA / CD3 bispecific binding molecules can be measured in a variety of ways. Effector cells can be, for example, stimulated and concentrated (human) CD8-positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMC). If the target cell is of macaque origin or expresses macaque BCMA or is transfected with macaque BCMA, the effector cells should also be of macaque origin, eg macaque T cell line, eg 4119LnPx. Target cells should express BCMA, eg, human or macaque BCMA (at least the extracellular domain). The target cell can be a cell line stably or transiently transfected with BCMA, eg, human or macaque BCMA (eg, CHO). Alternatively, the target cell can be a BCMA-positive naturally expressed cell line, such as the human multiple myeloma cell line L363 or NCI-H929. EC50 levels are usually thought to be lower for target cell lines that express high levels of BCMA on their cell surface. Effector vs. The target cell (E: T) ratio is usually about 10: 1, but this can be changed. The cytotoxic activity of the BCMA / CD3 bispecific binding molecule can be measured in a 51 chromium release assay (incubation time of about 18 hours) or in a FACS-based cytotoxicity assay (incubation time of about 48 hours). The incubation time of the assay (cytotoxic reaction) can also be varied. Other methods of measuring cytotoxicity are well known to those of skill in the art and include MTT or MTS assays, ATP-based assays, including bioluminescence assay, sulforhodamine B (SRB) assay, WST assay, cloning assay and ECIS technology. including. The cytotoxic activity mediated by the BCMA / CD3 bispecific binding molecule of the invention is preferably measured in a cell-based cytotoxic assay. It corresponds to the half-effect concentration (the concentration of binding molecule that induces an intermediate cytotoxic reaction between baseline and maximum).₅₀Represented by a value.

0107A method for the treatment or amelioration of B cell disorders that correlate with (over) expression of BCMA, such as plasma cell disorders and / or autoimmune diseases, in which the binding molecule of the invention is administered to a subject in need thereof. Methods involving steps are also provided by the present invention. Autoimmune diseases are, for example, systemic lupus erythematosus or rheumatoid arthritis. In plasma cell damage, one clone of plasma cells proliferates indefinitely. As a result, this clone produces a huge amount of a single (monoclonal) antibody known as the M protein. In some cases, for example in the case of monoclonal immunoglobulinemia, the antibody produced is incomplete and consists of only light chains or only heavy chains. These abnormal plasma cells and the antibodies they produce are usually limited to one type. Preferably, plasmacytosis is multiple myeloma, plasmacytoma, plasmacytotic leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary bone plasmacytoma, extramedullary plasma cells. It is selected from the group consisting of tumors, osteosclerotic myeloma, heavy chain disease, unclear monoclonal gamma globulinemia, and smoldering multiple myeloma.

0108In another aspect, a kit comprising a binding molecule of the invention, a nucleic acid molecule of the invention, a vector of the invention or a host cell of the invention is provided. The kit may include one or more vials containing the binding molecule and instructions for use. The kit also provides means for administering the binding molecule of the invention, eg, syringes, pumps, injectors, etc., for reconstructing the binding molecule of the invention and / or for diluting the binding molecule of the invention. May include means. In another aspect of the invention, the second binding domain can bind to CD3 epsilon. In yet another aspect of the invention, the second binding domain can bind to human CD3 and macaque CD3, preferably human CD3 epsilon and macaque CD3 epsilon. Additional or alternative, the second binding domain can bind to the CD3 epsilon of common marmosets, cotton-top tamarins and / or common squirrel monkeys. According to these embodiments, the binding domains of one or both of the binding molecules of the present invention are preferably cross-species relative to members of the order Mammalia of Primates. specific). Interspecific CD3 binding domains are described, for example, in WO 2008/119567.

0109A second binding domain that can bind to the T cell CD3 receptor complex (a) CDR-L1 shown in SEQ ID NO: 27 of WO 2008/119567, CDR-L2 shown in SEQ ID NO: 28 of WO 2008/119567, and SEQ ID NO: 29 of WO 2008/119567. CDR-L3; (b) CDR-L1 shown in SEQ ID NO: 117 of WO 2008/119567, CDR-L2 shown in SEQ ID NO: 118 of WO 2008/119567, and SEQ ID NO: 119 of WO 2008/119567. CDR-L3; and (c) CDR-L1 shown in SEQ ID NO: 153 of WO 2008/119567, CDR-L2 shown in SEQ ID NO: 154 of WO 2008/119567 and SEQ ID NO: 155 of WO 2008/119567 CDR-L3 The binding molecule of the present invention containing a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from is particularly preferred.

0110In a preferred embodiment of the binding molecule of the present invention, the second binding domain capable of binding to the T cell CD3 receptor complex is: (a) CDR-H1 shown in SEQ ID NO: 12 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 13 of WO 2008/119567, and SEQ ID NO: 14 of WO 2008/119567. CDR-H3; (b) CDR-H1 shown in SEQ ID NO: 30 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 31 of WO 2008/119567, and SEQ ID NO: 32 of WO 2008/119567. CDR-H3; (c) CDR-H1 shown in SEQ ID NO: 48 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 49 of WO 2008/119567, and SEQ ID NO: 50 of WO 2008/119567. CDR-H3; (d) CDR-H1 shown in SEQ ID NO: 66 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 67 of WO 2008/119567, and SEQ ID NO: 68 of WO 2008/119567. CDR-H3; (e) CDR-H1 shown in SEQ ID NO: 84 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 85 of WO 2008/119567, and SEQ ID NO: 86 of WO 2008/119567. CDR-H3; (f) CDR-H1 shown in SEQ ID NO: 102 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 103 of WO 2008/119567, and SEQ ID NO: 104 shown in WO 2008/119567. CDR-H3; (g) CDR-H1 shown in SEQ ID NO: 120 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 121 of WO 2008/119567, and SEQ ID NO: 122 of WO 2008/119567. CDR-H3; (h) CDR-H1 shown in SEQ ID NO: 138 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 139 of WO 2008/119567, and SEQ ID NO: 140 shown in WO 2008/119567. CDR-H3; (i) CDR-H1 shown in SEQ ID NO: 156 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 157 of WO 2008/119567, and SEQ ID NO: 158 of WO 2008/119567. CDR-H3; and (j) CDR-H1 shown in SEQ ID NO: 174 of WO 2008/119567, CDR-H2 shown in SEQ ID NO: 175 of WO 2008/119567 and SEQ ID NO: 176 of WO 2008/119567. CDR-H3 Contains the VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from.

0111A second binding domain capable of binding to the T cell CD3 receptor complex is selected from the group consisting of the VL region shown in SEQ ID NO: 35, 39, 125, 129, 161 or 165 of WO 2008/119567. Binding molecules of the invention that include the region are more preferred.

0112The second binding domain capable of binding to the T cell CD3 receptor complex is WO 2008/119567 SEQ ID NO: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, Alternatively, it is preferred to include a VH region selected from the group consisting of the VH regions shown in 123, 127, 141, 145, 159, 163, 177 or 181.

0113More preferably, the binding molecule of the present invention is: (a) VL region indicated by SEQ ID NO: 17 or 21 of WO 2008/119567 and VH region indicated by SEQ ID NO: 15 or 19 of WO 2008/119567; (b) VL region indicated by SEQ ID NO: 35 or 39 of WO 2008/119567 and VH region indicated by SEQ ID NO: 33 or 37 of WO 2008/119567; (c) VL region indicated by SEQ ID NO: 53 or 57 of WO 2008/119567 and VH region indicated by SEQ ID NO: 51 or 55 of WO 2008/119567; (d) VL region indicated by SEQ ID NO: 71 or 75 of WO 2008/119567 and VH region indicated by SEQ ID NO: 69 or 73 of WO 2008/119567; (e) VL region indicated by SEQ ID NO: 89 or 93 of WO 2008/119567 and VH region indicated by SEQ ID NO: 87 or 91 of WO 2008/119567; (f) VL region indicated by SEQ ID NO: 107 or 111 of WO 2008/119567 and VH region indicated by SEQ ID NO: 105 or 109 of WO 2008/119567; (g) VL region indicated by SEQ ID NO: 125 or 129 of WO 2008/119567 and VH region indicated by SEQ ID NO: 123 or 127 of WO 2008/119567; (h) VL region indicated by SEQ ID NO: 143 or 147 of WO 2008/119567 and VH region indicated by SEQ ID NO: 141 or 145 of WO 2008/119567; (i) VL region indicated by SEQ ID NO: 161 or 165 of WO 2008/119567 and VH region indicated by SEQ ID NO: 159 or 163 of WO 2008/119567; (j) VL region indicated by SEQ ID NO: 179 or 183 of WO 2008/119567 and VH region indicated by SEQ ID NO: 177 or 181 of WO 2008/119567; It is characterized by a second binding domain capable of binding to the T cell CD3 receptor complex containing the VL and VH regions selected from the group consisting of.

0114According to the preferred embodiment of the binding molecule of the present invention, particularly the second binding domain capable of binding to the T cell CD3 receptor complex, the pair of VH and VL regions is in the form of a single chain antibody (scFv). The VH and VL regions are arranged in the order of VH-VL or VL-VH. The VH region is preferably located on the N-terminal side of the linker sequence. The VL region is located on the C-terminal side of the linker sequence.

0115A preferred embodiment of the binding molecule of the present invention described above is WO 2008/119567 SEQ ID NO: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149. , 151, 167, 169, 185 or 187, characterized by a second binding domain capable of binding to a T cell CD3 receptor complex containing an amino acid sequence selected from the group.

0116In one embodiment, the first and second binding domains are or are obtained from antibodies. In another embodiment, both binding domains are or are obtained from the antibody.

0117In the binding molecule of the present invention, the first and second domains are (scFv).₂, (Single domain mAb)₂, ScFv-It is also preferred to form a molecule selected from the group consisting of single domain mAbs, diabodies or oligomers thereof.

0118It is further envisioned that the BCMA / CD3 bispecific binding molecule of the present invention can exert therapeutic efficacy or antitumor activity. This can be evaluated, for example, in the following examples, for example in the study disclosed in Example A19 (advanced human tumor xenograft model). Those skilled in the art will achieve significant reproducible results while certain parameters of this study, such as the number of tumor cells to be injected, the site of injection, the number of human T cells to be transplanted, and the BCMA / CD3 double to be administered. We know how to modify or adapt the amount and time series of specific binding molecules. Preferably, the tumor growth inhibitory T / C [%] is 70 or 60 or less, more preferably 50 or 40 or less, even more preferably 30 or 20 or less, and most preferably 10 or less. 5 or less, 2.5 or even less.

0119Preferably, the BCMA / CD3 bispecific binding molecule of the invention does not induce / mediate lysis of BCMA negative cells such as HL60, MES-SA and SNU-16, or essentially does not induce / mediate lysis. The terms "do not induce lysis", "essentially do not induce lysis", "do not mediate lysis" or "do not essentially mediate lysis" are used by the binding molecules of the present invention in BCMA-positive cell lines, eg. With 100% dissolution of NCI-H929, L-363 or OPM-2, greater than 30%, preferably greater than 20%, more preferably greater than 10%, particularly preferably 9%, 8%, 7%, 6% or It means that it does not induce or mediate the lysis of more than 5% BCMA-negative cells. This applies for concentrations of binding molecules up to at least 500 nM. Those skilled in the art know how to measure cytolysis and require no additional effort. In addition, the specification provides specific instructions on how to measure cytolysis; see, for example, Example A20 below.

0120The present invention also includes SEQ ID NO: Provided are binding molecules comprising any one of the amino acid sequences shown in 1-1000 and 1022-1093. Preferably, the binding molecule is three VH CDR sequences derived from the binding molecule represented by "BCMA- (X)" where X is 1-100 (see column 2 of the attached sequence listing). (See column 4 of the attached sequence listing, named "VH CDR1", "VH CDR2", "VH CDR3") and / or in BCMA-X where X is 1-100. Three VL CDR sequences (named "VL CDR1", "VH CDR2", "VH CDR3") derived from the bound molecule represented (see column 2 of the attached sequence listing), appendix See column 4 of the sequence list). Preferably, the binding molecule comprises the VH and / or VL sequences shown in the appendix sequence listing (see column 4 of the appendix sequence listing; "VH" and "VL"). Preferably, the binding molecule comprises the scFv sequence shown in the appendix sequence listing (see column 4 of the appendix sequence listing; "scFv"). Preferably, the binding molecule comprises the bispecific molecular sequence shown in the appendix sequence listing (see column 4 of the appendix sequence listing; "two-characteristic molecule").

0121The present invention also includes a first binding domain and a second binding domain, the first binding domain binding to the B cell maturation antigen BCMA and the second binding domain binding to CD3, at least two binding domains. Regarding bispecific binding agents containing (general item 1), and also including those of the following general item (GI): GI 2 A bispecific binding agent of general item 1, in which the first binding domain binds to the extracellular domain of BCMA and the second binding domain binds to the ε chain of CD3. GI 3 Bispecific binder of general item 1 or 2 in the form of a full-length antibody or antibody fragment. GI 4 A bispecific binding agent of general item 3 in the form of a full-length antibody, in which the first BCMA binding domain is derived from mice and the second CD3 binding domain is derived from rats. GI 5 The bispecific of general item 3, which is a form of antibody fragment in the form of a diabody, comprising a heavy chain variable domain linked to a light chain variable domain on the same polypeptide so that the two domains do not form a pair. Sex binder. A bispecific binder of general item 1 or 2, which is a form of bispecific single chain antibody consisting of two scFv molecules linked through a GI 6 linker peptide or by a human serum albumin molecule. The GI 7 heavy chain region (VH) and the corresponding variable light chain region (VL) move from the N-terminus to the C-terminus. VH (BCMA) -VL (BCMA) -VH (CD3) -VL (CD3), VH (CD3) -VL (CD3) -VH (BCMA) -VL (BCMA) or VH CD3) -VL (CD3) -VL (BCMA) -VH (BCMA) The bispecific binder of general item 6, which is arranged in the order of. GI 8 A bispecific binding agent of general item 1 or 2, which is a form of single domain immunoglobulin domain selected from VHH and VH. GI 9 A form of Fv molecule with four antibody variable domains with at least two binding domains, at least one binding domain specific for human BCMA and at least one binding domain specific for human CD3. , General item 1 or 2 bispecific binder. GI 10 BCMA-specific first binding domain, constant subregion located on the C-terminal side of the first binding domain, scorpion linker located on the C-terminal side of the constant subregion, and C of the constant subregion Bispecific binding agent of general item 1 or 2, which is a form of a single chain binding molecule consisting of a second binding domain specific to CD3 located at the terminal side. GI 11 An antibody-like antibody that binds to BCMA through two heavy / light chain Fvs of an antibody or antibody fragment and binds to CD3 through a processed binding domain that integrates into the non-CDR loop of the heavy or light chain of the antibody or antibody fragment. Bispecific binding agent of general item 1 or 2, which is in the form of a molecule. GI 12 Bispecific bispecific binding agent of general item 1, which is a form of ankyrin repeat molecule. GI 13 The first binding domain has a format selected from the format defined in any of general items 3-12, and the second binding domain has a format defined in any of general items 3-12. The bispecific binding agent of General Item 1 having a different form of choice. GI 14 Bicyclic peptide, general item 1 bispecific binder. GI 15 A pharmaceutical composition comprising at least one bispecific binder according to any one of the general items 1-14. GI 16 A bispecific binder according to any of general items 1 to 14 and general item 14 for the treatment of plasma cell disorders or other B cell disorders that correlate with BCMA expression and for the treatment of autoimmune diseases. Pharmaceutical composition. GI 17 Plasmacytoma, plasmacytotic leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstram macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, A bispecific binding agent according to any of general items 1 to 14 for the treatment of plasma cell disorders selected from heavy chain disease, unclear monoclonal gamma globulinemia, and smoldering multiple myeloma. General item 15 pharmaceutical composition. Variations on the above items can be derived from EP-Nr. 10 191 418.2, which are also included in the present invention.

0122Detailed explanation The present invention specifically relates to a group of binding molecules that are grouped as follows. The above definitions, embodiments and / or aspects may apply to the following groups of binding molecules.

0123<u style="single">First binding molecule group (A)</u> The first binding molecule group is a first and second binding domain in which the first binding domain can bind to epitope cluster 3 and epitope cluster 4 of BCMA and the second binding domain can bind to the T cell CD3 receptor complex. Includes binding domain.

0124Thus, in the first aspect, the invention is a binding molecule that is at least bispecific, comprising the first and second binding domains, where. (a) The first binding domain can bind to epitope cluster 3 and epitope cluster 4 of BCMA, and (b) The second binding domain can bind to the T cell CD3 receptor complex, Here, the epitope cluster 3 of BCMA corresponds to the amino acid residues 24 to 41 of the sequence shown in SEQ ID NO: 1002, and the epitope cluster 4 of BCMA corresponds to the amino acid residues 42 to 42 of the sequence shown in SEQ ID NO: 1002. Provided are binding molecules, corresponding to 54.

0125It is also envisioned that the first binding domain of the invention can simultaneously bind to epitope clusters 3 (SEQ ID NO: 1016) and 4 (SEQ ID NO: 1019) of human BCMA.

0126In a further aspect, the first binding domain of the binding molecule of the invention cannot bind to the chimeric extracellular domain of BCMA shown in SEQ ID NO: 1015. In other words, epitope cluster E7, or more specifically amino acid residue 39 (arginine) of SEQ ID NO: 1002, plays an important antigenic role in the binding of the first binding domain to BCMA. When this amino acid is replaced with another amino acid, preferably by a non-conservative substitution, such as a substitution with proline or alanine, the first binding domain of the binding molecule of the invention no longer binds to the extracellular domain of BCMA. Can not.

0127The term "unable to bind" means that the first binding domain of the binding molecule of the invention does not bind to the human / mouse chimeric BCMA, eg, SEQ ID NO: 1015, ie, eg, SEQ ID NO: 1015. For human / mouse chimeric BCMA, preferably over 30%, preferably over 20%, more preferably over 10%, particularly preferably over 9%, 8 under the conditions applied in Appendix Example A. Means that it does not show reactivity greater than%, 7%, 6% or 5%.

0128In one aspect, the first binding domain of the invention can bind to epitope clusters 3 and 4 of human BCMA, preferably human BCMA ECD. Thus, each epitope cluster in the human BCMA protein is exchanged for each epitope cluster of the mouse BCMA antigen (thus forming a construct containing human BCMA in which human epitope clusters 3 and / or 4 are replaced by each mouse epitope cluster. As an example, SEQ ID NO: (See 1011 and 1012), a decrease in binding in the binding domain can occur. The reduction is preferably at least 10%, 20%, 30%, 40%, 50%; compared to each epitope cluster of human BCMA protein, with 100% binding of human BCMA protein to each epitope cluster. Preferably, it is at least 60%, 70%, 80%, 90%, 95%, or even 100%. It is envisioned that the above human BCMA / mouse BCMA chimera will be expressed in CHO cells. It is also envisioned to fuse at least one of the human BCMA / mouse BCMA chimeras, preferably the mouse E3 / human BCMA chimera, with a transmembrane domain and / or cytoplasmic domain of a different membrane binding protein, such as EpCAM; checking ... This method of testing the loss of binding due to the exchange of non-human (eg, mouse) BCMA antigens with each epitope cluster is described in Appendix A, especially Examples A1-A3. A further method for determining the contribution of a particular residue of a target antigen to recognition by a given binding molecule or binding domain is alanine scanning (eg, Morrison) in which each residue to be analyzed is replaced with alanine, eg, through site-directed mutagenesis. KL &amp; Weiss GA. Cur Opin Chem Biol. 2001 Jun; 5 (3): See 302-7). The reason alanine is used is that it is not bulky, is chemically inert, and yet has a methyl functional group that mimics the reference secondary structure of many other amino acids. ,. Occasionally, bulky amino acids such as valine or leucine may be used in examples where it is desired to preserve the size of the residue to be mutated. Alanine scanning is a mature technique that has been used for a long time. In one aspect, the first binding domain of the invention binds to macaque BCMA epitope clusters 3 and 4, and further macaque BCMA, preferably macaque BCMA epitope clusters 3 and / or 4 (SEQ ID NO:, respectively). It can bind to 1020 and 1021), such as those from Macaca mulatta or Macaca fascicularis. It is assumed that the first binding domain binds or does not bind to mouse BCMA.

0129Thus, in one embodiment, the epitope cluster 3 and the extracellular protein domain of BCMA formed by amino acid residues 24-41 and 42-54 of the human sequence set forth in SEQ ID NO: 1002, respectively, and human BCMA, respectively. The binding domain that binds to 4 is also an epitope of the extracellular protein domain of macaque BCMA, in particular BCMA formed by amino acid residues 24-41 and 42-54 of the macaque BCMA sequence shown in SEQ ID NO: 1006, respectively. Also bind to clusters 3 and / or 4.

0130In one embodiment, the first binding domain of the binding molecule can bind to epitope clusters 3 and 4 of BCMA, where epitope clusters 3 and 4 of BCMA are SEQ ID NO: 1002 (human BCMA full length polypeptide) or SEQ ID. Corresponds to amino acid residues 24-40 and 41-53 of the sequence shown in NO: 1007 (human BCMA extracellular domain: SEQ ID NO: 1002 amino acids 1-54), respectively.

0131In one aspect of the invention, the first binding domain of the binding molecule is, additionally or alternatively, of common marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus oedipus) and / or common squirrel monkeys (Saimiri sciureus). It can bind to epitope clusters 3 and / or 4 of BCMA.

0132The affinity of the first binding domain for human BCMA is preferably 40nM, more preferably 35nM, 15nM, or 10nM, even more preferably 5nM, even more preferably 1nM, even more preferably 0.5 nM, even more preferably 0.1 nM, and most preferably 0.05 nM. The affinity of the first binding domain for macaque BCMA is preferably 15nM, more preferably 10nM, even more preferably 5nM, even more preferably 1nM, even more preferably 0.5nM, even more preferably. 0.1 nM, and most preferably 0.05 nM and even 0.01 nM. Affinity can be measured, for example, in the Biacore assay or in the Scatchard assay, as described in the Examples. Macaque BCMA binding pair The affinity gap for binding to human BCMA is preferably [1: 10 to 1: 5] or [5: 1 to 10: 1], more preferably [1: 5 to 5: 1], and most preferably. It is [1: 2 ~ 3: 1] and even [1: 1 ~ 3: 1]. Other methods of determining affinity are well known to those of skill in the art.

0133Cytotoxicity mediated by BCMA / CD3 bispecific binding molecules can be measured in a variety of ways. Effector cells can be, for example, stimulated and concentrated (human) CD8-positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMC). If the target cell is of macaque origin or expresses macaque BCMA or is transfected with macaque BCMA, the effector cells should also be of macaque origin, eg macaque T cell line, eg 4119LnPx. Target cells should express BCMA, eg, human or macaque BCMA (at least the extracellular domain). The target cell can be a cell line stably or transiently transfected with BCMA, eg, human or macaque BCMA (eg, CHO). Alternatively, the target cell can be a BCMA-positive naturally expressed cell line, such as the human multiple myeloma cell line L363 or NCI-H929. EC50 levels are usually thought to be lower for target cell lines that express high levels of BCMA on their cell surface. Effector vs. The target cell (E: T) ratio is usually about 10: 1, but this can be changed. The cytotoxic activity of the BCMA / CD3 bispecific binding molecule can be measured in a 51 chromium release assay (incubation time of about 18 hours) or in a FACS-based cytotoxicity assay (incubation time of about 48 hours). The incubation time of the assay (cytotoxic reaction) can also be varied. Other methods of measuring cytotoxicity are well known to those of skill in the art and include MTT or MTS assays, ATP-based assays, including bioluminescence assay, sulforhodamine B (SRB) assay, WST assay, cloning assay and ECIS technology. including. The cytotoxic activity mediated by the BCMA / CD3 bispecific binding molecule of the invention is preferably measured in a cell-based cytotoxic assay. It corresponds to the half-effect concentration (the concentration of binding molecule that induces an intermediate cytotoxic reaction between baseline and maximum).₅₀Represented by a value. Preferably, the EC of the BCMA / CD3 bispecific binding molecule₅₀The values are 20.000 pg / ml, more preferably 5000 pg / ml, even more preferably 1000 pg / ml, even more preferably 500 pg / ml, even more preferably 250 pg / ml, even more. It is preferably 100 pg / ml, even more preferably 50 pg / ml, even more preferably 10 pg / ml, and most preferably 5 pg / ml.

0134EC above₅₀Any of the values can be combined with any one of the indicated scenarios of cell-based cytotoxicity assays. For example, when (human) CD8-positive T cells or Makaku T cell lines are used as effector cells, the EC of the BCMA / CD3 bispecific binding molecule₅₀The values are preferably 1000 pg / ml, more preferably 500 pg / ml, even more preferably 250 pg / ml, even more preferably 100 pg / ml, even more preferably 50 pg / ml, Even more preferably 10 pg / ml, and most preferably 5 pg / ml. If the target cell in this assay is a (human or mackerel) BCMA-transfected cell, such as a CHO cell, then the EC of the BCMA / CD3 bispecific binding molecule₅₀The values are preferably 150 pg / ml, more preferably 100 pg / ml, even more preferably 50 pg / ml, even more preferably 30 pg / ml, even more preferably 10 pg / ml, And most preferably 5 pg / ml. EC if the target cell is a BCMA-positive naturally expressed cell line₅₀The values are preferably 250 pg / ml, even more preferably 200 pg / ml, even more preferably 100 pg / ml, even more preferably 150 pg / ml, even more preferably 100 pg / ml. , And most preferably 50 pg / ml or less. EC of BCMA / CD3 bispecific binding molecule when (human) PBMC is used as an effector cell₅₀The values are preferably 1000 pg / ml, more preferably 750 pg / ml, more preferably 500 pg / ml, even more preferably 250 pg / ml, even more preferably 100 pg / ml, and Most preferably 50 pg / ml or less.

0135The difference in cytotoxic activity between the monomeric and dimeric isoforms of individual BCMA / CD3 bispecific binding molecules (eg, antibodies) is referred to as the "potency gap." This efficacy gap is, for example, the EC of the monomer of the molecule.₅₀Value and dimer EC₅₀It can be calculated as a ratio between values. The potency gap of the tested BCMA / CD3 bispecific binding molecule of the present invention is preferably 5, more preferably 4, even more preferably 3, even more preferably 2 and most preferably 1. Is.

0136Preferably, the BCMA / CD3 bispecific binding molecules of the invention neither bind to nor cross-react with human BAFF-R and / or human TACI. Methods for detecting cross-reactivity with human BAFF-R and / or human TACI are disclosed in Example A9.

0137It is also preferred that the BCMA / CD3 bispecific binding molecules of the invention have a dimer ratio equal to or less than 1.5%, preferably equal to or less than 0.8%, after multiple freeze / thaw cycles. .. The freezing / thawing cycle and determination of the dimer ratio can be carried out according to Example A16.

0138The BCMA / CD3 bispecific binding molecule (eg, antibody) of the present invention exhibits good thermal stability, preferably above 60 ° C, more preferably at melting temperatures of 62 ° C to 63 ° C. (See Example A17).

0139Plasma interference tests can be performed to determine potential interactions between BCMA / CD3 bispecific binding molecules (eg, antibodies) and human plasma proteins (see, eg, Example A18). In a preferred embodiment, the reduction in target binding of the BCMA / CD3 bispecific binding molecule mediated by plasma protein is not significant. The relative plasma interference value is preferably 2.

0140In one embodiment, the first binding domain of the binding molecule of the invention is: (a) CDR-H1 shown in SEQ ID NO: 231, CDR-H2 shown in SEQ ID NO: 232, CDR-H3 shown in SEQ ID NO: 233, CDR-L1 shown in SEQ ID NO: 234. , SEQ ID NO: 235, CDR-L2 and SEQ ID NO: 236, CDR-L3; (b) CDR-H1 shown in SEQ ID NO: 241, CDR-H2 shown in SEQ ID NO: 242, CDR-H3 shown in SEQ ID NO: 243, and CDR-L1 shown in SEQ ID NO: 244. , SEQ ID NO: 245, CDR-L2 and SEQ ID NO: 246, CDR-L3; (c) CDR-H1 shown in SEQ ID NO: 251, CDR-H2 shown in SEQ ID NO: 252, CDR-H3 shown in SEQ ID NO: 253, CDR-L1 shown in SEQ ID NO: 254. , SEQ ID NO: 255, CDR-L2 and SEQ ID NO: 256, CDR-L3; (d) CDR-H1 shown in SEQ ID NO: 261, CDR-H2 shown in SEQ ID NO: 262, CDR-H3 shown in SEQ ID NO: 263, and CDR-L1 shown in SEQ ID NO: 264. , SEQ ID NO: 265, CDR-L2 and SEQ ID NO: 266, CDR-L3; (e) CDR-H1 shown in SEQ ID NO: 271, CDR-H2 shown in SEQ ID NO: 272, CDR-H3 shown in SEQ ID NO: 273, CDR-L1 shown in SEQ ID NO: 274. , SEQ ID NO: 275, CDR-L2 and SEQ ID NO: 276, CDR-L3; (f) CDR-H1 shown in SEQ ID NO: 281, CDR-H2 shown in SEQ ID NO: 282, CDR-H3 shown in SEQ ID NO: 283, CDR-L1 shown in SEQ ID NO: 284. , SEQ ID NO: 285, CDR-L2 and SEQ ID NO: 286, CDR-L3; (g) CDR-H1 shown in SEQ ID NO: 291, CDR-H2 shown in SEQ ID NO: 292, CDR-H3 shown in SEQ ID NO: 293, CDR-L1 shown in SEQ ID NO: 294. , SEQ ID NO: 295, CDR-L2 and SEQ ID NO: 296, CDR-L3; (h) CDR-H1 shown in SEQ ID NO: 301, CDR-H2 shown in SEQ ID NO: 302, CDR-H3 shown in SEQ ID NO: 303, and CDR-L1 shown in SEQ ID NO: 304. , SEQ ID NO: 305, CDR-L2 and SEQ ID NO: 306, CDR-L3; (i) CDR-H1 shown in SEQ ID NO: 391, CDR-H2 shown in SEQ ID NO: 392, CDR-H3 shown in SEQ ID NO: 393, CDR-L1 shown in SEQ ID NO: 394. , SEQ ID NO: 395, CDR-L2 and SEQ ID NO: 396, CDR-L3; (k) CDR-H1 shown in SEQ ID NO: 401, CDR-H2 shown in SEQ ID NO: 402, CDR-H3 shown in SEQ ID NO: 403, CDR-L1 shown in SEQ ID NO: 404. , SEQ ID NO: 405, CDR-L2 and SEQ ID NO: 406, CDR-L3; (l) CDR-H1 shown in SEQ ID NO: 411, CDR-H2 shown in SEQ ID NO: 412, CDR-H3 shown in SEQ ID NO: 413, CDR-L1 shown in SEQ ID NO: 414. , SEQ ID NO: 415, CDR-L2 and SEQ ID NO: 416, CDR-L3; (m) CDR-H1 shown in SEQ ID NO: 421, CDR-H2 shown in SEQ ID NO: 422, CDR-H3 shown in SEQ ID NO: 423, CDR-L1 shown in SEQ ID NO: 424. , SEQ ID NO: 425, CDR-L2 and SEQ ID NO: 426, CDR-L3; (n) CDR-H1 shown in SEQ ID NO: 431, CDR-H2 shown in SEQ ID NO: 432, CDR-H3 shown in SEQ ID NO: 433, CDR-L1 shown in SEQ ID NO: 434. , SEQ ID NO: 435, CDR-L2 and SEQ ID NO: 436, CDR-L3; (o) CDR-H1 shown in SEQ ID NO: 441, CDR-H2 shown in SEQ ID NO: 442, CDR-H3 shown in SEQ ID NO: 443, CDR-L1 shown in SEQ ID NO: 444. , SEQ ID NO: 445, CDR-L2 and SEQ ID NO: 446, CDR-L3; (p) CDR-H1 shown in SEQ ID NO: 451, CDR-H2 shown in SEQ ID NO: 452, CDR-H3 shown in SEQ ID NO: 453, CDR-L1 shown in SEQ ID NO: 454. , SEQ ID NO: 455, CDR-L2 and SEQ ID NO: 456, CDR-L3; (q) CDR-H1 shown in SEQ ID NO: 461, CDR-H2 shown in SEQ ID NO: 462, CDR-H3 shown in SEQ ID NO: 463, CDR-L1 shown in SEQ ID NO: 464. , SEQ ID NO: 465, CDR-L2 and SEQ ID NO: 466, CDR-L3; (r) CDR-H1 shown in SEQ ID NO: 471, CDR-H2 shown in SEQ ID NO: 472, CDR-H3 shown in SEQ ID NO: 473, CDR-L1 shown in SEQ ID NO: 474. , SEQ ID NO: 475, CDR-L2 and SEQ ID NO: 476, CDR-L3; (s) CDR-H1 shown in SEQ ID NO: 481, CDR-H2 shown in SEQ ID NO: 482, CDR-H3 shown in SEQ ID NO: 483, CDR-L1 shown in SEQ ID NO: 484. , SEQ ID NO: 485, CDR-L2 and SEQ ID NO: 486, CDR-L3; (t) CDR-H1 shown in SEQ ID NO: 491, CDR-H2 shown in SEQ ID NO: 492, CDR-H3 shown in SEQ ID NO: 493, CDR-L1 shown in SEQ ID NO: 494. , SEQ ID NO: 495, CDR-L2 and SEQ ID NO: 496; and (u) CDR-H1 shown in SEQ ID NO: 501, CDR-H2 shown in SEQ ID NO: 502, CDR-H3 shown in SEQ ID NO: 503, CDR-L1 shown in SEQ ID NO: 504. , SEQ ID NO: 505, CDR-L2 and SEQ ID NO: 506, CDR-L3 Includes a VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of and a VL region containing CDR-L1, CDR-L2 and CDR-L3.

0141In yet another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 237, SEQ ID NO: 247, SEQ ID NO: 257, SEQ ID NO: 267, SEQ ID NO: 277, SEQ ID NO:: 287, SEQ ID NO: 297, SEQ ID NO: 307, SEQ ID NO: 397, SEQ ID NO: 407, SEQ ID NO: 417, SEQ ID NO: 427, SEQ ID NO: 437, SEQ ID NO: 447, The VH region selected from the group consisting of the VH regions shown in SEQ ID NO: 457, SEQ ID NO: 467, SEQ ID NO: 477, SEQ ID NO: 487, SEQ ID NO: 497 and SEQ ID NO: 507. Including.

0142In another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 238, SEQ ID NO: 248, SEQ ID NO: 258, SEQ ID NO: 268, SEQ ID NO: 278, SEQ ID NO: 288. , SEQ ID NO: 298, SEQ ID NO: 308, SEQ ID NO: 398, SEQ ID NO: 408, SEQ ID NO: 418, SEQ ID NO: 428, SEQ ID NO: 438, SEQ ID NO: 448, SEQ Includes a VL region selected from the group consisting of the VL regions shown in ID NO: 458, SEQ ID NO: 468, SEQ ID NO: 478, SEQ ID NO: 488, SEQ ID NO: 498 and SEQ ID NO: 508. ..

0143In one embodiment, the first binding domain of the binding molecule is: (a) VH region shown by SEQ ID NO: 237 and VL region shown by SEQ ID NO: 238; (b) VH region shown by SEQ ID NO: 247 and VL region shown by SEQ ID NO: 248; (c) VH region shown by SEQ ID NO: 257 and VL region shown by SEQ ID NO: 258; (d) VH region shown by SEQ ID NO: 267 and VL region shown by SEQ ID NO: 268; (e) VH region shown by SEQ ID NO: 277 and VL region shown by SEQ ID NO: 278; (f) VH region shown by SEQ ID NO: 287 and VL region shown by SEQ ID NO: 288; (g) VH region shown by SEQ ID NO: 297 and VL region shown by SEQ ID NO: 298; (h) VH region shown by SEQ ID NO: 307 and VL region shown by SEQ ID NO: 308; (i) VH region shown by SEQ ID NO: 397 and VL region shown by SEQ ID NO: 398; (k) VH region shown by SEQ ID NO: 407 and VL region shown by SEQ ID NO: 408; (l) VH region shown by SEQ ID NO: 417 and VL region shown by SEQ ID NO: 418; (m) VH region shown by SEQ ID NO: 427 and VL region shown by SEQ ID NO: 428; (n) VH region shown by SEQ ID NO: 437 and VL region shown by SEQ ID NO: 438; (o) VH region shown by SEQ ID NO: 447 and VL region shown by SEQ ID NO: 448; (p) VH region shown by SEQ ID NO: 457 and VL region shown by SEQ ID NO: 458; (q) VH region shown by SEQ ID NO: 467 and VL region shown by SEQ ID NO: 468; (r) VH region shown by SEQ ID NO: 477 and VL region shown by SEQ ID NO: 478; (s) VH region shown by SEQ ID NO: 487 and VL region shown by SEQ ID NO: 488; (t) VH region shown in SEQ ID NO: 497 and VL region shown in SEQ ID NO: 498; and (u) VH region shown by SEQ ID NO: 507 and VL region shown by SEQ ID NO: 508 Includes VH and VL regions selected from the group consisting of.

0144In one example, the first binding domain is SEQ ID NO: 239, SEQ ID NO: 249, SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279, SEQ ID NO: 289, SEQ ID. NO: 299, SEQ ID NO: 309, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419, SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449, SEQ ID NO: Includes an amino acid sequence selected from the group consisting of 459, SEQ ID NO: 469, SEQ ID NO: 479, SEQ ID NO: 489, SEQ ID NO: 499 and SEQ ID NO: 509.

0145Preferred CDR-H1 is represented by the amino acid sequence DYYIN. Preferred CDR-H2 has an amino acid sequence<img id="000003" he="4" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Indicated by. Preferred CDR-H3 has an amino acid sequence<img id="000004" he="4" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Indicated by. Preferred CDR-L1 has an amino acid sequence<img id="000005" he="4" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Indicated by. Preferred CDR-L2 is represented by the amino acid sequence KVSNRFS. Preferred CDR-L2 is indicated by the amino acid sequence AETSHVPWT or SQSSIYPWT.

0146A binding molecule having the amino acid sequence shown in SEQ ID NO: 300 is preferred. A binding molecule having the amino acid sequence shown in SEQ ID NO: 500 is also preferred.

0147Furthermore, the present invention relates to the use of epitope clusters 3 and 4 of BCMA, preferably human BCMA, to generate binding molecules capable of binding BCMA, preferably human BCMA, preferably antibodies. Epitope clusters 3 and 4 of BCMA preferably correspond to amino acid residues 24-41 and 42-54 of the sequence shown in SEQ ID NO: 1002, respectively.

0148In addition, the present invention is a method of producing an antibody capable of binding BCMA, preferably human BCMA, preferably a bispecific binding molecule. (a) A step of immunizing an animal with a polypeptide containing BCMA, preferably human BCMA epitope clusters 3 and 4, wherein the BCMA epitope clusters 3 and 4 are SEQ ID NO: Steps, corresponding to amino acid residues 24-41 and 42-54 of the sequence shown in 1002, (b) The step of obtaining the antibody, and (c) Optionally, the step of converting the antibody into a bispecific binding molecule capable of binding to human BCMA and preferably the T cell CD3 receptor complex. Provide a method including. Preferably, step (b) involves testing the resulting antibody as follows: Each epitope cluster of the human BCMA protein has been exchanged with each epitope cluster of the mouse BCMA antigen (thus human). If epitope clusters 3 and / or 4 are replaced with their respective mouse epitope clusters to produce a construct containing human BCMA), reduced antibody binding can occur. The reduction is preferably at least 10%, 20%, 30%, 40%, 50% compared to each epitope cluster of human BCMA protein, with 100% binding to each epitope cluster 3 and 4 of human BCMA protein. Is; more preferably at least 60%, 70%, 80%, 90%, 95% or even 100%. It is envisioned that the above human BCMA / mouse BCMA chimera will be expressed in CHO cells. It is also envisioned to fuse at least one of the human BCMA / mouse BCMA chimeras with a different membrane binding protein, such as the transmembrane domain and / or cytoplasmic domain of EpCAM; see Example 2a. This method of testing the loss of binding due to exchange of a non-human (eg, mouse) BCMA antigen with each epitope cluster is described in Example A below, in particular Examples A1-3. In this method, the antibody further binds to epitope clusters 3 and 4 of human BCMA, and further epitope cluster 3 of macaque BCMA, such as macaque Murata or BCMA derived from macaque fasciculalis (SEQ ID NO: 1017 and 121). And / or may include testing whether it can bind to 4.

0149<u style="single">Second binding molecule group (B)</u> The second binding molecule group contains the first and second binding domains, the first binding domain can bind to the extracellular domain of human BCMA and the extracellular domain of mouse BCMA, and the second binding domain is T cells. For bispecific binding molecules capable of binding to the CD3 receptor complex.

0150Thus, the first aspect of the second group of the invention is, here, a binding molecule that is at least bispecific, including the first and second binding domains. (a) The first binding domain can bind to the extracellular domain of human BCMA and the extracellular domain of mouse BCMA, and (b) The second binding domain can bind to the T cell CD3 receptor complex, Here, the extracellular domain of human BCMA corresponds to the amino acid sequence shown in SEQ ID NO: 1007, and the extracellular domain of mouse BCMA provides a binding molecule, which corresponds to the amino acid sequence shown in SEQ ID NO: 1008. To do.

0151The first binding domain can bind to the extracellular domain of human BCMA and the extracellular domain of mouse BCMA. The extracellular domain of human BCMA (amino acid sequence shown in SEQ ID NO: 1007) corresponds to amino acid residues 1-54 of the human BCMA full-length polypeptide shown in SEQ ID NO: 1002. The extracellular domain of mouse BCMA (amino acid sequence shown in SEQ ID NO: 1008) corresponds to amino acid residues 1-49 of the mouse BCMA full length polypeptide shown in SEQ ID NO: 1004.

0152In one aspect, the first binding domain of the invention further binds to macaque BCMA, eg, BCMA from macaque Murata (SEQ ID NO: 1017) or macaque fascicularis (SEQ ID NO: 1017). To do.

0153In one aspect of the invention, the first binding domain of the binding molecule can additionally or optionally bind to the BCMA of common marmosets, cotton-top tamarins and / or common squirrel monkeys.

0154The affinity of the first binding domain for human BCMA is preferably 15nM, more preferably 10nM, even more preferably 5nM, most preferably 1nM.

0155In one embodiment, the first binding domain of the binding molecule of the invention is: (a) CDR-H1 shown in SEQ ID NO: 81, CDR-H2 shown in SEQ ID NO: 82, CDR-H3 shown in SEQ ID NO: 83, and CDR-L1 shown in SEQ ID NO: 84. , SEQ ID NO: 85, CDR-L2 and SEQ ID NO: 86, CDR-L3; (b) CDR-H1 shown in SEQ ID NO: 91, CDR-H2 shown in SEQ ID NO: 92, CDR-H3 shown in SEQ ID NO: 93, and CDR-L1 shown in SEQ ID NO: 94. , CDR-L2 shown at SEQ ID NO: 95 and CDR-L3 shown at SEQ ID NO: 96; (c) CDR-H1 shown in SEQ ID NO: 101, CDR-H2 shown in SEQ ID NO: 102, CDR-H3 shown in SEQ ID NO: 103, and CDR-L1 shown in SEQ ID NO: 104. , SEQ ID NO: 105, CDR-L2 and SEQ ID NO: 106, CDR-L3; (d) CDR-H1 shown in SEQ ID NO: 111, CDR-H2 shown in SEQ ID NO: 112, CDR-H3 shown in SEQ ID NO: 113, and CDR-L1 shown in SEQ ID NO: 114. , CDR-L2 shown in SEQ ID NO: 115 and CDR-L3 shown in SEQ ID NO: 116; (e) CDR-H1 shown in SEQ ID NO: 121, CDR-H2 shown in SEQ ID NO: 122, CDR-H3 shown in SEQ ID NO: 123, and CDR-L1 shown in SEQ ID NO: 124. , CDR-L2 shown in SEQ ID NO: 125 and CDR-L3 shown in SEQ ID NO: 126; (f) CDR-H1 shown in SEQ ID NO: 131, CDR-H2 shown in SEQ ID NO: 132, CDR-H3 shown in SEQ ID NO: 133, and CDR-L1 shown in SEQ ID NO: 134. , SEQ ID NO: 135, CDR-L2 and SEQ ID NO: 136, CDR-L3; (g) CDR-H1 shown in SEQ ID NO: 141, CDR-H2 shown in SEQ ID NO: 142, CDR-H3 shown in SEQ ID NO: 143, CDR-L1 shown in SEQ ID NO: 144. , SEQ ID NO: 145, CDR-L2 and SEQ ID NO: 146, CDR-L3; (h) CDR-H1 shown in SEQ ID NO: 151, CDR-H2 shown in SEQ ID NO: 152, CDR-H3 shown in SEQ ID NO: 153, and CDR-L1 shown in SEQ ID NO: 154. , SEQ ID NO: 155, CDR-L2 and SEQ ID NO: 156, CDR-L3 Includes a VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of and a VL region containing CDR-L1, CDR-L2 and CDR-L3.

0156In yet another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO: 107, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: Includes a VH region selected from the group consisting of the VH regions shown in 137, SEQ ID NO: 147 and SEQ ID NO: 157.

0157In another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO: 108, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 138. , SEQ ID NO: 148 and SEQ ID NO: 158, including the VL region selected from the group consisting of the VL regions.

0158In one embodiment, the first binding domain of the binding molecule is: (a) VH region shown by SEQ ID NO: 87 and VL region shown by SEQ ID NO: 88; (b) VH region shown by SEQ ID NO: 97 and VL region shown by SEQ ID NO: 98; (c) VH region shown by SEQ ID NO: 107 and VL region shown by SEQ ID NO: 108; (d) VH region shown by SEQ ID NO: 117 and VL region shown by SEQ ID NO: 118; (e) VH region shown by SEQ ID NO: 127 and VL region shown by SEQ ID NO: 128; (f) VH region shown by SEQ ID NO: 137 and VL region shown by SEQ ID NO: 138; (g) VH region shown in SEQ ID NO: 147 and VL region shown in SEQ ID NO: 148; (h) VH region shown by SEQ ID NO: 157 and VL region shown by SEQ ID NO: 158 Includes VH and VL regions selected from the group consisting of.

0159In one embodiment, the first binding domain is SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID. Contains an amino acid sequence selected from the group consisting of NO: 149 and SEQ ID NO: 159.

0160<u style="single">The second binding molecule group is also related to the following items:</u>1. A binding molecule containing the first and second binding domains, where (a) The first binding domain can bind to the extracellular domain of human BCMA and the extracellular domain of mouse BCMA, and (b) The second binding domain can bind to the T cell CD3 receptor complex, Here, the extracellular domain of human BCMA corresponds to the amino acid sequence shown in SEQ ID NO: 1007, and the extracellular domain of mouse BCMA corresponds to the amino acid sequence shown in SEQ ID NO: 1008. 2. The binding molecule of item 1 in which the first binding domain can further bind to macaque BCMA. 3. A binding molecule of item 1 or 2 in which the second binding domain can bind to CD3 epsilon, preferably human CD3 epsilon. 4. The binding molecule of any of items 1-3, wherein the first and / or second binding domain is derived from an antibody. 5. (scFv)₂, (Single domain mAb)₂, ScFv-A binding molecule of item 4 selected from the group consisting of single domain mAbs, diabodies and oligomers thereof. 6. The first binding domain is (a) CDR-H1 shown in SEQ ID NO: 81, CDR-H2 shown in SEQ ID NO: 82, CDR-H3 shown in SEQ ID NO: 83, and CDR-L1 shown in SEQ ID NO: 84. , SEQ ID NO: 85, CDR-L2 and SEQ ID NO: 86, CDR-L3; (b) CDR-H1 shown in SEQ ID NO: 91, CDR-H2 shown in SEQ ID NO: 92, CDR-H3 shown in SEQ ID NO: 93, and CDR-L1 shown in SEQ ID NO: 94. , CDR-L2 shown at SEQ ID NO: 95 and CDR-L3 shown at SEQ ID NO: 96; (c) CDR-H1 shown in SEQ ID NO: 101, CDR-H2 shown in SEQ ID NO: 102, CDR-H3 shown in SEQ ID NO: 103, and CDR-L1 shown in SEQ ID NO: 104. , SEQ ID NO: 105, CDR-L2 and SEQ ID NO: 106, CDR-L3; (d) CDR-H1 shown in SEQ ID NO: 111, CDR-H2 shown in SEQ ID NO: 112, CDR-H3 shown in SEQ ID NO: 113, and CDR-L1 shown in SEQ ID NO: 114. , CDR-L2 shown in SEQ ID NO: 115 and CDR-L3 shown in SEQ ID NO: 116; (e) CDR-H1 shown in SEQ ID NO: 121, CDR-H2 shown in SEQ ID NO: 122, CDR-H3 shown in SEQ ID NO: 123, and CDR-L1 shown in SEQ ID NO: 124. , CDR-L2 shown in SEQ ID NO: 125 and CDR-L3 shown in SEQ ID NO: 126; (f) CDR-H1 shown in SEQ ID NO: 131, CDR-H2 shown in SEQ ID NO: 132, CDR-H3 shown in SEQ ID NO: 133, and CDR-L1 shown in SEQ ID NO: 134. , SEQ ID NO: 135, CDR-L2 and SEQ ID NO: 136, CDR-L3; (g) CDR-H1 shown in SEQ ID NO: 141, CDR-H2 shown in SEQ ID NO: 142, CDR-H3 shown in SEQ ID NO: 143, CDR-L1 shown in SEQ ID NO: 144. , SEQ ID NO: 145, CDR-L2 and SEQ ID NO: 146, CDR-L3; (h) CDR-H1 shown in SEQ ID NO: 151, CDR-H2 shown in SEQ ID NO: 152, CDR-H3 shown in SEQ ID NO: 153, and CDR-L1 shown in SEQ ID NO: 154. , SEQ ID NO: 155, CDR-L2 and SEQ ID NO: 156, CDR-L3 A binding molecule of any of the above items, comprising a VH region containing CDR-H1, CDR-H2 and CDR-H3 and a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of. 7. The first binding domain is SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO: 107, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 137, SEQ ID NO: 147 and SEQ. A binding molecule of any of the above items, comprising a VH region selected from the group consisting of the VH region shown in ID NO: 157. 8. The first binding domain is SEQ ID NO: 88, SEQ ID NO: 98, SEQ ID NO: 108, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 138, SEQ ID NO: 148 And SEQ ID NO: A binding molecule of any of the above items comprising a VL region selected from the group consisting of the VL region shown in 158. 9. The first binding domain is (a) VH region shown by SEQ ID NO: 87 and VL region shown by SEQ ID NO: 88; (b) VH region shown by SEQ ID NO: 97 and VL region shown by SEQ ID NO: 98; (c) VH region shown by SEQ ID NO: 107 and VL region shown by SEQ ID NO: 108; (d) VH region shown by SEQ ID NO: 117 and VL region shown by SEQ ID NO: 118; (e) VH region shown by SEQ ID NO: 127 and VL region shown by SEQ ID NO: 128; (f) VH region shown by SEQ ID NO: 137 and VL region shown by SEQ ID NO: 138; (g) VH region shown in SEQ ID NO: 147 and VL region shown in SEQ ID NO: 148; (h) VH region shown by SEQ ID NO: 157 and VL region shown by SEQ ID NO: 158 A binding molecule of any of the above items, comprising a VH region and a VL region selected from the group consisting of. Ten. The first binding domain is SEQ ID NO: 89, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 139, SEQ ID NO: 149 and SEQ. A binding molecule of item 9 containing an amino acid sequence selected from the group consisting of ID NO: 159. 11. Nucleic acid sequence encoding any of the binding molecules of items 1-10. 12. A vector containing the nucleic acid sequence of item 11. 13. Host cells transformed or transfected with the nucleic acid sequence of item 11 or the vector of item 12. 14. Item 1 includes the step of culturing the host cell of item 13 under conditions that allow the expression of any of the binding molecules of items 1 to 10 and the step of recovering the produced binding molecule from the culture. The process of making any of ~ 10 bound molecules. 15. A pharmaceutical composition comprising a binding molecule of any of items 1-10 or a binding molecule produced by the process of item 14. 16. A binding molecule of any of items 1-10 for use in the prevention, treatment or remission of a disease selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases. Bound molecule produced by the process of item 14. 17. A method for the treatment or remission of a disease selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases, item 1 for subjects in need of it. A method comprising administering a binding molecule of any of ~ 10 or a binding molecule produced by the process of item 14. 18. Plasma cell disorders include multiple myeloma, plasmacytoma, plasmacytotic leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma Item 17 method selected from the group consisting of osteosclerotic myeloma, heavy chain disease, unclear monoclonal gamma globulinemia, and smoldering multiple myeloma. 19. The method of item 17, wherein the autoimmune disease is systemic lupus erythematosus or rheumatoid arthritis. 20. A kit comprising a binding molecule of any of items 1-10, a nucleic acid molecule of item 11, a vector of item 12 or a host cell of item 13.

0161<u style="single">Third binding molecule group (C)</u> The third binding molecule group of the present invention is a binding molecule that is at least bispecific, including the first and second binding domains, in which the first binding domain binds to epitope clusters 1 and 4 of BCMA. With respect to a binding molecule, which can and can bind a second binding domain to the T cell CD3 receptor complex.

0162Thus, in the first aspect, the invention is a binding molecule that is at least bispecific and comprises first and second binding domains. (a) Epitope clusters 1 (MLQMAGQ) (SEQ ID NO: 1018) and 4 in which the first binding domain is BCMA<img id="000006" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Can be combined with (b) The second binding domain can bind to the T cell CD3 receptor complex, Here, epitope cluster 1 of BCMA corresponds to amino acid residues 1 to 7 of the sequence shown in SEQ ID NO: 1002, and epitope cluster 4 of BCMA corresponds to amino acid residues 42 to of the sequence shown in SEQ ID NO: 1002. Provided are binding molecules, corresponding to 54. It is also envisioned that the first binding domain of the invention can simultaneously bind to epitope clusters 1 and 4 of human BCMA.

0163In one aspect, the first binding domain of the invention can bind to epitope clusters 1 and 4 of human BCMA, preferably human BCMA ECD. Thus, each epitope cluster in the human BCMA protein is exchanged for each epitope cluster of the mouse BCMA antigen (thus forming a construct containing human BCMA in which human epitope clusters 1 and / or 4 are replaced by each mouse epitope cluster. As an example, SEQ ID NO: In the case of (see 1009 and 1012), a decrease in the binding of the binding domain can occur. The reduction is preferably at least 10%, 20%, 30%, 40%, 50%; compared to each epitope cluster of human BCMA protein, with 100% binding of human BCMA protein to each epitope cluster. Preferably, it is at least 60%, 70%, 80%, 90%, 95%, or even 100%. It is envisioned that the above human BCMA / mouse BCMA chimera will be expressed in CHO cells. It is also envisioned to fuse at least one of the human BCMA / mouse BCMA chimeras, preferably the mouse E1 / human BCMA chimera, with a transmembrane domain and / or cytoplasmic domain of a different membrane binding protein, such as EpCAM; checking ... This method of testing the loss of binding due to the exchange of non-human (eg, mouse) BCMA antigens with each epitope cluster is described in the accompanying Examples, in particular Examples C1-3.

0164In one aspect, the first binding domain of the invention binds to epitope clusters 1 and 4 of human BCMA and further to epitope clusters 1 and / or 4 of macaque BCMA (SEQ ID NO: 1020 and 1021, respectively). For example, it can be bound to Macaque Murata or those derived from Macaque Fascilalis. It is also assumed that the first binding domain does not bind to mouse BCMA.

0165Thus, in one embodiment, the extracellular protein domains of human BCMA, particularly BCMA formed by amino acid residues 1-7 and 42-54 of the human sequence set forth in SEQ ID NO: 1002, respectively, epitope cluster 1 and The binding domain that binds to 4 is also an epitope of Macaku BCMA, in particular the extracellular protein domain of BCMA formed by amino acid residues 1-7 and 41-53 of the Macak BCMA sequence shown in SEQ ID NO: 1006, respectively. Also bind to clusters 1 and / or 4. In one embodiment, the first binding domain of the binding molecule can bind to epitope clusters 1 and 4 of BCMA, where epitope clusters 1 and 4 of BCMA are SEQ ID NO: 1002 (human BCMA full length polypeptide) or SEQ ID. NO: 1007 (Human BCMA extracellular domain: SEQ ID NO: Corresponds to amino acid residues 1-7 and 42-54, respectively, in the sequences shown in amino acids 1-54) of 1002. In one aspect of the invention, the first binding domain of the binding molecule can additionally or optionally bind to epitope clusters 1 and / or 4 of BCMA in common marmosets, cotton-top tamarins and / or common squirrel monkeys. ..

0166The affinity of the first binding domain for human BCMA is preferably 15nM, more preferably 10nM, even more preferably 5nM, most preferably 1nM.

0167In one embodiment, the first binding domain of the binding molecule of the invention is: (a) CDR-H1 shown in SEQ ID NO: 511, CDR-H2 shown in SEQ ID NO: 512, CDR-H3 shown in SEQ ID NO: 513, CDR-L1 shown in SEQ ID NO: 514. , SEQ ID NO: 515, CDR-L2 and SEQ ID NO: 516, CDR-L3; (b) CDR-H1 shown in SEQ ID NO: 521, CDR-H2 shown in SEQ ID NO: 522, CDR-H3 shown in SEQ ID NO: 523, CDR-L1 shown in SEQ ID NO: 524. , SEQ ID NO: 525, CDR-L2 and SEQ ID NO: 526, CDR-L3; (c) CDR-H1 shown in SEQ ID NO: 531, CDR-H2 shown in SEQ ID NO: 532, CDR-H3 shown in SEQ ID NO: 533, CDR-L1 shown in SEQ ID NO: 534. , SEQ ID NO: 535, CDR-L2 and SEQ ID NO: 536, CDR-L3; (d) CDR-H1 shown in SEQ ID NO: 541, CDR-H2 shown in SEQ ID NO: 542, CDR-H3 shown in SEQ ID NO: 543, CDR-L1 shown in SEQ ID NO: 544. , SEQ ID NO: 545, CDR-L2 and SEQ ID NO: 546, CDR-L3; (e) CDR-H1 shown in SEQ ID NO: 551, CDR-H2 shown in SEQ ID NO: 552, CDR-H3 shown in SEQ ID NO: 553, CDR-L1 shown in SEQ ID NO: 554. , SEQ ID NO: 555, CDR-L2 and SEQ ID NO: 556, CDR-L3; (f) CDR-H1 shown in SEQ ID NO: 561, CDR-H2 shown in SEQ ID NO: 562, CDR-H3 shown in SEQ ID NO: 563, and CDR-L1 shown in SEQ ID NO: 564. , SEQ ID NO: 565 for CDR-L2 and SEQ ID NO: 566 for CDR-L3; (g) CDR-H1 shown in SEQ ID NO: 571, CDR-H2 shown in SEQ ID NO: 572, CDR-H3 shown in SEQ ID NO: 573, CDR-L1 shown in SEQ ID NO: 574. , SEQ ID NO: 575, CDR-L2 and SEQ ID NO: 576, CDR-L3 Includes a VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of and a VL region containing CDR-L1, CDR-L2 and CDR-L3.

0168In yet another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 517, SEQ ID NO: 527, SEQ ID NO: 537, SEQ ID NO: 547, SEQ ID NO: 557, SEQ ID NO:: Includes a VH region selected from the group consisting of the VH regions shown in 567 and SEQ ID NO: 577.

0169In another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 518, SEQ ID NO: 528, SEQ ID NO: 538, SEQ ID NO: 548, SEQ ID NO: 558, SEQ ID NO: 568. And SEQ ID NO: 578 contains the VL region selected from the group consisting of the VL regions.

0170In one embodiment, the first binding domain of the binding molecule is: (a) VH region shown by SEQ ID NO: 517 and VL region shown by SEQ ID NO: 518; (b) VH region shown by SEQ ID NO: 527 and VL region shown by SEQ ID NO: 528; (c) VH region shown by SEQ ID NO: 537 and VL region shown by SEQ ID NO: 538; (d) VH region shown by SEQ ID NO: 547 and VL region shown by SEQ ID NO: 548; (e) VH region shown by SEQ ID NO: 557 and VL region shown by SEQ ID NO: 558; (f) VH region shown in SEQ ID NO: 567 and VL region shown in SEQ ID NO: 568; (g) VH region shown by SEQ ID NO: 577 and VL region shown by SEQ ID NO: 578 Includes VH and VL regions selected from the group consisting of. In one example, the first binding domain is SEQ ID NO: 519, SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549, SEQ ID NO: 559, SEQ ID NO: 569 and SEQ ID. NO: Contains an amino acid sequence selected from the group consisting of 579. Furthermore, the present invention relates to the use of BCMA, preferably human BCMA epitope clusters 1 and 4, for the production of binding molecules capable of binding BCMA, preferably human BCMA, preferably antibodies. Epitope clusters 1 and 4 of BCMA preferably correspond to amino acid residues 1-7 and 42-54 of the sequence shown in SEQ ID NO: 1002, respectively.

0171In addition, the present invention is a method of producing an antibody capable of binding BCMA, preferably human BCMA, preferably a bispecific binding molecule. (a) A step of immunizing an animal with a polypeptide containing BCMA, preferably human BCMA epitope clusters 1 and 4, wherein the BCMA epitope clusters 1 and 4 are SEQ ID NO: Steps, corresponding to amino acid residues 1-7 and 42-54 of the sequence shown in 1002, (b) The step of obtaining the antibody, and (c) Optionally, the step of converting the antibody into a bispecific binding molecule capable of binding to human BCMA and preferably the T cell CD3 receptor complex. Provide a method including. Preferably, step (b) involves testing the resulting antibody as follows: Each epitope cluster of human BCMA protein is exchanged with each epitope cluster of mouse BCMA antigen (thus human epitope). If clusters 1 and / or 4 are replaced with each mouse epitope cluster to produce a construct containing human BCMA), reduced antibody binding can occur. The reduction is preferably at least 10%, 20%, 30%, 40%, 50 compared to each epitope cluster of human BCMA protein, with 100% binding to each of the epitope clusters 1 and 4 of the human BCMA protein. %; More preferably at least 60%, 70%, 80%, 90%, 95% or even 100%.

0172The method may further include testing whether the antibody can bind to epitope clusters 1 and 4 of human BCMA and further to epitope clusters 1 and / or 4 of macaque BCMA.

0173The third binding molecule group is<u style="single">The following items</u>Also related to: 1. A binding molecule that is at least bispecific and contains the first and second binding domains. (a) Epitope clusters 1 (MLQMAGQ) and 4 whose first binding domain is BCMA<img id="000007" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Can be combined with (b) The second binding domain can bind to the T cell CD3 receptor complex, Here, epitope cluster 1 of BCMA corresponds to amino acid residues 1 to 7 of the sequence shown in SEQ ID NO: 1002, and epitope cluster 4 of BCMA corresponds to amino acid residues 42 to of the sequence shown in SEQ ID NO: 1002. Binding molecule corresponding to 54. 2. The first binding domain is macaque BCMA epitope clusters 1 (MLQMARQ) and 4<img id="000008" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The binding molecule of item 1 that can be further bound to. 3. A binding molecule of item 1 or 2 to which the second binding domain can bind to CD3 epsilon. 4. A binding molecule of any of the above items, wherein the second binding domain can bind to human CD3 and macaque CD3. 5. A binding molecule of any of the above items, wherein the first and / or second binding domain is derived from an antibody. 6. (scFv)₂, (Single domain mAb)₂, ScFv-A binding molecule of item 5, selected from the group consisting of single domain mAbs, diabodies and oligomers thereof. 7. The first binding domain is (a) CDR-H1 shown in SEQ ID NO: 511, CDR-H2 shown in SEQ ID NO: 512, CDR-H3 shown in SEQ ID NO: 513, CDR-L1 shown in SEQ ID NO: 514. , SEQ ID NO: 515, CDR-L2 and SEQ ID NO: 516, CDR-L3; (b) CDR-H1 shown in SEQ ID NO: 521, CDR-H2 shown in SEQ ID NO: 522, CDR-H3 shown in SEQ ID NO: 523, CDR-L1 shown in SEQ ID NO: 524. , SEQ ID NO: 525, CDR-L2 and SEQ ID NO: 526, CDR-L3; (c) CDR-H1 shown in SEQ ID NO: 531, CDR-H2 shown in SEQ ID NO: 532, CDR-H3 shown in SEQ ID NO: 533, CDR-L1 shown in SEQ ID NO: 534. , SEQ ID NO: 535, CDR-L2 and SEQ ID NO: 536, CDR-L3; (d) CDR-H1 shown in SEQ ID NO: 541, CDR-H2 shown in SEQ ID NO: 542, CDR-H3 shown in SEQ ID NO: 543, CDR-L1 shown in SEQ ID NO: 544. , SEQ ID NO: 545, CDR-L2 and SEQ ID NO: 546, CDR-L3; (e) CDR-H1 shown in SEQ ID NO: 551, CDR-H2 shown in SEQ ID NO: 552, CDR-H3 shown in SEQ ID NO: 553, CDR-L1 shown in SEQ ID NO: 554. , SEQ ID NO: 555, CDR-L2 and SEQ ID NO: 556, CDR-L3; (f) CDR-H1 shown in SEQ ID NO: 561, CDR-H2 shown in SEQ ID NO: 562, CDR-H3 shown in SEQ ID NO: 563, and CDR-L1 shown in SEQ ID NO: 564. , SEQ ID NO: 565 for CDR-L2 and SEQ ID NO: 566 for CDR-L3; (g) CDR-H1 shown in SEQ ID NO: 571, CDR-H2 shown in SEQ ID NO: 572, CDR-H3 shown in SEQ ID NO: 573, CDR-L1 shown in SEQ ID NO: 574. , SEQ ID NO: 575, CDR-L2 and SEQ ID NO: 576, CDR-L3 A binding molecule of any of the above items, comprising a VH region containing CDR-H1, CDR-H2 and CDR-H3 and a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of. 8. 8. The first binding domain is shown in SEQ ID NO: 517, SEQ ID NO: 527, SEQ ID NO: 537, SEQ ID NO: 547, SEQ ID NO: 557, SEQ ID NO: 567 and SEQ ID NO: 577. A binding molecule of any of the above items, comprising a VH region selected from the group consisting of VH regions. 9. The first binding domain is SEQ ID NO: 518, SEQ ID NO: 528, SEQ ID NO: 538, SEQ ID NO: 548, SEQ ID NO: 558, SEQ ID NO: 568 and SEQ ID NO: 578. A binding molecule of any of the above items, comprising a VL region selected from the group consisting of the VL regions shown in. Ten. The first binding domain is (a) VH region shown by SEQ ID NO: 517 and VL region shown by SEQ ID NO: 518; (b) VH region shown by SEQ ID NO: 527 and VL region shown by SEQ ID NO: 528; (c) VH region shown by SEQ ID NO: 537 and VL region shown by SEQ ID NO: 538; (d) VH region shown by SEQ ID NO: 547 and VL region shown by SEQ ID NO: 548; (e) VH region shown by SEQ ID NO: 557 and VL region shown by SEQ ID NO: 558; (f) VH region shown in SEQ ID NO: 567 and VL region shown in SEQ ID NO: 568; (g) VH region shown by SEQ ID NO: 577 and VL region shown by SEQ ID NO: 578 A binding molecule of any of the above items, comprising a VH region and a VL region selected from the group consisting of. 11. 11. The first binding domain consists of SEQ ID NO: 519, SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549, SEQ ID NO: 559, SEQ ID NO: 569 and SEQ ID NO: 579. The binding molecule of item 10, which comprises an amino acid sequence selected from the group. 12. Nucleic acid sequence encoding any of the binding molecules of items 1-11. 13. A vector containing the nucleic acid sequence of item 12. 14. Host cells transformed or transfected with the nucleic acid sequence of item 12 or the vector of item 13. 15. Item 1 includes the step of culturing the host cell of item 14 under conditions that allow the expression of any of the binding molecules of items 1 to 11 and the step of recovering the produced binding molecule from the culture. The process of manufacturing any of the binding molecules of ~ 11. 16. A pharmaceutical composition comprising a binding molecule of any of items 1-11 or a binding molecule produced by the process of item 15. 17. 17. A binding molecule of any of items 1-11 for use in the prevention, treatment or remission of a disease selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases. Bound molecule produced by the process of item 15. 18. A method for the treatment or remission of a disease selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases, item 1 for subjects in need of it. A method comprising administering a binding molecule of any of ~ 11 or a binding molecule produced by the process of item 15. 19. Plasma cell disorders include multiple myeloma, plasmacytoma, plasmacytotic leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma Item 18 method selected from the group consisting of osteosclerotic myeloma, heavy chain disease, unclear monoclonal gamma globulinemia, and smoldering multiple myeloma. 20. The method of item 18, wherein the autoimmune disease is systemic lupus erythematosus. twenty one. A kit comprising a binding molecule of any of items 1-11, a nucleic acid molecule of item 12, a vector of item 13, and / or a host cell of item 14. 22. Use of epitope cluster 1 and epitope cluster 4 of BCMA for the production of binding molecules capable of binding BCMA, preferably for the production of antibodies, wherein the epitope cluster 1 of BCMA is of the sequence shown in SEQ ID NO: 1002. Corresponding to amino acid residues 1-7, epitope cluster 4 of BCMA corresponds to amino acid residues 42-54 of the sequence shown in SEQ ID NO: 1002, used. twenty three. An antibody capable of binding BCMA, preferably a method for producing a bispecific binding molecule. (a) In the step of immunizing an animal with a polypeptide containing epitope cluster 1 and epitope cluster 4 of BCMA, epitope cluster 1 of BCMA corresponds to amino acid residues 1 to 7 of the sequence shown in SEQ ID NO: 1002. However, the epitope cluster 4 of BCMA corresponds to amino acid residues 42 to 54 of the sequence shown in SEQ ID NO: 1002, step. (b) The step of obtaining the antibody, and (c) Optionally, the step of converting the antibody into a bispecific binding molecule capable of binding to human BCMA and preferably the T cell CD3 receptor complex. How to include.

0174<u style="single">Fourth bound molecule group (D)</u> The fourth binding molecule group of the present invention is a bispecific binding molecule containing at least the first and second binding domains, wherein the first binding domain is the extracellular domain of human BCMA and human BCMA. It can bind to at least one chimeric extracellular domain of BCMA produced by exchanging epitope clusters or amino acids of the antigen with each epitope cluster or amino acid of a non-human BCMA antigen, with a second binding domain being the T cell CD3 receptor complex. Regarding binding molecules, which can bind to the body.

0175Thus, in the first aspect, the invention is a binding molecule that is at least bispecific and comprises first and second binding domains. (a) The first binding domain is (i) The extracellular domain of human BCMA corresponding to the amino acid sequence shown in SEQ ID NO: 1007, as well as (ii) In the amino acid sequence shown in SEQ ID NO: 1009, SEQ ID NO: 1010, SEQ ID NO: 1011, SEQ ID NO: 1012, SEQ ID NO: 1013, SEQ ID NO: 1014 and SEQ ID NO: 1015. At least one of the BCMA chimeric extracellular domains selected from the group consisting of the corresponding BCMA domains, Can be combined with (b) The second binding domain can bind to the T cell CD3 receptor complex, To provide a binding molecule.

0176In a preferred embodiment, the first binding domain as defined in (a) (ii) herein can bind at least the chimeric extracellular domain of BCMA set forth in SEQ ID NO: 1011. In another embodiment according to the invention, the first binding domain is SEQ ID NO: 1009, SEQ ID NO: 1010, SEQ ID NO: 1011, SEQ ID NO: 1012, SEQ ID NO: 1013, SEQ ID NO: It can bind to 2, 3, 4, 5, 6 or all of the chimeric extracellular domains of BCMA shown in 1014 and SEQ ID NO: 1015. In this aspect, it is preferred to bind to the chimeric extracellular domain of BCMA shown in SEQ ID NO: 1011 along with one or more other chimeric extracellular domains.

0177In one aspect, the first binding domain of the invention can bind to epitope clusters 1-7 of human BCMA, preferably human BCMA ECD. For example, each epitope cluster in the human BCMA protein is exchanged for each epitope cluster of the mouse BCMA antigen (eg, a construct comprising human BCMA in which, for example, human epitope clusters 1 and / or 4 are replaced by each mouse epitope cluster. Are formed; as an example, SEQ ID NO: (See 1009 and 1012), but this binding domain can still be bound. It is envisioned that the first binding domain can bind to a chimeric BCMA construct that contains one or more of the above mouse epitope clusters in a human BCMA ECD in each possible combination. For example, each epitope cluster in the human BCMA protein is exchanged for each epitope cluster of the mouse BCMA antigen (eg, thereby forming a construct containing human BCMA in which, for example, human epitope cluster 3 is replaced by each mouse epitope cluster. As an example, SEQ ID NO: (See 1011), but this binding domain can still be bound. When two or more epitope clusters in human BCMA ECD are replaced by each mouse epitope cluster, it is preferred that at least one epitope cluster in the chimera is still derived from human BCMA ECD, preferably epitope clusters 1, 2. , 3 and 4 selected from at least one, two, three or four epitope clusters from human BCMA ECD.

0178It is envisioned that the above human BCMA / mouse BCMA chimera will be expressed in CHO cells. It is also envisioned that at least one of the human BCMA / mouse BCMA chimeras, such as the mouse E1 / human BCMA chimera, will be fused to the transmembrane and / or cytoplasmic domains of different membrane binding proteins, such as EpCAM; see Figure 2a. That thing. Methods for testing binding due to exchange of non-human (eg, mouse) BCMA antigens with each epitope cluster are described in the accompanying Examples, especially Examples D1-3.

0179In one aspect, it is preferred that the first binding domain of the binding molecule according to the invention cannot bind to the extracellular domain of mouse BCMA corresponding to the amino acid sequence set forth in SEQ ID NO: 1008.

0180The term "unable to bind" is applied to mouse BCMA, preferably in the accompanying examples, where the first binding domain of the binding molecule of the invention does not bind to mouse BCMA (SEQ ID NO: 1008). Means that they do not exhibit reactivity greater than 30%, preferably greater than 20%, more preferably greater than 10%, particularly preferably greater than 9%, 8%, 7%, 6% or greater than 5% under these conditions. To do.

0181Specific binding is believed to be brought about by specific motifs within the binding domain and amino acid sequence of the antigen. Therefore, binding is achieved as a result of their primary, secondary and / or tertiary structures and as a result of secondary modifications of these structures. The specific interaction between the antigen interaction site and its specific antigen can result in mere binding of the site to the antigen. Further, the specific interaction between the antigen interaction site and the specific antigen thereof can initiate the signal alternative or additionally, for example, by inducing a change in the three-dimensional structure of the antigen, oligomerization of the antigen, or the like.

0182In one aspect, the first binding domain of the invention can further bind to the BCMA of macaques such as macaque murata or macaque fasciculalis (SEQ ID NO: 1020 and 1021 respectively). It is also assumed that the first binding domain does not bind to mouse BCMA.

0183The affinity of the first binding domain for human BCMA is preferably 15nM, more preferably 10nM, even more preferably 5nM, most preferably 1nM.

0184In one embodiment, the first binding domain of the binding molecule of the invention is: (a) CDR-H1 shown in SEQ ID NO: 841, CDR-H2 shown in SEQ ID NO: 842, CDR-H3 shown in SEQ ID NO: 843, and CDR-L1 shown in SEQ ID NO: 844. , SEQ ID NO: 845, CDR-L2 and SEQ ID NO: 846, CDR-L3; (b) CDR-H1 shown in SEQ ID NO: 851, CDR-H2 shown in SEQ ID NO: 852, CDR-H3 shown in SEQ ID NO: 853, CDR-L1 shown in SEQ ID NO: 854. , SEQ ID NO: 855, CDR-L2 and SEQ ID NO: 856, CDR-L3; (c) CDR-H1 shown in SEQ ID NO: 861, CDR-H2 shown in SEQ ID NO: 862, CDR-H3 shown in SEQ ID NO: 863, CDR-L1 shown in SEQ ID NO: 864. , CDR-L2 shown in SEQ ID NO: 865 and CDR-L3 shown in SEQ ID NO: 866; (d) CDR-H1 shown in SEQ ID NO: 871, CDR-H2 shown in SEQ ID NO: 872, CDR-H3 shown in SEQ ID NO: 873, CDR-L1 shown in SEQ ID NO: 874. , CDR-L2 shown in SEQ ID NO: 875 and CDR-L3 shown in SEQ ID NO: 876; (e) CDR-H1 shown in SEQ ID NO: 881, CDR-H2 shown in SEQ ID NO: 882, CDR-H3 shown in SEQ ID NO: 883, CDR-L1 shown in SEQ ID NO: 884. , CDR-L2 shown in SEQ ID NO: 885 and CDR-L3 shown in SEQ ID NO: 886; (f) CDR-H1 shown in SEQ ID NO: 891, CDR-H2 shown in SEQ ID NO: 892, CDR-H3 shown in SEQ ID NO: 893, CDR-L1 shown in SEQ ID NO: 894. , CDR-L2 shown in SEQ ID NO: 895 and CDR-L3 shown in SEQ ID NO: 896; (g) CDR-H1 shown in SEQ ID NO: 901, CDR-H2 shown in SEQ ID NO: 902, CDR-H3 shown in SEQ ID NO: 903, CDR-L1 shown in SEQ ID NO: 904. , CDR-L2 shown in SEQ ID NO: 905 and CDR-L3 shown in SEQ ID NO: 906; (h) CDR-H1 shown in SEQ ID NO: 911, CDR-H2 shown in SEQ ID NO: 912, CDR-H3 shown in SEQ ID NO: 913, CDR-L1 shown in SEQ ID NO: 914. , SEQ ID NO: 915, CDR-L2 and SEQ ID NO: 916, CDR-L3; (i) CDR-H1 shown in SEQ ID NO: 921, CDR-H2 shown in SEQ ID NO: 922, CDR-H3 shown in SEQ ID NO: 923, CDR-L1 shown in SEQ ID NO: 924. , SEQ ID NO: 925, CDR-L2 and SEQ ID NO: 926, CDR-L3; (k) CDR-H1 shown in SEQ ID NO: 931, CDR-H2 shown in SEQ ID NO: 932, CDR-H3 shown in SEQ ID NO: 933, CDR-L1 shown in SEQ ID NO: 934. , CDR-L2 shown in SEQ ID NO: 935 and CDR-L3 shown in SEQ ID NO: 936; (l) CDR-H1 shown in SEQ ID NO: 941, CDR-H2 shown in SEQ ID NO: 942, CDR-H3 shown in SEQ ID NO: 943, CDR-L1 shown in SEQ ID NO: 944. , CDR-L2 shown in SEQ ID NO: 945 and CDR-L3 shown in SEQ ID NO: 946; (m) CDR-H1 shown in SEQ ID NO: 951, CDR-H2 shown in SEQ ID NO: 952, CDR-H3 shown in SEQ ID NO: 953, CDR-L1 shown in SEQ ID NO: 954. , SEQ ID NO: 955, CDR-L2 and SEQ ID NO: 956, CDR-L3 Includes a VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of and a VL region containing CDR-L1, CDR-L2 and CDR-L3.

0185In yet another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 847, SEQ ID NO: 857, SEQ ID NO: 867, SEQ ID NO: 877, SEQ ID NO: 887, SEQ ID NO:: VH selected from the group consisting of the VH regions shown in 897, SEQ ID NO: 907, SEQ ID NO: 917, SEQ ID NO: 927, SEQ ID NO: 937, SEQ ID NO: 947 and SEQ ID NO: 957. Includes area.

0186In another embodiment, the first binding domain of the binding molecule is SEQ ID NO: 848, SEQ ID NO: 858, SEQ ID NO: 868, SEQ ID NO: 878, SEQ ID NO: 888, SEQ ID NO: 898. , SEQ ID NO: 908, SEQ ID NO: 918, SEQ ID NO: 928, SEQ ID NO: 938, SEQ ID NO: 948 and SEQ ID NO: 958. including.

0187In one embodiment, the first binding domain of the binding molecule is: (a) VH region shown by SEQ ID NO: 847 and VL region shown by SEQ ID NO: 848; (b) VH region shown by SEQ ID NO: 857 and VL region shown by SEQ ID NO: 858; (c) VH region shown by SEQ ID NO: 867 and VL region shown by SEQ ID NO: 868; (d) VH region shown by SEQ ID NO: 877 and VL region shown by SEQ ID NO: 878; (e) VH region shown by SEQ ID NO: 887 and VL region shown by SEQ ID NO: 888; (f) VH region shown in SEQ ID NO: 897 and VL region shown in SEQ ID NO: 898; (g) VH region shown by SEQ ID NO: 907 and VL region shown by SEQ ID NO: 908; (h) VH region shown by SEQ ID NO: 917 and VL region shown by SEQ ID NO: 918; (i) VH region shown by SEQ ID NO: 927 and VL region shown by SEQ ID NO: 928; (k) VH region shown by SEQ ID NO: 937 and VL region shown by SEQ ID NO: 938; (l) VH region shown in SEQ ID NO: 947 and VL region shown in SEQ ID NO: 948; and (m) VH region shown by SEQ ID NO: 957 and VL region shown by SEQ ID NO: 958 Includes VH and VL regions selected from the group consisting of.

0188In one example, the first binding domain is SEQ ID NO: 849, SEQ ID NO: 859, SEQ ID NO: 869, SEQ ID NO: 879, SEQ ID NO: 889, SEQ ID NO: 899, SEQ ID. Contains an amino acid sequence selected from the group consisting of NO: 909, SEQ ID NO: 919, SEQ ID NO: 929, SEQ ID NO: 939, SEQ ID NO: 949 and SEQ ID NO: 959.

0189The fourth binding molecule group is also<u style="single">The following items</u>Also related to: 1. A binding molecule containing the first and second binding domains (a) The first binding domain is (i) The extracellular domain of human BCMA corresponding to the amino acid sequence shown in SEQ ID NO: 1007, as well as (ii) In the amino acid sequence shown in SEQ ID NO: 1009, SEQ ID NO: 1010, SEQ ID NO: 1011, SEQ ID NO: 1012, SEQ ID NO: 1013, SEQ ID NO: 1014 and SEQ ID NO: 1015. At least one of the BCMA chimeric extracellular domains selected from the group consisting of the corresponding BCMA domains, Can be combined with (b) The second binding domain can bind to the T cell CD3 receptor complex, Bonding molecule. 2. Item 1 binding molecule to which the first binding domain can further bind to macaque BCMA. 3. A binding molecule of item 1 or 2 to which the second binding domain can bind to CD3 epsilon. 4. A binding molecule of any of the above items, wherein the second binding domain can bind to human CD3 and macaque CD3. Five. A binding molecule of any of the above items, wherein the first binding domain is unable to bind to the extracellular domain of mouse BCMA corresponding to the amino acid sequence shown in SEQ ID NO: 1008. 6. A binding molecule of any of the above items, wherein the first and / or second binding domain is derived from an antibody. 7. (scFv)₂, (Single domain mAb)₂, ScFv-A binding molecule of item 6 selected from the group consisting of single domain mAbs, diabodies and oligomers thereof. 8. 8. The first binding domain is (a) CDR-H1 shown in SEQ ID NO: 841, CDR-H2 shown in SEQ ID NO: 842, CDR-H3 shown in SEQ ID NO: 843, and CDR-L1 shown in SEQ ID NO: 844. , SEQ ID NO: 845, CDR-L2 and SEQ ID NO: 846, CDR-L3; (b) CDR-H1 shown in SEQ ID NO: 851, CDR-H2 shown in SEQ ID NO: 852, CDR-H3 shown in SEQ ID NO: 853, CDR-L1 shown in SEQ ID NO: 854. , SEQ ID NO: 855, CDR-L2 and SEQ ID NO: 856, CDR-L3; (c) CDR-H1 shown in SEQ ID NO: 861, CDR-H2 shown in SEQ ID NO: 862, CDR-H3 shown in SEQ ID NO: 863, CDR-L1 shown in SEQ ID NO: 864. , CDR-L2 shown in SEQ ID NO: 865 and CDR-L3 shown in SEQ ID NO: 866; (d) CDR-H1 shown in SEQ ID NO: 871, CDR-H2 shown in SEQ ID NO: 872, CDR-H3 shown in SEQ ID NO: 873, CDR-L1 shown in SEQ ID NO: 874. , CDR-L2 shown in SEQ ID NO: 875 and CDR-L3 shown in SEQ ID NO: 876; (e) CDR-H1 shown in SEQ ID NO: 881, CDR-H2 shown in SEQ ID NO: 882, CDR-H3 shown in SEQ ID NO: 883, CDR-L1 shown in SEQ ID NO: 884. , CDR-L2 shown in SEQ ID NO: 885 and CDR-L3 shown in SEQ ID NO: 886; (f) CDR-H1 shown in SEQ ID NO: 891, CDR-H2 shown in SEQ ID NO: 892, CDR-H3 shown in SEQ ID NO: 893, CDR-L1 shown in SEQ ID NO: 894. , CDR-L2 shown in SEQ ID NO: 895 and CDR-L3 shown in SEQ ID NO: 896; (g) CDR-H1 shown in SEQ ID NO: 901, CDR-H2 shown in SEQ ID NO: 902, CDR-H3 shown in SEQ ID NO: 903, CDR-L1 shown in SEQ ID NO: 904. , CDR-L2 shown in SEQ ID NO: 905 and CDR-L3 shown in SEQ ID NO: 906; (h) CDR-H1 shown in SEQ ID NO: 911, CDR-H2 shown in SEQ ID NO: 912, CDR-H3 shown in SEQ ID NO: 913, CDR-L1 shown in SEQ ID NO: 914. , SEQ ID NO: 915, CDR-L2 and SEQ ID NO: 916, CDR-L3; (i) CDR-H1 shown in SEQ ID NO: 921, CDR-H2 shown in SEQ ID NO: 922, CDR-H3 shown in SEQ ID NO: 923, CDR-L1 shown in SEQ ID NO: 924. , SEQ ID NO: 925, CDR-L2 and SEQ ID NO: 926, CDR-L3; (k) CDR-H1 shown in SEQ ID NO: 931, CDR-H2 shown in SEQ ID NO: 932, CDR-H3 shown in SEQ ID NO: 933, CDR-L1 shown in SEQ ID NO: 934. , CDR-L2 shown in SEQ ID NO: 935 and CDR-L3 shown in SEQ ID NO: 936; (l) CDR-H1 shown in SEQ ID NO: 941, CDR-H2 shown in SEQ ID NO: 942, CDR-H3 shown in SEQ ID NO: 943, CDR-L1 shown in SEQ ID NO: 944. , CDR-L2 shown in SEQ ID NO: 945 and CDR-L3 shown in SEQ ID NO: 946; (m) CDR-H1 shown in SEQ ID NO: 951, CDR-H2 shown in SEQ ID NO: 952, CDR-H3 shown in SEQ ID NO: 953, CDR-L1 shown in SEQ ID NO: 954. , SEQ ID NO: 955, CDR-L2 and SEQ ID NO: 956, CDR-L3 A binding molecule of any of the above items, comprising a VH region containing CDR-H1, CDR-H2 and CDR-H3 and a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of. 9. The first binding domain is SEQ ID NO: 847, SEQ ID NO: 857, SEQ ID NO: 867, SEQ ID NO: 877, SEQ ID NO: 887, SEQ ID NO: 897, SEQ ID NO: 907, SEQ Any of the above items, including the VH region selected from the group consisting of the VH regions shown in ID NO: 917, SEQ ID NO: 927, SEQ ID NO: 937, SEQ ID NO: 947 and SEQ ID NO: 957. Binding molecule. 10. The first binding domain is SEQ ID NO: 848, SEQ ID NO: 858, SEQ ID NO: 868, SEQ ID NO: 878, SEQ ID NO: 888, SEQ ID NO: 898, SEQ ID NO: 908. , SEQ ID NO: 918, SEQ ID NO: 928, SEQ ID NO: 938, SEQ ID NO: 948 and SEQ ID NO: 958. Either binding molecule. 11. 11. The first binding domain is (a) VH region shown by SEQ ID NO: 847 and VL region shown by SEQ ID NO: 848; (b) VH region shown by SEQ ID NO: 857 and VL region shown by SEQ ID NO: 858; (c) VH region shown by SEQ ID NO: 867 and VL region shown by SEQ ID NO: 868; (d) VH region shown by SEQ ID NO: 877 and VL region shown by SEQ ID NO: 878; (e) VH region shown by SEQ ID NO: 887 and VL region shown by SEQ ID NO: 888; (f) VH region shown in SEQ ID NO: 897 and VL region shown in SEQ ID NO: 898; (g) VH region shown by SEQ ID NO: 907 and VL region shown by SEQ ID NO: 908; (h) VH region shown by SEQ ID NO: 917 and VL region shown by SEQ ID NO: 918; (i) VH region shown by SEQ ID NO: 927 and VL region shown by SEQ ID NO: 928; (k) VH region shown by SEQ ID NO: 937 and VL region shown by SEQ ID NO: 938; (l) VH region shown in SEQ ID NO: 947 and VL region shown in SEQ ID NO: 948; and (m) VH region shown by SEQ ID NO: 957 and VL region shown by SEQ ID NO: 958 A binding molecule of any of the above items, comprising a VH region and a VL region selected from the group consisting of. 12. The first binding domain is SEQ ID NO: 849, SEQ ID NO: 859, SEQ ID NO: 869, SEQ ID NO: 879, SEQ ID NO: 889, SEQ ID NO: 899, SEQ ID NO: 909. , SEQ ID NO: 919, SEQ ID NO: 929, SEQ ID NO: 939, SEQ ID NO: 949 and SEQ ID NO: 959. 13. Nucleic acid sequence encoding any of the binding molecules of items 1-12. 14. A vector containing the nucleic acid sequence of item 13. 15. 15. Host cells transformed or transfected with the nucleic acid sequence of item 13 or the vector of item 14. 16. Item 1 includes the step of culturing the host cell of item 15 under conditions that allow the expression of any of the binding molecules of items 1 to 12, and the step of recovering the produced binding molecule from the culture. Manufacturing process of any of ~ 12 bound molecules. 17. A pharmaceutical composition comprising a binding molecule of any of items 1-12 or a binding molecule produced by the process of item 16. 18. Binding of any of items 1-12 for use in the prevention, treatment or remission of diseases selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases. Molecules or bound molecules produced by the process of item 16. 19. A method for the treatment or remission of a disease selected from the group consisting of plasma cell disorders, other B cell disorders that correlate with BCMA expression, and autoimmune diseases, item 1 for subjects in need of it. A method comprising administering a binding molecule of any of ~ 12 or a binding molecule produced by the process of item 16. 20. Plasma cell disorders include multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, bone Item 19. Method selected from the group consisting of sclerosing myeloma, heavy chain disease, unclear monoclonal gamma globulinemia, and smoldering multiple myeloma. 21. The method of item 19, wherein the autoimmune disease is systemic lupus erythematosus. 22. A kit comprising the binding molecule of any of items 1-12, the nucleic acid molecule of item 13, the vector of item 14 or the host cell of item 15.

0190It should be understood that the present invention is not limited to specific methodologies, protocols or reagents and they can be modified. The discussions and examples provided herein are provided solely for the purpose of illustrating individual aspects and are not intended to limit the scope of the invention, the scope of the invention is attached. It is defined only by the scope of claims. All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) cited throughout the document of this specification, both above and below. All of them are incorporated herein by reference. Nothing in this specification should be considered an authorization not to claim a date prior to such disclosure because the invention is a prior invention. To the extent that the documents incorporated by reference conflict with or conflict with this specification, this specification supersedes any such document.

0191The following drawings and notations of Examples represent the association with one of the groups (A)-(D) of binding molecules described herein above. In other words, Figure A [+ number] and Example A [+ number] relate to group (A), and Figure B [+ number] and Example B [+ number] refer to group (B). Figure C [+ number] and Example C [+ number] relate to group (C), and Figure D [+ number] and Example D [+ number] relate to group (D). Is.

0192<figref num="1">Sequence alignment of the extracellular domain (ECD) of human BCMA (amino acid residues 1-54 of full-length protein) and mouse BCMA (amino acid residues 1-49 of full-length protein). Highlighted are the regions (domains or amino acid residues) exchanged in the chimeric construct designated for epitope clustering. Cysteine is indicated by a black square. Disulfide bonds are shown.</figref><figref num="2">Epitope mapping of BCMA constructs. Human and mouse BCMA expressed on the surface of CHO cells (Fig. 2a) and various chimeric human-mouse BCMA constructs (Fig. 2b), as shown by flow cytometry. Expression of human BCMA in CHO was detected with a monoclonal anti-human BCMA antibody. Expression of mouse BCMA was detected with a monoclonal anti-mouse BCMA antibody. The bound monoclonal antibody was detected with an anti-rat IgG-Fc-γ specific antibody conjugated to phycoerythrin.</figref><figref num="A3">Examples of epitope clusters E3 and E4 specific binding molecules detected in the epitope mapping of the chimeric BCMA construct (see Example A3). Some binding molecules can also bind to arginine, the amino acid residue at position 39 ("E7") of human BCMA.</figref><figref num="B3">Examples of binding molecules specific for human and mouse BCMA (see Example B3).</figref><figref num="C3">Examples of epitope clusters E1 and E4 specific binding molecules detected in the epitope mapping of the chimeric BCMA construct (see Example C3).</figref><figref num="D3">Examples of binding molecules detected in epitope mapping that bind to human BCMA, do not cross-react with mouse BCMA, and additionally bind to various chimeric BCMA constructs (see Example D3).</figref><figref num="A4">Determination of binding constants for bispecific binding molecules (anti-BCMA x anti-CD3) to human and macaque BCMA using the Biacore system. The antigen was immobilized on a CM5 chip to a low to medium density (100 RU). A dilution of the conjugate was floated on the chip surface and binding was determined using BiaEval software. Each offrate and binding constant (KD) for each conjugate is shown below each graph.</figref><figref num="B4">The functionality and binding strength of affinity-maturated scFv molecules were analyzed in FACS using human BCMA-transfected CHO cells. The results are shown as a FACS histogram of a 1: 3 serial dilution of E. coli cell periplasmic extract, plotting the log-to-relative cell count of fluorescence intensity.</figref><figref num="C4">The functionality and binding strength of affinity-maturated scFv molecules were analyzed in FACS using human BCMA and macaque BCMA-transfected CHO cells. The results are shown as a FACS histogram of a 1: 3 serial dilution of E. coli cell periplasmic extract, plotting the log-to-relative cell count of fluorescence intensity.</figref><figref num="D4">Determination of binding constants for bispecific binding molecules (anti-BCMA x anti-CD3) to human and macaque BCMA using the Biacore system. The antigen was immobilized on a CM5 chip to a low to medium density (100 RU). A dilution of the conjugate was floated on the chip surface and binding was determined using BiaEval software. Each offrate and binding constant (KD) for each conjugate is shown below each graph.</figref><figref num="A5">18 hours⁵¹Cytotoxic activity of BCMA bispecific antibody as measured in chromium release assay. Effector cells: Stimulated and concentrated human CD8 T cells. Target cells: Human BCMA-transfected CHO cells (left figure) and Macaque BCMA-transfected CHO cells (right figure). Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="D5">18 hours⁵¹Cytotoxic activity of BCMA bispecific antibody as measured in chromium release assay. Effector cells: Stimulated and concentrated human CD8 T cells. Target cells: Human BCMA-transfected CHO cells (left figure) and Macaque BCMA-transfected CHO cells (right figure). Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="A6">Determination of BCMA / CD3 bispecific antibody binding constants for epitope clusters E3 / E4 ± E7 for human and macaque BCMA and for human and macaque CD3 using the Biacore system. The antigen was immobilized on a CM5 chip at a low to medium density (100 to 200 RU). A dilution of the bispecific antibody was floated on the chip surface and binding was determined using BiaEval software. The respective on-rate and off-rate of each bispecific antibody and the resulting binding constant (KD) are shown below each graph.</figref><figref num="A7">Cell Lines Shown: 1) Human BCMA Transfected CHO Cells, 2) Human CD3 Positive Human T Cell Lines HBP-ALL, 3) Makaku BCMA Transfected CHO Cells, 4) Makaku T Cell Lines 4119 LnPx, 5) BCMA FACS analysis of BCMA / CD3 bispecific antibody of epitope cluster E3 / E4 ± E7 against positive human multiple myeloma cell lines NCI-H929 and 6) untransfected CHO cells. Negative control [1) ~ 6)]: Detection antibody that does not contain the above BCMA / CD3 bispecific antibody.</figref><figref num="A8">Scatchard analysis of BCMA / CD3 bispecific antibody against BCMA-expressing cells. Cells were incubated with increasing concentrations of monomeric antibody until saturated. Antibodies were detected by flow cytometry. To demonstrate that a valid concentration range was used, the values of the triple measurements were plotted as a hyperbola and as a sigmoid curve. The maximum binding was determined using Scatchard evaluation and each KD value was calculated.</figref><figref num="A9">Cytotoxic activity of BCMA / CD3 bispecific antibody of epitope cluster E3 / E4 ± E7 against human BCMA-transfected CHO cells as measured in an 18-hour 51-chromium release assay. Effector cells: Stimulated and concentrated human CD8 T cells. Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="A10">Cytotoxic activity of BCMA / CD3 bispecific antibody of epitope cluster E3 / E4 ± E7 as measured in a 48-hour FACS-based cytotoxicity assay. Effector cells: Unstimulated human PBMC. Target cells: CHO cells transfected with human BCMA. Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="A11">FACS analysis of BCMA / CD3 bispecific antibodies of epitope clusters E3 / E4 ± E7 against BAFF-R and TACI-transfected CHO cells. Cell Line: 1) Human BAFF-R Transfected CHO Cell, 2) Human TACI Transfected CHO Cell, 3) Multiple Myeloma Cell Line L363; Negative Control: Detection without BCMA / Cd3 Bispecific Antibody Above antibody. Positive control: BAFF-R detection: Goat anti-human BAFF-R (R &amp; D AF1162; 1:20) TACI detection detected by anti-goat antibody PE (Jackson 705-116-147; 1:50): Goat anti-rabbit antibody PE Rabbit anti-TACI antibody detected by (Sigma P9757; 1:20) (abcam AB 79023; 1: 100).</figref><figref num="A12">Cytotoxic activity of BCMA / CD3 bispecific antibody as measured in 18 hour 51 chromium release assay. Effector cells: Stimulated and concentrated human CD8 T cells. Target cells: BCMA-positive human multiple myeloma cell line L363 (ie, naturally expressed cells). Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="A13">Cytotoxic activity of BCMA / CD3 bispecific antibody as measured in a 48-hour FACS-based cytotoxic assay. Effector cells: Unstimulated human PBMC. Target cells: Human multiple myeloma cell line L363 (natural BCMA expressor). Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="A14">Cytotoxic activity of BCMA / CD3 bispecific antibody as measured in a 48-hour FACS-based cytotoxic assay. Effector cells: Unstimulated human PBMC. Target cells: BCMA-positive human multiple myeloma cell line NCI-H929. Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="A15">Cytotoxic activity of BCMA / CD3 bispecific antibody as measured in a 48-hour FACS-based cytotoxic assay. Effector cells: Macaque T cell line 4119LnPx. Target cells: CHO cells transfected with macaque BCMA. Effector to target cell (E: T) ratio: 10: 1.</figref><figref num="A16">Antitumor activity of BCMA / CD3 bispecific antibody of epitope cluster E3 / E4 ± E7 in advanced NCI-H929 xenograft model (see Example A16).</figref><figref num="A17">FACS-based cytotoxicity assay (48 hours; E: T = 10: 1) using human multiple myeloma cell lines NCI-H929, L-363 and OPM-2 as target cells and human PBMC as effector cells. The figure shows the cytokine levels determined for IL-2, IL-6, IL-10, TNF and IFN-γ under BCMA / CD3 bispecific antibody of increasing concentrations of epitope clusters E3 / E4 ± E7 [ pg / ml] (see Example A22).</figref>

0193The present invention will be described with reference to the following examples. These examples should not be considered as limiting the scope of the invention. The examples are included for illustration purposes only, and the present invention is limited only by the appended claims.

0194<u style="single">Example</u>A<u style="single">Example A1</u>Generation of CHO cells expressing chimeric BCMA To construct a chimeric epitope mapping molecule, the amino acid sequence or single amino acid residue of each epitope domain of human BCMA was converted to a mouse sequence. We constructed the following molecules:

0195-Human BCMA ECD / E1 mouse (SEQ ID NO: 1009) Chimeric extracellular BCMA domain: Epitope cluster 1 (SEQ ID NO: 1002 or 1007 amino acid residues 1-7) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 1-4). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 1-3 and mutation of G6Q in 1002 or 1007

0196-Human BCMA ECD / E2 mouse (SEQ ID NO: 1010) Chimeric extracellular BCMA domain: Epitope cluster 2 (SEQ ID NO: 1002 or 1007 amino acid residues 8-21) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 5-18). Human extracellular BCMA domain SEQ ID NO: Mutations of S9F, Q10H and N11S in 1002 or 1007

0197-Human BCMA ECD / E3 mouse (SEQ ID NO: 1011) Chimeric extracellular BCMA domain: Epitope cluster 3 (SEQ ID NO: 1002 or 1007 amino acid residues 24-41) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 21-36). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 31 and 32 at 1002 or 1007 and mutation of Q25H, S30N, L35A and R39P

0198-Human BCMA ECD / E4 mouse (SEQ ID NO: 1012) Chimeric extracellular BCMA domain: Epitope cluster 4 (SEQ ID NO: 1002 or 1007 amino acid residues 42-54) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 37-49). Human extracellular BCMA domain SEQ ID NO: Mutations of N42D, A43P, N47S, N53Y and A54T in 1002 or 1007

0199-Human BCMA ECD / E5 mouse (SEQ ID NO: 1013) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 22 (isoleucine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (lysine, position 19) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of I22K in 1002 or 1007

0200-Human BCMA ECD / E6 mouse (SEQ ID NO: 1014) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 25 (glutamine) at SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (histidine, position 22) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of Q25H in 1002 or 1007

0201-Human BCMA ECD / E7 mouse (SEQ ID NO: 1015) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 39 (arginine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (proline, position 34) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of R39P in 1002 or 1007.

0202A) The cDNA construct was cloned into the mammalian expression vector pEF-DHFR and stably transfected into CHO cells. Expression of human BCMA in CHO cells was verified in a FACS assay using a monoclonal anti-human BCMA antibody. Expression of mouse BCMA was demonstrated using a monoclonal anti-mouse BCMA antibody. The concentration of BCMA antibody used was 10 μg / ml in PBS / 2% FCS. Bound monoclonal antibody was detected using anti-rat IgG-Fcγ-PE (1: 100 in PBS / 2% FCS; Jackson-Immuno-Research # 112-116-071). As a negative control, cells were incubated with PBS / 2% FCS in place of the first antibody.Samples from FACSCanto II equipment (Becton) Measured by flow cytometry in Dickinson) and analyzed by FlowJo software (version 7.6). Transfected CHO cells were analyzed and confirmed for surface expression of human-mouse BCMA chimeras in flow cytometry assays using various anti-BCMA antibodies (Fig. 2).

0203B) Code sequences for human, macaque, mouse BCMA and human-mouse BCMA chimeras for the generation of CHO cells expressing human, macaque, mouse and human / mouse chimera transmembrane BCMA (GenBank accession number NM_001192 [human]] (BCMA sequences published as NM_011608 [mouse] and XM_001106892 [macaque]) were obtained by gene synthesis according to standard methods. If the gene synthesis fragment is a chimera in which each epitope domain of the human sequence is exchanged for the mouse sequence, it first obtains the Kozak site for eukaryotic expression of the construct and the coding sequence of the 19 amino acid immunoglobulin leader peptide, respectively. It was then designed to contain the coding sequence for the BCMA protein in-frame.

0204Except for human BCMA ECD / E4 mice and human BCMA constructs, the extracellular domain coding sequence of BCMA protein is followed by the intracellular Ser1-Gly4-Ser1 linker coding sequence in frame, followed by the intracellular domain of human EpCAM (amino acids). 226 ~ 314; the sequence published as GenBank accession number NM_002354).

0205All coding sequences are followed by a stop codon. In addition, the gene synthesis fragment was designed so that an appropriate restriction site was introduced. The gene synthesis fragment was cloned into a plasmid called pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). All of the above steps were performed according to standard methods (Sambrook, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). For each antigen, clones with sequence-validated nucleotide sequences were transfected into DHFR-deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR-deficient CHO cells is described in Kaufman RJ (1990) Methods Enzymol. It was done as described in 185, 537-566. Gene amplification of the construct was induced by increasing the concentration of methotrexate (MTX) to a final concentration of up to 20 nM MTX.

0206<u style="single">Example A2</u>2.1 Transient expression in HEK293 cells Clone expression plasmids with sequence-validated nucleotide sequences were used for transfection and protein expression in the FreeStyle 293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's protocol. A supernatant containing the expressed protein was obtained, cells were removed by centrifugation and the supernatant was stored at -20 ° C.

02072.2 Stable expression in CHO cells Clone expression plasmids with sequence-verified nucleotide sequences were transfected into DHFR-deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR-deficient CHO cells was performed as described in Kaufman RJ (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX. After two passages in static culture, cells contain nucleoside-free HyQ PF CHO liquid soybean medium (containing 4.0 mM L-glutamine, containing 0.1% Pluronic F-68; HyClone) for 7 days prior to collection. Growing in roller bottles. Cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -20 ° C.

02082.3 Protein purification Purification of the soluble BCMA protein was carried out as follows. Akta® Explorer System (GE Healthcare) and Unicorn® software were used for chromatography. Immobilized metal affinity chromatography ("IMAC") was performed using a ZnCl2-filled Fractogel EMD chelate® (Merck) according to a protocol provided by the manufacturer. The column was equilibrated with buffer A (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCl) and filtered (0.2 μm) cell culture supernatant was applied to the column (10 ml) at a flow rate of 3 ml / min. The column was washed with buffer A to remove unbound samples. The bound protein was eluted with 2-step gradient buffer B (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCl, 0.5 M imidazole) according to the following procedure: Stage 1: 10% buffer B in 6 column volumes Second stage: 6 column volumes of 100% buffer B. The protein fraction eluted by the second step was collected for further purification. All compounds are of research grade and were purchased from Sigma (Deisenhofen) or Merck (Darmstadt). Gel filtration chromatography equilibrates with Equi buffer (10 mM citrate for proteins expressed in HEK cells, 25 mM lysine HCl, pH 7.2, PBS pH 7.4 for proteins expressed in CHO cells). The HiLoad 16/60 Superdex 200 preparative grade column (GE / Amersham) was used. Eluted protein samples (flow rate 1 ml / min) were subjected to standard SDS-PAGE and Western blots for detection. Protein concentration was determined using OD 280 nm. The protein obtained through transient expression in HEK293 cells was used for immunological treatment. Proteins obtained through stable expression in CHO cells were used for conjugate selection and binding measurements.

0209<u style="single">Example A3</u>Epitope clustering of mouse scFv fragments Cells transfected with human or mouse BCMA or chimeric BCMA molecules were stained with a crude undiluted periplasmic extract containing scFv that binds to human / macaque BCMA. Bound scFv was detected with 1 μg / ml anti-FLAG antibody (Sigma F1804) and R-PE labeled anti-mouse Fcγ-specific antibody (1: 100; Dianova # 115-116-071). All antibodies were diluted with PBS containing 2% FCS. As a negative control, cells were incubated with PBS / 2% FCS instead of periplasmic extract. Samples were measured by flow cytometry on a FACSCanto II instrument (Becton Dickinson) and analyzed by FlowJo software (version 7.6).

0210<u style="single">Example A4</u>Obtaining different recombinant forms of soluble human and macaque BCMA A) Code sequences for human and rhesus monkey BCMA (published at GenBank, accession numbers NM_001192 [human], XM_001106892 [rhesus monkey]), human albumin, human Fcγ1 and mouse albumin coding sequences, respectively for human and macaque BCMA and human albumin. , A soluble fusion protein of human IgG1 Fc and mouse albumin, respectively, and an artificial cDNA sequence encoding a soluble protein containing only the extracellular domain of BCMA. To generate constructs for the expression of soluble human and macaque BCMA proteins, cDNA fragments were obtained by PCR mutagenesis and molecular cloning of the above full-length BCMA cDNA according to standard protocols. For fusions with human albumin, they first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, respectively. And rhesus monkey BCMA protein coding sequence, then in-frame coding sequence of artificial Ser1-Gly4-Ser1 linker, then in-frame coding sequence of human serum albumin, then in-frame coding sequence of Flag tag, then in-frame Frame-modified histidine tag<img id="000009" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For fusions with mouse IgG1, humans first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, followed by the coding sequence of the human IgG1 hinge and Fcγ moiety in frame, and then the hexahistidine tag code in frame. Modified cDNA fragments were designed to include sequences and stop codons. For fusions with mouse albumin, humans each contain amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, first the Kozak site for eukaryotic expression of the construct. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, then the coding sequence of mouse serum albumin in frame, then the coding sequence of the Flag tag in frame, and then in frame. Frame-modified histidine tag<img id="000010" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For soluble extracellular domain constructs, first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively, human and Macaque BCMA protein coding sequence, then in-frame artificial Ser1-Gly1 linker coding sequence, then in-frame FLAG tag coding sequence, then in-frame modified histidine tag<img id="000011" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. The cDNA fragment was also designed to introduce restriction sites at the beginning and end of the fragment. The introduced restriction sites, EcoRI at the 5'end and SalI at the 3'end, were used in the cloning procedure below. The cDNA fragment was cloned through EcoRI and SalI into a plasmid called pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). All of the above steps were performed according to standard protocols (Sambrook, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)).

0211B) The above human and macaque BCMA coding sequences and human albumin, human Fcγ1, mouse Fcγ1, mouse Fcγ2a, mouse albumin, rat albumin, rat Fcγ1 and rat Fcγ2b coding sequences, respectively, and human albumin, For the construction of artificial cDNA sequences encoding human IgG1 Fc, mouse IgG1 Fc, mouse IgG2a Fc, mouse albumin, rat IgG1 Fc, rat IgG2b and rat albumin soluble fusion proteins, and soluble proteins containing only the extracellular domain of BCMA. used. To generate constructs for the expression of soluble human and macaque BCMA proteins, cDNA fragments were obtained by PCR mutagenesis and molecular cloning of the above full-length BCMA cDNA according to standard protocols. For albumin fusions, first the Kozak site for eukaryotic expression of the construct and the coding sequence of the 19 amino acid immunoglobulin leader peptide, then in-frame the coding sequence of the extracellular domain of each BCMA protein, and then In-frame code sequence of artificial Ser1-Gly4-Ser1 linker, then in-frame code sequence of each serum albumin, then in-frame code sequence of Flag tag, then in-frame modified histidine tag<img id="000012" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For fusions with IgG Fc, first the Kozak site for eukaryotic expression of the construct and the coding sequence of the 19 amino acid immunoglobulin leader peptide, then in-frame the coding sequence of the extracellular domain of each BCMA protein, Then in-frame the coding sequence of the artificial Ser1-Gly4-Ser1 linker, but in the case of human IgG1 Fc, use the artificial Ser1-Gly1 linker, then in-frame the coding sequence of the hinge and Fcγ moieties of each IgG, and then In-frame code sequence of Flag tags, followed by in-frame modified histidine tags<img id="000013" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For soluble extracellular domain constructs, first the Kozak site for eukaryotic expression of the construct and the coding sequence of the 19 amino acid immunoglobulin leader peptide, then in-frame the coding sequence of the extracellular domain of each BCMA protein, and then In-frame in-frame code sequence of the artificial Ser1-Gly1 linker, then in-frame code sequence of the Flag tag, then in-frame modified histidine tag<img id="000014" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. Appropriate restriction sites were introduced for cloning the construct. All cDNA fragments were cloned into a plasmid called pEF-DHFR (pEF-DHFR is Raum et al. Described in 2001). All of the above steps were performed according to the standard protocol (Sambrook, 2001). The following constructs were designed to allow panning directed to different epitopes. The coding sequences for the mouse-human BCMA chimera and the mouse-Makaku BCMA chimera (the sequences for mouse, human and Makaku BCMA are as described above) and the coding sequences for mouse albumin and mouse Fcγ1 can be found in the mouse-human and mouse-Makaku BCMA chimeric It was used to construct artificial cDNA sequences encoding the respective soluble fusion proteins of mouse IgG1 Fc and mouse albumin, respectively. Mouse BCMA (amino acids 1-49) containing each epitope domain mutated for each of its human and macaque sequences to generate constructs for the expression of soluble mouse human and mouse macaque BCMA chimeras. The cDNA fragment of was obtained by gene synthesis according to a standard protocol. Cloning the construct as described above and standard protocol (Sambrook, I went according to 2001). We constructed the following molecules: . Amino acids 1-4 Human, mouse IgG1 Fc . Amino acids 1-4 human, mouse albumin . Amino acids 1 to 4 rhesus monkeys, mouse IgG1 Fc . Amino acids 1 to 4 rhesus monkeys, mouse albumin . Amino acids 5-18 Human, mouse IgG1 Fc . Amino acids 5-18 human, mouse albumin . Amino acids 5-18 rhesus monkeys, mouse IgG1 Fc . Amino acids 5-18 rhesus monkeys, mouse albumin . Amino acids 37-49 Human, mouse IgG1 Fc . Amino acids 37-49 Human, mouse albumin . Amino acids 37-49 rhesus monkey, mouse IgG1 Fc . Amino acids 37-49 rhesus monkeys, mouse albumin

0212<u style="single">Example A5</u>5.1 Biacore-based determination of bispecific antibody affinity for human and macaque BCMA and CD3 Biacore analytical experiments were performed with recombinant BCMA fusion proteins containing human serum albumin (ALB) to determine target binding to BCMA. To measure the affinity for CD3, a recombinant fusion protein having the N-terminal 27 amino acids of CD3 epsilon (CD3e) fused to the Fc portion of the human antibody was used. This recombinant protein is available in human CD3e 1-27 and cynomolgus monkey CD3e versions, both of which carry the epitope of the CD3 binding portion of the bispecific antibody. More specifically, approximately 100-150 RU of each recombinant antigen was immobilized on a CM5 sensor chip (GE Healthcare) using acetate buffer pH 4.5 according to the manufacturer's manual. Bispecific antibody sample, HBS-EP electrophoresis buffer (GE Five concentrations diluted with Healthcare): filled with 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.13 nM. The flow rate was 30-35 μl / min for 3 minutes, after which HBS-EP electrophoresis buffer was applied again at a flow rate of 30-35 μl / ml for 8 minutes. Chip regeneration was performed with 10 mM glycine 0.5 M NaCl pH 2.45. The dataset was analyzed using BiaEval software (see Figure A4). Usually, two independent experiments were performed.

02135.2 Binding affinity for human and macaque BCMA The binding affinity of BCMA / CD3 bispecific antibody for human and macaque BCMA was determined by Biacore analysis using a recombinant BCMA fusion protein containing mouse albumin (ALB).

0214More specifically, approximately 150-200 RU of each recombinant antigen was immobilized on a CM5 sensor chip (GE Healthcare) using acetate buffer pH 4.5 according to the manufacturer's manual. Bispecific antibody samples were loaded with 5 concentrations diluted with HBS-EP electrophoresis buffer (GE Healthcare): 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.13 nM. For determination of BCMA affinity, the flow rate was 35 μl / min for 3 minutes, after which HBS-EP electrophoresis buffer was applied again at a flow rate of 35 μl / ml for 10, 30 or 60 minutes. Chip regeneration was performed using a buffer consisting of a 1: 1 mixture of 10 mM glycine 0.5 M NaCl pH 1.5 and 6 M guanidine chloride solution. The dataset was analyzed using BiaEval software (see Figure A6). Usually, two independent experiments were performed.

0215Confirmation of binding to human and macaque CD3 epsilon was performed in a single experiment using the same concentration applied to BCMA binding; off-rate determinations were made with a dissociation time of 10 minutes.

0216All BCMA / CD3 bispecific antibodies according to the invention, i.e. the antibodies of the epitope cluster "E3 / E4 ± E7", showed high affinity for human BCMA in the nanomolar range. The binding to macaque BCMA was also commensurate with it, showing affinities in the nanomolar range. The affinities and affinity gaps for BCMA / CD3 bispecific antibodies are shown in Table 2.

0217(Table 2) Affinity of BCMA / CD3 bispecific antibody of epitope cluster E3 / E4 ± E7 to human and macaque BCMA determined by Biacore analysis and calculated affinity gap (macaque BCMA: human BCMA)<img id="000015" he="144" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

02185.3 Biacore-based determination of bispecific antibody affinity for human and macaque BCMA Biacore measurements of BCMA / CD3 bispecific antibody affinity for recombinant soluble BCMA on CM5 chips using a longer dissociation period (60 minutes instead of the 10 minutes used in previous experiments) were used for KD and especially Repeated to reconfirm the off-rate. All of the BCMA / CD3 bispecific antibodies tested were subjected to two independent affinity measurements using 5 different concentrations each.

0219The affinity of the BCMA / CD3 bispecific antibody for epitope clusters E3 / E4 ± E7 was clearly below nanomoles, see the example in Table 3.

0220(Table 3) Affinity of BCMA / CD3 2 characteristic antibody of epitope cluster E3 / E4 ± E7 in Biacore experiment using long dissociation time (KD) (2 independent experiments each)<img id="000016" he="20" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0221<u style="single">Example A6</u>Bispecific binding and interspecific cross-reactivity To confirm binding to human and macaque BCMA and CD3, CHO cells transfected with bispecific antibody in human and macaque BCMA, respectively, human multiple myeloma cell line expressing native human BCMA NCI- Flow using H929, CD3 expressing human T cell leukemia cell line HPB-ALL (DSMZ, Braunschweig, ACC483) and CD3 expressing Makaku T cell line 4119LnPx (Knappe A, et al., Blood, 2000, 95, 3256-3261) Tested by cytometry. In addition, untransfected CHO cells were used as a negative control.

0222For flow cytometry, 200,000 cells from each cell line were incubated with 50 μl of purified bispecific antibody at a concentration of 5 μg / ml for 30 minutes on ice. Cells were washed twice with PBS / 2% FCS and construct binding was detected with mouse PentaHis antibody (Qiagen; 50 μl PBS / 2% FCS 1:20 dilution). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS / 2% FCS. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson).

0223BCMA / CD3 bispecific antibodies of epitope cluster E3 / E4 ± E7 are human and macaque BCMA-transfected CHO cells, human BCMA-expressing multiple myeloma cell line NCI-H929 and human and macaque T cells. Was stained. In addition, untransfected CHO cells were not stained (see Figure A7).

0224<u style="single">Example A7</u>Scatchard-based determination of bispecific antibody affinity for human and macaque BCMA In Scatchard analysis, saturated binding experiments are performed using a monovalent detection system (anti-His Fab / Alexa 488) developed by Micromet to accurately determine the monovalent binding of bispecific antibodies to each cell line. Each cell line (CHO cell line that recombinantly expresses human BCMA, CHO cell line that recombinantly expresses macaque BCMA) 2 x 10⁴Cells are incubated with 50 μl each of a triple dilution series (1: 2 8-step dilution) of each BCMA bispecific antibody starting at 100 nM, followed by incubation at 4 ° C for 16 hours with stirring, 1 Perform multiple residue cleaning steps. The cells are then incubated with 30 μl of anti-His Fab / Alexa 488 solution (Micromet; 30 μg / ml) for an additional 30 minutes. After one wash step, cells are resuspended in 150 μl FACS buffer containing 3.5% formaldehyde, incubated for an additional 15 minutes, centrifuged, resuspended in FACS buffer, and FACS Canto II machine and FACS. Analyze using Diva software. Data are obtained from two independent experimental sets. The values are plotted as a combined curve of hyperbolas. Calculations are performed to estimate the maximum binding (Bmax) from each Scatchard analysis. Half-maximal that reflects each KD Determine the concentration of bispecific antibody at binding). Plot the values of the triple measurements as a hyperbola. The maximum binding is determined using the Scatchard evaluation and each KD is calculated.

0225The affinity of BCMA / CD3 bispecific antibodies for CHO cells transfected with human or macaque BCMA is the most reliable method for measuring the potential affinity gap between human and macaque BCMA. , Determined by Scatchard analysis.

0226Cells expressing BCMA antigen were incubated while increasing the concentration of each monomeric BCMA / CD3 bispecific antibody until saturation was reached (16 hours). Bound bispecific antibody was detected by flow cytometry. The concentration of BCMA / CD3 bispecific antibody at maximum half-dose binding, reflecting each KD, was determined.

0227The values of the triple measurements were plotted as a hyperbola and an S-curve to demonstrate a suitable concentration range from minimal to optimal coupling. The maximum binding (Bmax) was determined using the Scatchard evaluation (Fig. A8) and the respective KDs were calculated. The values shown in Table 4 were obtained from two independent experiments for each BCMA / CD3 bispecific antibody.

0228Cell-based Scatchard analysis showed that the BCMA / CD3 bispecific antibody of epitope cluster E3 / E4 ± E7 was less than nanomolar in affinity for human BCMA and had a small interspecific BCMA affinity gap of 1.9 to 2.9. confirmed.

0229Within epitope clustering (see Examples A1 and A2), another group of antibodies capable of binding to epitope clusters 1 and 4 ("E1 / E4") of BCMA was identified. Epitope cluster 1 is MLQMAGQ (SEQ ID NO: 1018) and epitope cluster 4 is NASVTNSVKGTNA (SEQ ID NO: 1019). In contrast to the BCMA / CD3 bispecific antibody of the epitope cluster E3 / E4 ± E7, the antibody of the epitope cluster "E1 / E4" shows a higher human-macaque BCMA affinity gap of 3.9-4.5.

0230(Table 4) BCMA / CD3 bispecific antibody affinity (KD) of epitope clusters E3 / E4 ± E7 and calculated KD macaque BCMA / KD in cell-based Scatchard analysis (2 independent experiments each) Affinity gap in human BCMA<img id="000017" he="26" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0231<u style="single">Example A8</u>Cytotoxic activity 8.1 Chromium release assay with stimulated human T cells CD8⁺T cells enriched and stimulated for T cells were obtained as follows: Petri dishes (145 mm in diameter, Greiner bio-one GmbH, Kremsmunster) were coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) at a final concentration of 1 μg / ml at 37 ° C. for 1 hour. Unbound proteins were removed by a single wash step with PBS. 3-5 x 10 in 120 ml RPMI 1640 containing stabilized glutamine / 10% FCS / IL-2 20 U / ml (Proleukin®, Chiron)⁷Human PBMCs were added to pre-coated Petri dishes and stimulated for 2 days. On day 3, cells were harvested and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U / ml and the cells were again cultured in the same cell culture medium as above for 1 day. CD8⁺Cytotoxic T lymphocytes (CTL) CD4 using Dyna-Beads according to the manufacturer's protocol⁺T cells and CD56⁺Concentrated by depleting NK cells.

0232Macaque or human BCMA-transfected CHO target cells (BCMA-positive target cells) were washed twice with PBS and 11.1 MBq in a final volume of 100 μl RPMI containing 50% FCS.⁵¹Labeled with Cr at 37 ° C for 60 minutes. The labeled target cells were then washed 3 times with 5 ml RPMI, which was then used in the cytotoxicity assay. The assay was performed in 96-well plates at a 10: 1 E: T ratio in supplemented RPMI with a total volume of 200 μl. Purified bispecific antibodies with a starting concentration of 0.01-1 μg / ml and 3-fold dilutions thereof were used. The incubation time in the assay was 18 hours. Cytotoxicity was determined as the relative value of chromium released in the supernatant relative to the difference between maximal lysis (with Triton-X added) and spontaneous lysis (without effector cells). All measurements were made in quadruples. Chromium activity in the supernatant was measured at the Wizard 3''γ counter (Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of experimental data is available for Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, California, USA) was used. EC50 values calculated from the sigmoid dose response curve by the analytical program were used to compare cytotoxic activity (see Figure A5).

02338.2 Efficacy to redirect stimulated human effector T cells against human BCMA-transfected CHO cells The cytotoxic activity of the BCMA / CD3 bispecific antibody was obtained by using human BCMA-transfected CHO cells as target cells and human CD8 T cells stimulated and concentrated as effector cells, 51 chromium (⁵¹Cr) Analyzed in a released cytotoxicity assay. This experiment was performed as described in Example A8.1.

0234All BCMA / CD3 bispecific antibodies of epitope cluster E3 / E4 ± E7 have strong cytotoxic activity with EC50 values in the range of single digit pg / ml to double digit pg / ml against human BCMA-transfected CHO cells. (Fig. A9 and Table 5). Thus, the epitope clusters E3 / E4 ± E7 provide a very favorable epitope-activity relationship that supports a very strong bispecific antibody-mediated cytotoxic activity.

0235(Table 5) 51 chromium (Table 5) using human BCMA-transfected CHO cells as target cells and human CD8 T cells stimulated and concentrated as effector cells.⁵¹Cr) EC50 value of BCMA / CD3 bispecific antibody of epitope cluster E3 / E4 ± E7 analyzed in released cytotoxicity assay [pg / ml]<img id="000018" he="139" wi="158" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0236<u style="single">8.3 FACS-based cytotoxic assay with unstimulated human PBMC</u>Isolation of effector cells Human peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient centrifugation from concentrated lymphocyte preparations (soft membranes), a by-product of blood collected by blood banks for transfusion. The buffy coat was provided by a domestic blood bank and PBMCs were prepared on the same day as blood collection. After thorough Ficoll density centrifugation and thorough washing with Dulbecco PBS (Gibco), the remaining erythrocytes are lysed with erythrocyte lysis buffer (155 mM NH).₄Cl, 10mM KHCO₃, 100 μM EDTA) removed from PBMC. Platelets were removed through the supernatant by centrifugation at 100 xg of PBMC. The remaining lymphocytes mainly contain B and T lymphocytes, NK cells and monocytes. PBMC in RPMI medium (Gibco) containing 10% FCS (Gibco) at 37 ° C / 5% CO₂It was maintained by the culture below.

0237CD14⁺And CD56⁺Cell depletion CD14⁺Human CD14 microbeads (Milteny Biotec, MACS, # 130-050-201) were used to deplete cells, and human CD56 microbeads (MACS, # 130-050-401) were used to deplete NK cells. It was used. PBMCs were counted and centrifuged at 300 xg for 10 minutes at room temperature. Discard the supernatant and dispose of the cell pellet in MACS isolation buffer [80 μL / 10⁷Cells; resuspended in PBS (Invitrogen, # 20012-043), 0.5% (v / v) FBS (Gibco, # 10270-106), 2 mM EDTA (Sigma-Aldrich, # E-6511)]. CD14 microbeads and CD56 microbeads (20 μL / 10)⁷Cells) were added and incubated at 4-8 ° C for 15 minutes. MACS isolation buffer (1-2 mL / 10) for cells⁷Washed with cells). After centrifugation (see above), discard the supernatant and isolate cells in MACS isolation buffer (500 μL / 10).⁸Resuspended in cells). Next, CD14 / CD56 negative cells were isolated using an LS column (Miltenyi Biotec, # 130-042-401). PBMC without CD14 + / CD56 + cells in RPMI complete medium, ie, 10% FBS (Biochrom AG, # S0115), 1 x non-essential amino acids (Biochrom AG, # K0293), in an incubator at 37 ° C until required. RPMI1640 (Biochrom AG, # FG1215) supplemented with 10 mM Hepes buffer (Biochrom AG, # L1613), 1 mM sodium pyruvate (Biochrom AG, # L0473) and 100 U / mL penicillin / streptomycin (Biochrom AG, # A2213). Cultured in.

0238Target cell labeling Fluorescent membrane dye DiOC for analysis of cytolysis in flow cytometry assays₁₈(DiO) (Molecular Probes, # V22886) can be used to label human BCMA or macaque BCMA-transfected CHO cells as target cells (human / macaque BCMA-positive target cells) so that they can be distinguished from effector cells. did. Briefly, cells are collected, washed once with PBS, and 2% (v / v) FBS and membrane dye DiO (5 μL / 10).⁶10 in PBS containing cells)⁶Adjusted to cells / mL. After incubation at 37 ° C for 3 minutes, the cells were washed twice in complete RPMI medium and the cell number was 1.25 x 10⁵Adjusted to cells / mL. Cell viability was determined using 0.5% (v / v) isotonic Eosin G solution (Roth, # 45380).

0239Flow cytometry-based analysis This assay was designed to quantify lysis of macaque or human BCMA-transfected CHO cells (or BCMA-positive target cells) in the presence of serial dilutions of BCMA / CD3 bispecific antibody. Equal amounts of DiO-labeled target cells and effector cells (ie, CD14)⁺Cell-free PBMCs) were mixed to give an E: T cell ratio of 10: 1. 160 μL of this suspension was transferred to each well of a 96-well plate. 40 μL of serial dilutions of BCMA / CD3 bispecific antibody and negative control bispecific antibody (CD3-based bispecific antibody that recognizes unrelated target antigens) or RPMI complete medium as additional negative control Was added. The cytotoxic response mediated by BCMA / CD3 bispecific antibody is 7% CO₂It was allowed to proceed for 48 hours in a humidified incubator. The cells were then transferred to a new 96-well plate and the loss of membrane integrity of the target cells was monitored by the addition of propidium iodide (PI) at a final concentration of 1 μg / mL. PI is a membrane-impermeable pigment that is normally eliminated from living cells, but dead cells take it up and become identifiable by fluorescent radiation. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). Target cells were identified as DiO positive cells. PI-negative target cells were classified as living target cells. The percentage of cytotoxicity was calculated according to the following formula. Cytotoxicity [%] = n (dead target cells) x 100 / n (target cells) n = number of events GraphPad Prism 5 Software (Graph Pad Software, Using San Diego), the percentage of cytotoxicity was plotted against the concentration of the corresponding bispecific antibody. Dose response curves were analyzed using a 4-parameter logistic regression model for evaluation of sigmoid dose response curves with a constant hill gradient, and EC50 values were calculated.

0240<u style="single">8.4 Unstimulated human PBMCs against human BCMA-transfected target cells</u> The cytotoxic activity of the BCMA / CD3 bispecific antibody was analyzed in a FACS-based cytotoxic assay using human BCMA-transfected CHO cells as target cells and unstimulated human PBMC as effector cells. This assay was performed as described above (Example A8.3).

0241The results of a FACS-based cytotoxicity assay using unstimulated human PBMCs as effector cells and human BCMA-transfected CHO cells as targets are shown in Figures A10 and Table 6.

0242(Table 6) BCMA of epitope clusters E3 / E4 ± E7 measured in a 48-hour FACS-based cytotoxicity assay using unstimulated human PBMC as effector cells and human BCMA-transfected CHO cells as target cells. / CD3 EC50 value of bispecific antibody [pg / ml]<img id="000019" he="20" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0243<u style="single">Example A9</u>9.1 Elimination of cross-reactivity with BAFF receptor For flow cytometry, 200,000 cells from each cell line were incubated with 50 μl of purified bispecific antibody at a concentration of 5 μg / ml for 30 minutes on ice. Cells were washed twice with PBS containing 2% FCS and construct binding was detected with mouse PentaHis antibody (Qiagen; diluted 1:20 with 50 μl PBS containing 2% FCS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS containing 2% FCS. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). The bispecific conjugate was shown to be non-cross-reactive with the BAFF receptor.

02449.2 Elimination of BCMA / CD3 bispecific antibodies that cross-react with human BAFF receptor (BAFF-R) and TACI To eliminate binding to human BAFF-R and TACI, BCMA / CD3 bispecific antibodies were tested by flow cytometry using CHO cells transfected with human BAFF-R and TACI, respectively. In addition, L363 multiple myeloma cells were used as positive controls for binding to human BCMA. Expression of BAFF-R and TACI antigens in CHO cells was confirmed with two positive control antibodies. Flow cytometry was performed as described in the above Examples.

0245Flow cytometric analysis confirmed that none of the BCMA / CD3 bispecific antibodies of epitope clusters E3 / E4 ± E7 cross-reacted with human BAFF-R or human TACI (see Figure A11). ..

0246<u style="single">Example A10</u>Cytotoxic activity The efficacy of human-like BCMA bispecific antibodies that redirect effector T cells to BCMA-expressing target cells is analyzed in five additional in vitro cytotoxicity assays: 1. The efficacy of BCMA bispecific antibodies that redirect stimulated human effector T cells to BCMA-positive (human) tumor cell lines is measured in a 51 chromium release assay. 2. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs to human BCMA-transfected CHO cells is measured in a FACS-based cytotoxicity assay. 3. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs to BCMA-positive (human) tumor cell lines is measured in a FACS-based cytotoxicity assay. 4. FACS-based cytotoxic assay using Makaku T cell lines as effector T cells to confirm that cross-reactive BCMA bispecific antibodies can redirect Makaku T cells to Makaku BCMA-transfected CHO cells. I do. Five. 51 chromium release assay using human BCMA-transfected CHO cells as target cells and human T cells stimulated as effector cells to open the potency gap between monomeric and dimeric forms of BCMA bispecific antibodies To decide in.

0247<u style="single">Example A11</u>Stimulated human T cells against BCMA-positive human multiple myeloma cell line L363 For the cytotoxic activity of BCMA / CD3 bispecific antibody, BCMA-positive human multiple myeloma cell line L363 (DSMZ No. ACC49) was used as the target cell source, and human CD8 T cells stimulated and concentrated as effector cells were used. There was 51 chrome (⁵¹Cr) Analyzed in a released cytotoxicity assay. The assay was performed as described in Example A8.1.

0248BCMA / CD3 bispecificity of epitope clusters E3 / E4 ± E7 according to the results of a 51 chromium release assay using human CD8 T lymphocytes stimulated and concentrated as effector cells and human BCMA-transfected CHO cells as targets. Antibodies have strong cytotoxic activity (Fig. A12 and Table 7).

0249Unexpectedly, however, the BCMA / CD3 bispecific antibody of epitope cluster E1 / E4 was potent in terms of cytotoxic activity against human BCMA-transfected CHO cells, but low native BCMA on the cell surface. It showed much weaker cytotoxicity against the densely expressed human multiple myeloma cell line L363 (Fig. A12 and Table 7). Without wishing to be constrained by the theory, we suspect that the E1 / E4 epitope of human BCMA may be less accessible in BCMA spontaneously expressing than BCMA-transfected cells.

0250(Table 7) BCMA-positive human multiple myeloma cell line L363 was used as the target cell source, and human CD8 T cells stimulated and concentrated as effector cells were used for 18 hours and 51 chromium (Table 7).⁵¹Cr) EC50 values of BCMA / CD3 bispecific antibodies of epitope clusters E1 / E4 (columns 1 and 2) and E3 / E4 ± E7 (columns 3 and 4) analyzed in the released cytotoxicity assay [ pg / ml]<img id="000020" he="34" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0251<u style="single">Example A12</u>Unstimulated human PBMC against BCMA-positive human multiple myeloma cell line L363 BCMA-positive human multiple myeloma cell lines that showed the weakest native BCMA surface expression of all the targeted T cell lines tested, using the cytotoxic activity of the BCMA / CD3 bispecific antibody as the target cell source. L363 (DSMZ, ACC49) was further analyzed in a FACS-based cytotoxicity assay using unstimulated human PBMC as effector cells. The assay was performed as described above (Example A8.3).

0252BCMA / CD3 bispecific antibodies of epitope clusters E1 / E4 were observed in a 51 chromium release assay using stimulated and concentrated human CD8 T lymphocytes against human multiple myeloma cell line L363. Again, in contrast to their strong cytotoxic activity against human BCMA-transfected CHO cells, the human multiple myeloma cell line L363, which also expresses native BCMA at low concentrations on the cell surface, has not yet been identified. It showed weak efficacy in reversing the cytotoxic activity of stimulated PBMC. This is consistent with the above theory, that the E1 / E4 epitopes of human BCMA are considered to be less accessible in BCMA spontaneously expressors than BCMA-transfected cells. BCMA / CD3 bispecific antibodies of epitope clusters E3 / E4 ± E7 showed EC50 values of 3 digits pg / ml in this assay (see Figures A13 and Table 8).

0253(Table 8) epitope clusters E1 / E4 measured in a 48-hour FACS-based cytotoxicity assay using unstimulated human PBMC as effector cells and human multiple myeloma cell line L363 as target cell source (row 1) And 2nd column) and EC50 value of BCMA / CD3 bispecific antibody of E3 / E4 ± E7 (3rd and 4th column) [pg / ml]<img id="000021" he="33" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0254As expected, EC50 values were higher in cytotoxicity assays using unstimulated PBMCs as effector cells than in cytotoxicity assays using concentrated and stimulated human CD8 T cells.

0255<u style="single">Example A13</u>Unstimulated human PBMC against BCMA-positive human multiple myeloma cell line NCI-H929 For the cytotoxic activity of BCMA / CD3 bispecific antibody, BCMA-positive human multiple myeloma cell line NCI-H929 (ATCC CRL-9068) was used as the target cell source, and unstimulated human PBMC was used as the effector cell. Analyzed in a FACS-based cytotoxicity assay. The assay was performed as described above (Example A8.3).

0256The results of this assay using another human multiple myeloma cell line expressing native BCMA on the cell surface (ie, NCI-H929) were obtained using the human multiple myeloma cell line L363. Is to confirm. Again, BCMA / CD3 bispecific antibodies of epitope clusters E1 / E4 to human polymyeloma cells, as opposed to their potent cytotoxic activity against human BCMA-transfected CHO cells. It showed low efficacy in reversing the cytotoxic activity of unstimulated PBMC, confirming the theory that human BCMA E1 / E4 epitopes may be less accessible in BCMA spontaneously expressing cells than BCMA-transfected cells. It was. No such activity gap between BCMA-transfected target cells and spontaneously expressed cells, as was seen with E1 / E4 conjugates, was found with E3 / E4 ± E7 conjugates. BCMA / CD3 of epitope cluster E3 / E4 ± E7 Bispecific antibodies showed double-digit pg / ml EC50 values, thus redirecting unstimulated PBMCs to NCI-H929 target cells with surprisingly good EC50 values (see Figures A14 and Table 9). ).

0257(Table 9) epitope clusters E1 / E4 measured in a 48-hour FACS-based cytotoxicity assay using unstimulated human PBMC as effector cells and human multiple myeloma cell line NCI-H929 as target cell source. EC50 values of BCMA / CD3 bispecific antibodies in rows 1 and 2) and E3 / E4 ± E7 (rows 3 and 4) [pg / ml]<img id="000022" he="34" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0258As expected, EC50 levels were higher in the human multiple myeloma cell line NCI-H929, which expresses high levels of BCMA on the cell surface compared to L363.

0259<u style="single">Example A14</u>Macaque T cells against macaque BCMA expression target cells Finally, the cytotoxic activity of the BCMA / CD3 bispecific antibody was tested in a FACS-based cytotoxic assay using Makaku BCMA-transfected CHO cells as target cells and Makaku T cell lines as effector cell sources. analyzed.

0260The macaque T cell line 4119LnPx (Knappe et al. Blood 95: 3256-61 (2000)) was used as an effector cell source. Flow cytometry-based analysis of target cell labeling and cytotoxic activity of macaque BCMA-transfected CHO cells was performed as described above.

0261Macaque T cells from cell line 4119LnPx were induced to effectively kill macaque BCMA-transfected CHO cells by BCMA / CD3 bispecific antibodies of epitope clusters E3 / E4 ± E7. This antibody showed very strong potency in the single digit pg / ml EC50 value in this assay, confirming that these antibodies are very active in the macaque system. On the other hand, BCMA / CD3 bispecific antibodies of epitope clusters E1 / E4 showed significantly weaker efficacy with EC50 values in the 2- to 3-digit pg / ml range (see Figure A15 and Table 10). Therefore, E3 / E4 ± E7-specific antibodies are about 20 to 100 times more potent in macaques.

0262(Table 10) epitope clusters E1 / E4 measured in a 48-hour FACS-based cytotoxicity assay using Makaku T cell line 4119LnPx as effector cells and Makaku BCMA-transfected CHO cells as target cells (row 1). And 2nd column) and EC50 value of BCMA / CD3 bispecific antibody of E3 / E4 ± E7 (3rd and 4th column) [pg / ml]<img id="000023" he="33" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0263<u style="single">Example A15</u>Efficacy gap between BCMA / CD3 bispecific antibody monomer and dimer To determine the difference in cytotoxic activity between individual BCMA / CD3 bispecific antibody monomers and dimer isoforms (referred to as the potency gap), the above (Example A8.1). A 51 chromium-releasing cytotoxicity assay, such as, was performed with purified BCMA / CD3 bispecific antibody monomers and dimers. The potency gap was calculated as the ratio between the monomeric bispecific antibody and the EC50 value of the dimer. The potency gap of the BCMA / CD3 bispecific antibody for the epitope clusters E3 / E4 ± E7 tested was between 0.2 and 1.2. Therefore, the dimer is not significantly more active than its respective monomer.

0264<u style="single">Example A16</u>Conversion of monomer to dimer after 3 freeze / thaw cycles Bispecific BCMA / CD3 antibody monomers were subjected to three freeze / thaw cycles followed by fast SEC to percentage of the antibody that was the initial monomer converted to the antibody dimer. It was determined.

0265The 15 μg monomeric antibody was adjusted to a concentration of 250 μg / ml in general buffer, then frozen at -80 ° C for 30 minutes and then thawed at room temperature for 30 minutes. After 3 freeze / thaw cycles, the dimer content was determined by HP-SEC. For this purpose, a 15 μg aliquot of the monomeric isoform of the antibody was thawed to a concentration of 250 μg / ml in initial SEC buffer (10 mM citrate 75 mM lysine HCl 4% trehalose pH 7.2), followed by Incubated at 37 ° C for 7 days. High resolution SEC column TSK Gel G3000 SWXL (Tosoh, Tokyo-Japan) was connected to the Akta Purifier 10 FPLC (GE Lifesciences) equipped with the A905 autosampler. The column equilibration / electrophoresis buffer consisted of 100 mM KH2PO4-200 mM Na2SO4 adjusted to pH 6.6. After 7 days of incubation, antibody solution (15 μg protein) was applied to the equilibrated column and eluted at a flow rate of 0.75 ml / min and a maximum pressure of 7 MPa. The entire electrophoresis was monitored by light absorption at 280, 254 and 210 nm. The analysis was performed by peak integration of the 210 nm signal recorded on the Akta Unicorn software migration evaluation sheet. The dimer content was calculated by dividing the area of the dimer peak by the total area of the monomer and the dimer peak.

0266BCMA / CD3 bispecific antibodies of epitope clusters E3 / E4 ± E7 show a dimer ratio of 0.8-1.5% after 3 freeze / thaw cycles, which is considered good. However, the dimer conversion rate of the BCMA / CD3 bispecific antibody in the epitope clusters E1 / E4 reached an unfavorably high value, exceeding the inconvenient dimeric threshold of 2.5% (each). , 4.7% and 3.8%), see Table 11.

0267(Table 11) Epitope clusters E1 / E4 (1st and 2nd columns) and E3 / E4 ± E7 (1st column) after 3 freeze / thaw cycles as determined by fast size exclusion chromatography (HP-SEC). 3 and 4 columns) BCMA / CD3 bispecific antibody monomer-to-dimer percentage<img id="000024" he="33" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0268<u style="single">Example A17</u>Thermal stability To determine the biophysical protein stability inherent in the BCMA / CD3 bispecific antibody, the melting temperature curve was determined by differential scanning calorimetry (DSC). These experiments were performed using MicroCal LLC (Northampton, MA, USA) VP-DSC devices. Energy uptake of samples containing BCMA / CD3 bispecific antibody was recorded from 20 ° C to 90 ° C compared to samples containing only prescription buffer for the antibody.

0269More specifically, BCMA / CD3 bispecific antibody was adjusted to a final concentration of 250 μg / ml with storage buffer. 300 μl of the prepared protein solution was transferred to a deep well plate and placed in the cooling autosampler rack position of the DSC device. Additional wells were filled with SEC electrophoresis buffer as a measurement reference material. In the measurement process, the protein solution was transferred to the capillary by an autosampler. Additional capillaries were filled with SEC electrophoresis buffer as a reference. For all samples, heating and recording of the thermal energy required to heat both capillaries to equal temperatures in the range 20-90 ° C were performed.

0270The overall temperature of the sample was raised stepwise to record each melting curve. At each temperature T, the energy uptake of the sample and prescription buffer references was recorded. The difference in energy uptake Cp (kcal / mole / ° C) minus the reference from the sample was plotted for each temperature. Melting temperature is defined as the temperature at which energy uptake is initially maximized.

0271BCMA / CD3 bispecific antibodies of all tested epitope clusters E3 / E4 ± E7 have good thermal stability of melting temperature above 60 ° C, more precisely between 62 ° C and 63 ° C. Showed sex.

0272<u style="single">Example A18</u>Elimination of plasma interference by flow cytometry A plasma interference test was constructed to determine the potential interaction of BCMA / CD3 bispecific antibodies with human plasma proteins. For this purpose, 10 μg / ml of each BCMA / CD3 bispecific antibody was incubated in 90% human plasma at 37 ° C for 1 hour. Subsequent binding to human BCMA-expressing CHO cells was determined by flow cytometry.

0273For flow cytometry, 200,000 cells from each cell line were incubated on ice for 30 minutes with 50 μl of purified antibody at a concentration of 5 μg / ml. Cells were washed twice with PBS / 2% FCS and construct binding was detected with mouse PentaHis antibody (Qiagen; 1:20 dilution with 50 μl PBS / 2% FCS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS / 2% FCS. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson).

0274The data obtained were compared to a control assay using PBS instead of human plasma. The relative join was calculated as follows: (PBS sample signal / signal without detection reagent) / (plasma sample signal / signal without detection reagent).

0275This experiment revealed that there was no significant reduction in target binding of each BCMA / CD3 bispecific antibody of the epitope clusters E3 / E4 ± E7 mediated by plasma proteins. Relative plasma interference values ranged from 1.28 ± 0.38 to 1.29 ± 0.31 (value "2" is considered the lower threshold of the interference signal).

0276<u style="single">Example A19</u>Therapeutic efficacy of BCMA / CD3 bispecific antibody in human tumor xenograft model 5 x 10 on the first day of study⁶The human cancer cell line NCI-H929 was subcutaneously injected into the right dorsal aspect of female NOD / SCID mice.

0277Average tumor volume is about 100 mm³Human CD3 deployed in vitro on day 9 when⁺ T cells into the abdominal cavity of this animal about 2 x 10⁷It was transplanted into mice by injection of cells. Mice in vehicle control group 1 (n = 5) were not fed effector cells and were compared with vehicle control group 2 (n = 10, effector cells fed) to monitor the effect of T cells alone on tumor growth. Used as an untransplanted control for.

0278Antibody treatment has an average tumor volume of about 200 mm³It started on the 13th day when it reached. The mean tumor size of each treatment group on the treatment start date was not statistically different from any other group (analysis of variance). Mice were treated with a 0.5 mg / kg / day BCMA / CD3 bispecific antibody BCMA-50 x CD3 (Group 3, n = 8) by intravenous bolus injection for 17 days.

0279During the study, tumors were measured by calipers and progression was assessed by group-to-group comparison of tumor volume (TV). Tumor growth inhibition T / C [%] was determined by calculating TV as T / C% = 100 x (median TV in analysis group) / (median TV in control group 2). The results are shown in Table 12 and Figure 16.

0280(Table 12) Median tumor volume (TV) and tumor growth inhibition (T / C) on days 13-30<img id="000025" he="79" wi="158" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0281<u style="single">Example A20</u>Elimination of target-negative cell lysis In vitro lysis assays were performed using BCMA-positive human multiple myeloma cell line NCI-H929 with an effector to target cell ratio of 5: 1 and purified T cells for a 24-hour incubation time. The BCMA / CD3 bispecific antibody (BCMA-50) of the epitope cluster E3 / E4 ± E7 showed high efficacy and efficacy for lysis of NCI-H929. However, BCMA-negative cell lines HL60 (AML / myeloblast morphology), MES-SA (uterine sarcoma, fibroblast morphology) and SNU-16 (gastric cancer, epithelial morphology) did not detect lysis with antibodies up to 500 nM. It was.

0282<u style="single">Example A21</u>Induction of T cell activation of different PBMC subsets FACS-based cytotoxicity assay (48h; E: T = 10: 1) with human multiple myeloma cell lines NCI-H929, L-363 and OPM-2 as target cells and different subsets of human PBMC as effector cells (CD4⁺/ CD8⁺/ CD25⁺/ CD69⁺) Was used. Results (see Table 13) are available in EC₅₀The values indicate that the degree of activation is essentially within the same range for the different PBMC subsets analyzed.

0283(Table 13) BCMA / of epitope clusters E3 / E4 ± E7 measured in a 48-hour FACS-based cytotoxicity assay using different subsets of human PBMC as effector cells and different human multiple myeloma cell lines as target cells. EC50 value of CD3 bispecific antibody BCMA-50 [ng / ml]<img id="000026" he="113" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0284<u style="single">Example A22</u>Induction of cytokine release FACS-based cytotoxicity assay (48h; E: T = 10: 1) with human multiple myeloma cell lines NCI-H929, L-363 and OPM-2 as target cells and human PBMC as effector cells went. The level of cytokine release [pg / ml] was determined under increasing concentrations of the epitope cluster E3 / E4 ± E7 under BCMA / CD3 bispecific antibody. The following cytokines were analyzed: Il-2, IL-6, IL-10, TNF and IFN-γ. The results are shown in Table 14 and Figure 17.

0285(Table 14) 2.5 μg / ml epitope cluster in a 48-hour FACS-based cytotoxicity assay (E: T = 10: 1) using human PBMC as effector cells and different human multiple myeloma cell lines as target cells. E3 / E4 ± E7 BCMA / CD3 bispecific antibody (BCMA-50) -induced release of IL-2, IL-6, IL-10, TNF and IFN-γ [pg / ml]<img id="000027" he="39" wi="159" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />

0286<u style="single">Example B</u><u style="single">Example B1</u>Generation of CHO cells expressing chimeric BCMA To construct a chimeric epitope mapping molecule, the amino acid sequence or single amino acid residue of each epitope domain of human BCMA was converted to a mouse sequence. We constructed the following molecules:

0287-Human BCMA ECD / E1 mouse (SEQ ID NO: 1009) Chimeric extracellular BCMA domain: Epitope cluster 1 (SEQ ID NO: 1002 or 1007 amino acid residues 1-7) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 1-4). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 1-3 and mutation of G6Q in 1002 or 1007

0288-Human BCMA ECD / E2 mouse (SEQ ID NO: 1010) Chimeric extracellular BCMA domain: Epitope cluster 2 (SEQ ID NO: 1002 or 1007 amino acid residues 8-21) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 5-18). Human extracellular BCMA domain SEQ ID NO: Mutations of S9F, Q10H and N11S in 1002 or 1007

0289-Human BCMA ECD / E3 mouse (SEQ ID NO: 1011) Chimeric extracellular BCMA domain: Epitope cluster 3 (SEQ ID NO: 1002 or 1007 amino acid residues 24-41) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 21-36). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 31 and 32 at 1002 or 1007 and mutation of Q25H, S30N, L35A and R39P

0290-Human BCMA ECD / E4 mouse (SEQ ID NO: 1012) Chimeric extracellular BCMA domain: Epitope cluster 4 (SEQ ID NO: 1002 or 1007 amino acid residues 42-54) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 37-49). Human extracellular BCMA domain SEQ ID NO: Mutations of N42D, A43P, N47S, N53Y and A54T in 1002 or 1007

0291-Human BCMA ECD / E5 mouse (SEQ ID NO: 1013) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 22 (isoleucine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (lysine, position 19) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of I22K in 1002 or 1007

0292-Human BCMA ECD / E6 mouse (SEQ ID NO: 1014) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 25 (glutamine) at SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (histidine, position 22) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of Q25H in 1002 or 1007

0293-Human BCMA ECD / E7 mouse (SEQ ID NO: 1015) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 39 (arginine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (proline, position 34) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of R39P in 1002 or 1007.

0294The cDNA construct was cloned into the mammalian expression vector pEF-DHFR and stably transfected into CHO cells. Expression of human BCMA in CHO cells was verified in a FACS assay using a monoclonal anti-human BCMA antibody. Expression of mouse BCMA was demonstrated using a monoclonal anti-mouse BCMA antibody. The concentration of BCMA antibody used was 10 μg / ml in PBS / 2% FCS. Bound monoclonal antibody was detected using anti-rat IgG-Fcy-PE (1: 100 in PBS / 2% FCS; Jackson-Immuno-Research # 112-116-071). As a negative control, cells were incubated with PBS / 2% FCS in place of the first antibody. Sample the FACSCanto II instrument (Becton) Measured by flow cytometry in Dickinson) and analyzed by FlowJo software (version 7.6). Transfected CHO cells were analyzed and confirmed for surface expression of human-mouse BCMA chimeras in flow cytometry assays using various anti-BCMA antibodies (Fig. 2).

0295<u style="single">Example B2</u>2.1 Transient expression in HEK293 cells Clone expression plasmids with sequence-validated nucleotide sequences were used for transfection and protein expression in the FreeStyle 293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's protocol. A supernatant containing the expressed protein was obtained, cells were removed by centrifugation and the supernatant was stored at -20 ° C.

02962.2 Stable expression in CHO cells Clone expression plasmids with sequence-verified nucleotide sequences were transfected into DHFR-deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR-deficient CHO cells was performed as described in Kaufman RJ (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX. After two passages in static culture, cells contain nucleoside-free HyQ PF CHO liquid soybean medium (containing 4.0 mM L-glutamine, containing 0.1% Pluronic F-68; HyClone) for 7 days prior to collection. Growing in roller bottles. Cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -20 ° C.

0297<u style="single">Example B3</u>Epitope clustering of mouse scFv fragments Cells transfected with human or mouse BCMA or chimeric BCMA molecules were stained with a crude undiluted periplasmic extract containing scFv that binds to human / macaque BCMA. Bound scFv was detected with 1 μg / ml anti-FLAG antibody (Sigma F1804) and R-PE labeled anti-mouse Fcγ-specific antibody (1: 100; Dianova # 115-116-071). All antibodies were diluted with PBS containing 2% FCS. As a negative control, cells were incubated with PBS / 2% FCS instead of periplasmic extract. Samples were measured by flow cytometry on a FACSCanto II instrument (Becton Dickinson) and analyzed by FlowJo software (version 7.6).

0298<u style="single">Example B4</u>Obtaining different recombinant forms of soluble human and macaque BCMA Code sequences for human and rhesus monkey BCMA (published at GenBank, accession numbers NM_001192 [human], XM_001106892 [rhesus monkey]), human albumin, human Fcγ1 and mouse albumin coding sequences, human and macaque BCMA, respectively, and human albumin, human It was used to construct an artificial cDNA sequence encoding a soluble fusion protein of IgG1 Fc and mouse albumin, respectively, as well as a soluble protein containing only the extracellular domain of BCMA. To generate constructs for the expression of soluble human and macaque BCMA proteins, cDNA fragments were obtained by PCR mutagenesis and molecular cloning of the above full-length BCMA cDNA according to standard protocols. For fusions with human albumin, they first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, respectively. And rhesus monkey BCMA protein coding sequence, then in-frame coding sequence of artificial Ser1-Gly4-Ser1 linker, then in-frame coding sequence of human serum albumin, then in-frame coding sequence of Flag tag, then in-frame Frame-modified histidine tag<img id="000028" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For fusions with mouse IgG1, humans first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, followed by the coding sequence of the human IgG1 hinge and Fcγ moiety in frame, and then the hexahistidine tag code in frame. Modified cDNA fragments were designed to include sequences and stop codons. For fusions with mouse albumin, humans each contain amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, first the Kozak site for eukaryotic expression of the construct. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, then the coding sequence of mouse serum albumin in frame, then the coding sequence of the Flag tag in frame, and then in frame. Frame-modified histidine tag<img id="000029" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For soluble extracellular domain constructs, first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively, human and Macaque BCMA protein coding sequence, then in-frame artificial Ser1-Gly1 linker coding sequence, then in-frame Flag tag coding sequence, then in-frame modified histidine tag<img id="000030" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. The cDNA fragment was also designed to introduce restriction sites at the beginning and end of the fragment. The introduced restriction sites, EcoRI at the 5'end and SalI at the 3'end, were used in the cloning procedure below. The cDNA fragment was cloned through EcoRI and SalI into a plasmid called pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). All of the above steps were performed according to standard protocols (Sambrook, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)).

0299<u style="single">Example B5</u>Biacore-based determination of bispecific antibody affinity for human and macaque BCMA and CD3 Biacore analytical experiments were performed with recombinant BCMA fusion proteins containing human serum albumin (ALB) to determine target binding to BCMA. To measure the affinity for CD3, a recombinant fusion protein having the N-terminal 27 amino acids of CD3 epsilon (CD3e) fused to the Fc portion of the human antibody was used. This recombinant protein is available in human CD3e 1-27 and cynomolgus monkey CD3e versions, both of which carry the CD3 conjugate epitope in the bispecific antibody. More specifically, approximately 100-150 RU of each recombinant antigen was immobilized on a CM5 sensor chip (GE Healthcare) using acetate buffer pH 4.5 according to the manufacturer's manual. Bispecific antibody sample, HBS-EP electrophoresis buffer (GE) Five concentrations diluted with Healthcare): filled with 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.13 nM. The flow rate was 30-35 μl / min for 3 minutes, after which HBS-EP electrophoresis buffer was applied again at a flow rate of 30-35 μl / min for 8 minutes. Chip regeneration was performed with 10 mM glycine 0.5 M NaCl pH 2.45. The dataset was analyzed using BiaEval software. Usually, two independent experiments were performed.

0300<u style="single">Example B6</u>Flow cytometric analysis The functionality and binding strength of affinity-maturated scFv molecules were analyzed in FACS using human and macaque BCMA-transfected CHO cells. Briefly, about 10⁵The cells were incubated with 50 μl of a 1: 3 serial dilution of E. coli cell periplasmic extract for 50 minutes on ice. After washing with PBS / 10% FCS / 0.05% sodium azide, cells are combined with 30 μl Flag-M2 IgG (Sigma, 1: 900 in PBS / 10% FCS / 0.05% sodium azide) on ice for 40 minutes. Incubated. After the second wash, cells are incubated with 30 μl R-phycoerythrin (PE) -labeled goat anti-mouse IgG (Jackson ImmunoResearch, 1: 100 in PBS / 10% FCS / 0.05% sodium azide) for 40 minutes on ice. did. The cells were then washed again and resuspended in 200 μl PBS / 10% FCS / 0.05% sodium azide. Relative fluorescence of stained cells was measured using a FACSCanto flow cytometer (BD). The results are shown as a FACS histogram plotting the logarithmic vs. relative cell count of fluorescence intensity (see Figure B4).

0301<u style="single">Example B7</u>Bispecific binding and interspecies cross-reactivity For flow cytometry, 200,000 cells from each cell line were incubated on ice for 30 minutes with 50 μl of purified bispecific molecule at a concentration of 5 μg / ml. Cells were washed twice with 2% FCS-containing PBS and construct binding was detected with mouse PentaHis antibody (Qiagen; 1:20 dilution with 50 μl 2% FCS-containing PBS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS containing 2% FCS.

0302<u style="single">Example B8</u>Scatchard-based determination of bispecific antibody affinity for human and macaque BCMA In Scatchard analysis, saturated binding experiments are performed using a monovalent detection system (anti-His Fab / Alexa 488) developed by Micromet to accurately determine the monovalent binding of bispecific antibodies to each cell line. Each cell line (CHO cell line that recombinantly expresses human BCMA, CHO cell line that recombinantly expresses macaque BCMA) 2 x 10⁴Cells are incubated with 50 μl each of a triple dilution series (1: 2 8-step dilution) of each BCMA bispecific antibody starting at 100 nM, followed by incubation at 4 ° C for 16 hours with stirring, 1 Perform multiple residue cleaning steps. The cells are then incubated with 30 μl of anti-His Fab / Alexa 488 solution (Micromet; 30 μg / ml) for an additional 30 minutes. After one wash step, cells are resuspended in 150 μl FACS buffer containing 3.5% formaldehyde, incubated for an additional 15 minutes, centrifuged, resuspended in FACS buffer, and FACS Canto II machine and FACS. Analyze using Diva software. Data are obtained from two independent experimental sets. The values are plotted as a combined curve of hyperbolas. Calculations are performed to estimate the maximum binding (Bmax) from each Scatchard analysis. Determine the concentration of bispecific antibody at maximum half-dose binding that reflects each KD. Plot the values of the triple measurements as a hyperbola. The maximum binding is determined using the Scatchard evaluation and each KD is calculated.

0303<u style="single">Example B9</u>Cytotoxic activity 9.1 Chromium release assay with stimulated human T cells CD8⁺T cells enriched and stimulated for T cells were obtained as follows. Petri dishes (145 mm in diameter, Greiner bio-one GmbH, Kremsmunster) were coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) at a final concentration of 1 μg / ml at 37 ° C. for 1 hour. Unbound proteins were removed by a single wash step with PBS. 3-5 x 10 in 120 ml RPMI 1640 containing stabilized glutamine / 10% FCS / IL-2 20 U / ml (Proleukin®, Chiron)⁷Human PBMCs were added to pre-coated Petri dishes and stimulated for 2 days. On day 3, cells were harvested and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U / ml and the cells were again cultured in the same cell culture medium as above for 1 day. CD8⁺Cytotoxic T lymphocytes (CTL) CD4 using Dyna-Beads according to the manufacturer's protocol⁺T cells and CD56⁺Concentrated by depleting NK cells. Macaque or human BCMA-transfected CHO target cells were washed twice with PBS and 11.1 MBq in a final volume of 100 μl RPMI containing 50% FCS.⁵¹Labeled with Cr at 37 ° C for 60 minutes. The labeled target cells were then washed 3 times with 5 ml RPMI, which was then used in the cytotoxicity assay. The assay was performed in 96-well plates at a 10: 1 E: T ratio in supplemented RPMI with a total volume of 200 μl. Purified bispecific antibodies with a starting concentration of 0.01-1 μg / ml and 3-fold dilutions thereof were used. The incubation time in the assay was 18 hours. Cytotoxicity was determined as the relative value of chromium released in the supernatant relative to the difference between maximal lysis (with Triton-X added) and spontaneous lysis (without effector cells). All measurements were made in quadruples. Chromium activity in the supernatant was measured at the Wizard 3''γ counter (Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of the results is based on Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, California, USA) was used. EC50 values calculated from the sigmoid dose response curve by the analytical program were used to compare cytotoxic activity.

03049.2 FACS-based cytotoxic assay with unstimulated human PBMC Isolation of effector cells Human peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient centrifugation from concentrated lymphocyte preparations (soft membranes), a by-product of blood collected by blood banks for transfusion. The buffy coat was provided by a domestic blood bank and PBMCs were prepared on the same day as blood collection. After thorough Ficoll density centrifugation and thorough washing with Dulbecco's PBS (Gibco), the remaining erythrocytes are removed with erythrocyte lysis buffer (155 mM NH).₄Cl, 10mM KHCO₃, 100 μM EDTA) removed from PBMC. Platelets were removed through the supernatant by centrifugation at 100 xg of PBMC. The remaining lymphocytes mainly contain B and T lymphocytes, NK cells and monocytes. PBMC in RPMI medium (Gibco) containing 10% FCS (Gibco) at 37 ° C / 5% CO₂It was maintained by the culture below.

0305CD14⁺And CD56⁺Cell depletion CD14⁺Human CD14 microbeads (Milteny Biotec, MACS, # 130-050-201) were used to deplete cells, and human CD56 microbeads (MACS, # 130-050-401) were used to deplete NK cells. It was used. PBMCs were counted and centrifuged at 300 xg for 10 minutes at room temperature. Discard the supernatant and dispose of the cell pellet in MACS isolation buffer [80 μL / 10⁷Cells; resuspended in PBS (Invitrogen, # 20012-043), 0.5% (v / v) FBS (Gibco, # 10270-106), 2 mM EDTA (Sigma-Aldrich, # E-6511)]. CD14 microbeads and CD56 microbeads (20 μL / 10)⁷Cells) were added and incubated at 4-8 ° C for 15 minutes. MACS isolation buffer (1-2 mL / 10) for cells⁷Washed with cells). After centrifugation (see above), discard the supernatant and isolate cells in MACS isolation buffer (500 μL / 10).⁸Resuspended in cells). Next, CD14 / CD56 negative cells were isolated using an LS column (Miltenyi Biotec, # 130-042-401). PBMC without CD14 + / CD56 + cells in RPMI complete medium, ie, 10% FBS (Biochrom AG, # S0115), 1 x non-essential amino acids (Biochrom AG, # K0293), in an incubator at 37 ° C until required. RPMI1640 (Biochrom AG, # FG1215) supplemented with 10 mM Hepes buffer (Biochrom AG, # L1613), 1 mM sodium pyruvate (Biochrom AG, # L0473) and 100 U / mL penicillin / streptomycin (Biochrom AG, # A2213). Cultured in.

0306Target cell labeling Fluorescent membrane dye DiOC for analysis of cytolysis in flow cytometry assays₁₈(DiO) (Molecular Probes, # V22886) was used to label human BCMA or macaque BCMA-transfected CHO cells as target cells so that they could be distinguished from effector cells. Briefly, cells are collected, washed once with PBS, and 2% (v / v) FBS and membrane dye DiO (5 μL / 10).⁶10 in PBS containing cells)⁶Adjusted to cells / mL. After incubation at 37 ° C for 3 minutes, the cells were washed twice in complete RPMI medium and the cell number was 1.25 x 10⁵Adjusted to cells / mL. Cell viability was determined using 0.5% (v / v) isotonic Eosin G solution (Roth, # 45380).

0307Flow cytometry-based analysis This assay was designed to quantify lysis of macaque or human BCMA-transfected CHO cells in the presence of serial dilutions of BCMA bispecific antibody. Equal amounts of DiO-labeled target cells and effector cells (ie, CD14)⁺Cell-free PBMCs) were mixed to give an E: T cell ratio of 10: 1. 160 μL of this suspension was transferred to each well of a 96-well plate. 40 μL of serial dilutions of BCMA bispecific antibody and negative control bispecific (CD3-based bispecific antibody recognizing unrelated target antigens) or RPMI complete medium as an additional negative control was added. .. Bispecific antibody-mediated cytotoxic response is 7% CO₂It was allowed to proceed for 48 hours in a humidified incubator. The cells were then transferred to a new 96-well plate and the loss of membrane integrity of the target cells was monitored by the addition of propidium iodide (PI) at a final concentration of 1 μg / mL. PI is a membrane-impermeable pigment that is normally eliminated from living cells, but dead cells take it up and become identifiable by fluorescent radiation. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). Target cells were identified as DiO positive cells. PI-negative target cells were classified as living target cells. The percentage of cytotoxicity is calculated by<img id="000031" he="18" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Calculated according to.

0308GraphPad Prism 5 software (Graph Pad Software, San Diego) was used to plot the percentage of cytotoxicity against the concentration of the corresponding bispecific antibody. Dose response curves were analyzed using a 4-parameter logistic regression model for evaluation of sigmoid dose response curves with a constant hill gradient, and EC50 values were calculated.

0309<u style="single">Example B10</u>Elimination of cross-reactivity with BAFF receptor For flow cytometry, 200,000 cells from each cell line were incubated on ice for 30 minutes with 50 μl of purified bispecific molecule at a concentration of 5 μg / ml. Cells were washed twice with PBS containing 2% FCS and construct binding was detected with mouse PentaHis antibody (Qiagen; diluted 1:20 with 50 μl PBS containing 2% FCS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS containing 2% FCS. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). The bispecific conjugate was shown to be non-cross-reactive with the BAFF receptor.

0310<u style="single">Example B11</u>Cytotoxic activity The efficacy of human-like BCMA bispecific antibodies that redirect effector T cells to BCMA-expressing target cells is analyzed in five additional in vitro cytotoxicity assays: 1. The efficacy of BCMA bispecific antibodies that redirect stimulated human effector T cells to BCMA-positive (human) tumor cell lines is measured in a 51 chromium release assay. 2. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs to human BCMA-transfected CHO cells is measured in a FACS-based cytotoxicity assay. 3. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs to BCMA-positive (human) tumor cell lines is measured in a FACS-based cytotoxicity assay. 4. FACS-based cytotoxic assay using Makaku T cell lines as effector T cells to confirm that cross-reactive BCMA bispecific antibodies can redirect Makaku T cells to Makaku BCMA-transfected CHO cells. I do. Five. The potency gap between the monomeric and dimeric forms of the BCMA bispecific antibody was determined in a 51 chromium release assay using human BCMA-transfected CHO cells as target cells and human T cells stimulated as effector cells. To do.

0311<u style="single">Example C</u><u style="single">Example C1</u>Generation of CHO cells expressing chimeric BCMA To construct a chimeric epitope mapping molecule, the amino acid sequence or single amino acid residue of each epitope domain of human BCMA was converted to a mouse sequence. We constructed the following molecules:

0312-Human BCMA ECD / E1 mouse (SEQ ID NO: 1009) Chimeric extracellular BCMA domain: Epitope cluster 1 (SEQ ID NO: 1002 or 1007 amino acid residues 1-7) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 1-4). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 1-3 and mutation of G6Q in 1002 or 1007

0313-Human BCMA ECD / E2 mouse (SEQ ID NO: 1010) Chimeric extracellular BCMA domain: Epitope cluster 2 (SEQ ID NO: 1002 or 1007 amino acid residues 8-21) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 5-18). Human extracellular BCMA domain SEQ ID NO: Mutations of S9F, Q10H and N11S in 1002 or 1007

0314-Human BCMA ECD / E3 mouse (SEQ ID NO: 1011) Chimeric extracellular BCMA domain: Epitope cluster 3 (SEQ ID NO: 1002 or 1007 amino acid residues 24-41) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 21-36). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 31 and 32 at 1002 or 1007 and mutation of Q25H, S30N, L35A and R39P

0315-Human BCMA ECD / E4 mouse (SEQ ID NO: 1012) Chimeric extracellular BCMA domain: Epitope cluster 4 (SEQ ID NO: 1002 or 1007 amino acid residues 42-54) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 37-49). Human extracellular BCMA domain SEQ ID NO: Mutations of N42D, A43P, N47S, N53Y and A54T in 1002 or 1007

0316-Human BCMA ECD / E5 mouse (SEQ ID NO: 1013) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 22 (isoleucine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (lysine, position 19) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of I22K in 1002 or 1007

0317-Human BCMA ECD / E6 mouse (SEQ ID NO: 1014) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 25 (glutamine) at SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (histidine, position 22) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of Q25H in 1002 or 1007

0318-Human BCMA ECD / E7 mouse (SEQ ID NO: 1015) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 39 (arginine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (proline, position 34) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of R39P in 1002 or 1007.

0319The cDNA construct was cloned into the mammalian expression vector pEF-DHFR and stably transfected into CHO cells. Expression of human BCMA in CHO cells was verified in a FACS assay using a monoclonal anti-human BCMA antibody. Expression of mouse BCMA was demonstrated using a monoclonal anti-mouse BCMA antibody. The concentration of BCMA antibody used was 10 μg / ml in PBS / 2% FCS. Bound monoclonal antibody was detected using anti-rat IgG-Fcy-PE (1: 100 in PBS / 2% FCS; Jackson-Immuno-Research # 112-116-071). As a negative control, cells were incubated with PBS / 2% FCS in place of the first antibody. Sample the FACSCanto II instrument (Becton) Measured by flow cytometry in Dickinson) and analyzed by FlowJo software (version 7.6). Transfected CHO cells were analyzed and confirmed for surface expression of human-mouse BCMA chimeras in flow cytometry assays using various anti-BCMA antibodies (Fig. 2).

0320<u style="single">Example C2</u>2.1 Transient expression in HEK293 cells Clone expression plasmids with sequence-validated nucleotide sequences were used for transfection and protein expression in the FreeStyle 293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's protocol. A supernatant containing the expressed protein was obtained, cells were removed by centrifugation and the supernatant was stored at -20 ° C.

03212.2 Stable expression in CHO cells Clone expression plasmids with sequence-verified nucleotide sequences were transfected into DHFR-deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR-deficient CHO cells was performed as described in Kaufman RJ (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX. After two passages in static culture, cells contain nucleoside-free HyQ PF CHO liquid soybean medium (containing 4.0 mM L-glutamine, containing 0.1% Pluronic F-68; HyClone) for 7 days prior to collection. Growing in roller bottles. Cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -20 ° C.

0322<u style="single">Example C3</u>Epitope clustering of mouse scFv fragments Cells transfected with human or mouse BCMA or chimeric BCMA molecules were stained with a crude undiluted periplasmic extract containing scFv that binds to human / macaque BCMA. Bound scFv was detected with 1 μg / ml anti-FLAG antibody (Sigma F1804) and R-PE labeled anti-mouse Fcγ-specific antibody (1: 100; Dianova # 115-116-071). All antibodies were diluted with PBS containing 2% FCS. As a negative control, cells were incubated with PBS / 2% FCS instead of periplasmic extract. Samples were measured by flow cytometry on a FACSCanto II instrument (Becton Dickinson) and analyzed by FlowJo software (version 7.6).

0323<u style="single">Example C4</u>Obtaining different recombinant forms of soluble human and macaque BCMA Code sequences for human and rhesus monkey BCMA (published at GenBank, accession numbers NM_001192 [human], XM_001106892 [rhesus monkey]), human albumin, human Fcγ1 and mouse albumin coding sequences, human and macaque BCMA, respectively, and human albumin, human It was used to construct an artificial cDNA sequence encoding a soluble fusion protein of IgG1 Fc and mouse albumin, respectively, as well as a soluble protein containing only the extracellular domain of BCMA. To generate constructs for the expression of soluble human and macaque BCMA proteins, cDNA fragments were obtained by PCR mutagenesis and molecular cloning of the above full-length BCMA cDNA according to standard protocols. For fusions with human albumin, they first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, respectively. And rhesus monkey BCMA protein coding sequence, then in-frame coding sequence of artificial Ser1-Gly4-Ser1 linker, then in-frame coding sequence of human serum albumin, then in-frame coding sequence of Flag tag, then in-frame Frame-modified histidine tag<img id="000032" he="4" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For fusions with mouse IgG1, humans first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, followed by the coding sequence of the human IgG1 hinge and Fcγ moiety in frame, and then the hexahistidine tag code in frame. Modified cDNA fragments were designed to include sequences and stop codons. For fusions with mouse albumin, humans each contain amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, first the Kozak site for eukaryotic expression of the construct. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, then the coding sequence of mouse serum albumin in frame, then the coding sequence of the Flag tag in frame, and then in frame. Frame-modified histidine tag<img id="000033" he="4" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For soluble extracellular domain constructs, first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively, human and Macaque BCMA protein coding sequence, then in-frame artificial Ser1-Gly1 linker coding sequence, then in-frame Flag tag coding sequence, then in-frame modified histidine tag<img id="000034" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. The cDNA fragment was also designed to introduce restriction sites at the beginning and end of the fragment. The introduced restriction sites, EcoRI at the 5'end and SalI at the 3'end, were used in the cloning procedure below. The cDNA fragment was cloned through EcoRI and SalI into a plasmid called pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). All of the above steps were performed according to standard protocols (Sambrook, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)).

0324<u style="single">Example C5</u>Biacore-based determination of bispecific antibody affinity for human and macaque BCMA and CD3 Biacore analytical experiments were performed with recombinant BCMA fusion proteins containing human serum albumin (ALB) to determine target binding to BCMA. To measure the affinity for CD3, a recombinant fusion protein having the N-terminal 27 amino acids of CD3 epsilon (CD3e) fused to the Fc portion of the human antibody was used. This recombinant protein is available in human CD3e 1-27 and cynomolgus monkey CD3e versions, both of which carry the CD3 conjugate epitope in the bispecific antibody. More specifically, approximately 100-150 RU of each recombinant antigen was immobilized on a CM5 sensor chip (GE Healthcare) using acetate buffer pH 4.5 according to the manufacturer's manual. Bispecific antibody sample, HBS-EP electrophoresis buffer (GE) Five concentrations diluted with Healthcare): filled with 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.13 nM. The flow rate was 30-35 μl / min for 3 minutes, after which HBS-EP electrophoresis buffer was applied again at a flow rate of 30-35 μl / min for 8 minutes. Chip regeneration was performed with 10 mM glycine 0.5 M NaCl pH 2.45. The dataset was analyzed using BiaEval software. Usually, two independent experiments were performed.

0325<u style="single">Example C6</u>Flow cytometric analysis The functionality and binding strength of affinity-maturated scFv molecules were analyzed in FACS using human and macaque BCMA-transfected CHO cells. Briefly, about 10⁵The cells were incubated with 50 μl of a 1: 3 serial dilution of E. coli cell periplasmic extract for 50 minutes on ice. After washing with PBS / 10% FCS / 0.05% sodium azide, cells are combined with 30 μl Flag-M2 IgG (Sigma, 1: 900 in PBS / 10% FCS / 0.05% sodium azide) on ice for 40 minutes. Incubated. After the second wash, cells are incubated with 30 μl R-phycoerythrin (PE) -labeled goat anti-mouse IgG (Jackson ImmunoResearch, 1: 100 in PBS / 10% FCS / 0.05% sodium azide) for 40 minutes on ice. did. The cells were then washed again and resuspended in 200 μl PBS / 10% FCS / 0.05% sodium azide. Relative fluorescence of stained cells was measured using a FACSCanto flow cytometer (BD). The results are shown as a FACS histogram plotting the log-to-relative cell count of fluorescence intensity (see Figure C4).

0326<u style="single">Example C7</u>Bispecific binding and cross-reactivity For flow cytometry, 200,000 cells from each cell line were incubated on ice for 30 minutes with 50 μl of purified bispecific molecule at a concentration of 5 μg / ml. Cells were washed twice with 2% FCS-containing PBS and construct binding was detected with mouse PentaHis antibody (Qiagen; 1:20 dilution with 50 μl 2% FCS-containing PBS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS containing 2% FCS.

0327<u style="single">Example C8</u>Scatchard-based determination of bispecific antibody affinity for human and macaque BCMA In Scatchard analysis, saturated binding experiments are performed using a monovalent detection system (anti-His Fab / Alexa 488) developed by Micromet to accurately determine the monovalent binding of bispecific antibodies to each cell line. Each cell line (CHO cell line that recombinantly expresses human BCMA, CHO cell line that recombinantly expresses macaque BCMA) 2 x 10⁴Cells are incubated with 50 μl each of a triple dilution series (1: 2 8-step dilution) of each BCMA bispecific antibody starting at 100 nM, followed by incubation at 4 ° C for 16 hours with stirring, 1 Perform multiple residue cleaning steps. The cells are then incubated with 30 μl of anti-His Fab / Alexa 488 solution (Micromet; 30 μg / ml) for an additional 30 minutes. After one wash step, cells are resuspended in 150 μl FACS buffer containing 3.5% formaldehyde, incubated for an additional 15 minutes, centrifuged, resuspended in FACS buffer, and FACS Canto II machine and FACS. Analyze using Diva software. Data are obtained from two independent experimental sets. The values are plotted as a combined curve of hyperbolas. Calculations are performed to estimate the maximum binding (Bmax) from each Scatchard analysis. Determine the concentration of bispecific antibody at maximum half-dose binding that reflects each KD. Plot the values of the triple measurements as a hyperbola. The maximum binding is determined using the Scatchard evaluation and each KD is calculated.

0328<u style="single">Example C9</u>Cytotoxic activity 9.1 Chromium release assay with stimulated human T cells CD8⁺T cells enriched and stimulated for T cells were obtained as follows. Petri dishes (145 mm in diameter, Greiner bio-one GmbH, Kremsmunster) were coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) at a final concentration of 1 μg / ml at 37 ° C. for 1 hour. Unbound proteins were removed by a single wash step with PBS. 3-5 x 10 in 120 ml RPMI 1640 containing stabilized glutamine / 10% FCS / IL-2 20 U / ml (Proleukin®, Chiron)⁷Human PBMCs were added to pre-coated Petri dishes and stimulated for 2 days. On day 3, cells were harvested and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U / ml and the cells were again cultured in the same cell culture medium as above for 1 day. CD8⁺Cytotoxic T lymphocytes (CTL) CD4 using Dyna-Beads according to the manufacturer's protocol⁺T cells and CD56⁺Concentrated by depleting NK cells. Macaque or human BCMA-transfected CHO target cells were washed twice with PBS and 11.1 MBq in a final volume of 100 μl RPMI containing 50% FCS.⁵¹Labeled with Cr at 37 ° C for 60 minutes. The labeled target cells were then washed 3 times with 5 ml RPMI, which was then used in the cytotoxicity assay. The assay was performed in 96-well plates at a 10: 1 E: T ratio in supplemented RPMI with a total volume of 200 μl. Purified bispecific antibodies with a starting concentration of 0.01-1 μg / ml and 3-fold dilutions thereof were used. The incubation time in the assay was 18 hours. Cytotoxicity was determined as the relative value of chromium released in the supernatant relative to the difference between maximal lysis (with Triton-X added) and spontaneous lysis (without effector cells). All measurements were made in quadruples. Chromium activity in the supernatant was measured at the Wizard 3''γ counter (Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of the results is based on Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, California, USA) was used. EC50 values calculated from the sigmoid dose response curve by the analytical program were used to compare cytotoxic activity.

03299.2 FACS-based cytotoxic assay with unstimulated human PBMC Isolation of effector cells Human peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient centrifugation from concentrated lymphocyte preparations (soft membranes), a by-product of blood collected by blood banks for transfusion. The buffy coat was provided by a domestic blood bank and PBMCs were prepared on the same day as blood collection. After thorough Ficoll density centrifugation and thorough washing with Dulbecco's PBS (Gibco), the remaining erythrocytes are removed with erythrocyte lysis buffer (155 mM NH).₄Cl, 10mM KHCO₃, 100 μM EDTA) removed from PBMC. Platelets were removed through the supernatant by centrifugation at 100 xg of PBMC. The remaining lymphocytes mainly contain B and T lymphocytes, NK cells and monocytes. PBMC in RPMI medium (Gibco) containing 10% FCS (Gibco) at 37 ° C / 5% CO₂It was maintained by the culture below.

0330CD14⁺And CD56⁺Cell depletion CD14⁺Human CD14 microbeads (Milteny Biotec, MACS, # 130-050-201) were used to deplete cells, and human CD56 microbeads (MACS, # 130-050-401) were used to deplete NK cells. It was used. PBMCs were counted and centrifuged at 300 xg for 10 minutes at room temperature. Discard the supernatant and dispose of the cell pellet in MACS isolation buffer [80 μL / 10⁷Cells; resuspended in PBS (Invitrogen, # 20012-043), 0.5% (v / v) FBS (Gibco, # 10270-106), 2 mM EDTA (Sigma-Aldrich, # E-6511)]. CD14 microbeads and CD56 microbeads (20 μL / 10)⁷Cells) were added and incubated at 4-8 ° C for 15 minutes. MACS isolation buffer (1-2 mL / 10) for cells⁷Washed with cells). After centrifugation (see above), discard the supernatant and isolate cells in MACS isolation buffer (500 μL / 10).⁸Resuspended in cells). Next, CD14 / CD56 negative cells were isolated using an LS column (Miltenyi Biotec, # 130-042-401). PBMC without CD14 + / CD56 + cells in RPMI complete medium, ie, 10% FBS (Biochrom AG, # S0115), 1 x non-essential amino acids (Biochrom AG, # K0293), in an incubator at 37 ° C until required. RPMI1640 (Biochrom AG, # FG1215) supplemented with 10 mM Hepes buffer (Biochrom AG, # L1613), 1 mM sodium pyruvate (Biochrom AG, # L0473) and 100 U / mL penicillin / streptomycin (Biochrom AG, # A2213). Cultured in.

0331Target cell labeling Fluorescent membrane dye DiOC for analysis of cytolysis in flow cytometry assays₁₈(DiO) (Molecular Probes, # V22886) was used to label human BCMA or macaque BCMA-transfected CHO cells as target cells so that they could be distinguished from effector cells. Briefly, cells are collected, washed once with PBS, and 2% (v / v) FBS and membrane dye DiO (5 μL / 10).⁶10 in PBS containing cells)⁶Adjusted to cells / mL. After incubation at 37 ° C for 3 minutes, the cells were washed twice in complete RPMI medium and the cell number was 1.25 x 10⁵Adjusted to cells / mL. Cell viability was determined using 0.5% (v / v) isotonic Eosin G solution (Roth, # 45380).

0332Flow cytometry-based analysis This assay was designed to quantify lysis of macaque or human BCMA-transfected CHO cells in the presence of serial dilutions of BCMA bispecific antibody. Equal amounts of DiO-labeled target cells and effector cells (ie, CD14)⁺Cell-free PBMCs) were mixed to give an E: T cell ratio of 10: 1. 160 μL of this suspension was transferred to each well of a 96-well plate. 40 μL of serial dilutions of BCMA bispecific antibody and negative control bispecific (CD3-based bispecific antibody recognizing unrelated target antigens) or RPMI complete medium as an additional negative control was added. .. Bispecific antibody-mediated cytotoxic response is 7% CO₂It was allowed to proceed for 48 hours in a humidified incubator. The cells were then transferred to a new 96-well plate and the loss of membrane integrity of the target cells was monitored by the addition of propidium iodide (PI) at a final concentration of 1 μg / mL. PI is a membrane-impermeable pigment that is normally eliminated from living cells, but dead cells take it up and become identifiable by fluorescent radiation. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). Target cells were identified as DiO positive cells. PI-negative target cells were classified as living target cells. The percentage of cytotoxicity is calculated by<img id="000035" he="18" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Calculated according to.

0333GraphPad Prism 5 software (Graph Pad Software, San Diego) was used to plot the percentage of cytotoxicity against the concentration of the corresponding bispecific antibody. Dose response curves were analyzed using a 4-parameter logistic regression model for evaluation of sigmoid dose response curves with a constant hill gradient, and EC50 values were calculated.

0334<u style="single">Example C10</u>Elimination of cross-reactivity with BAFF receptor For flow cytometry, 200,000 cells from each cell line were incubated on ice for 30 minutes with 50 μl of purified bispecific molecule at a concentration of 5 μg / ml. Cells were washed twice with PBS containing 2% FCS and construct binding was detected with mouse PentaHis antibody (Qiagen; diluted 1:20 with 50 μl PBS containing 2% FCS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS containing 2% FCS. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). The bispecific conjugate was shown to be non-cross-reactive with the BAFF receptor.

0335<u style="single">Example C11</u>Cytotoxic activity The efficacy of human-like BCMA bispecific antibodies that redirect effector T cells to BCMA-expressing target cells is analyzed in five additional in vitro cytotoxicity assays: 1. The efficacy of BCMA bispecific antibodies that redirect stimulated human effector T cells to BCMA-positive (human) tumor cell lines is measured in a 51 chromium release assay. 2. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs to human BCMA-transfected CHO cells is measured in a FACS-based cytotoxicity assay. 3. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs against BCMA-positive (human) tumor cell lines is measured in a FACS-based cytotoxicity assay. 4. FACS-based cytotoxic assay using Makaku T cell lines as effector T cells to confirm that cross-reactive BCMA bispecific antibodies can redirect Makaku T cells to Makaku BCMA-transfected CHO cells. I do. Five. The potency gap between the monomeric and dimeric forms of the BCMA bispecific antibody was determined in a 51 chromium release assay using human BCMA-transfected CHO cells as target cells and human T cells stimulated as effector cells. To do.

0336<u style="single">Example D</u><u style="single">Example D1</u>Generation of CHO cells expressing chimeric BCMA To construct a chimeric epitope mapping molecule, the amino acid sequence or single amino acid residue of each epitope domain of human BCMA was converted to a mouse sequence. We constructed the following molecules:

0337-Human BCMA ECD / E1 mouse (SEQ ID NO: 1009) Chimeric extracellular BCMA domain: Epitope cluster 1 (SEQ ID NO: 1002 or 1007 amino acid residues 1-7) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 1-4). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 1-3 and mutation of G6Q in 1002 or 1007

0338-Human BCMA ECD / E2 mouse (SEQ ID NO: 1010) Chimeric extracellular BCMA domain: Epitope cluster 2 (SEQ ID NO: 1002 or 1007 amino acid residues 8-21) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 5-18). Human extracellular BCMA domain SEQ ID NO: Mutations of S9F, Q10H and N11S in 1002 or 1007

0339-Human BCMA ECD / E3 mouse (SEQ ID NO: 1011) Chimeric extracellular BCMA domain: Epitope cluster 3 (SEQ ID NO: 1002 or 1007 amino acid residues 24-41) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 21-36). Human extracellular BCMA domain SEQ ID NO: Deletion of amino acid residues 31 and 32 at 1002 or 1007 and mutation of Q25H, S30N, L35A and R39P

0340-Human BCMA ECD / E4 mouse (SEQ ID NO: 1012) Chimeric extracellular BCMA domain: Epitope cluster 4 (SEQ ID NO: 1002 or 1007 amino acid residues 42-54) was replaced by each mouse cluster (SEQ ID NO: 1004 or 1008 amino acid residues 37-49). Human extracellular BCMA domain SEQ ID NO: Mutations of N42D, A43P, N47S, N53Y and A54T in 1002 or 1007

0341-Human BCMA ECD / E5 mouse (SEQ ID NO: 1013) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 22 (isoleucine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (lysine, position 19) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of I22K in 1002 or 1007

0342-Human BCMA ECD / E6 mouse (SEQ ID NO: 1014) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 25 (glutamine) at SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (histidine, position 22) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of Q25H in 1002 or 1007

0343-Human BCMA ECD / E7 mouse (SEQ ID NO: 1015) Chimeric extracellular BCMA domain: Human cells in which the amino acid residue at position 39 (arginine) of SEQ ID NO: 1002 or 1007 is replaced with the respective mouse amino acid residue (proline, position 34) at SEQ ID NO: 1004 or 1008. Outer BCMA domain SEQ ID NO: Mutation of R39P in 1002 or 1007.

0344The cDNA construct was cloned into the mammalian expression vector pEF-DHFR and stably transfected into CHO cells. Expression of human BCMA in CHO cells was verified in a FACS assay using a monoclonal anti-human BCMA antibody. Expression of mouse BCMA was demonstrated using a monoclonal anti-mouse BCMA antibody. The concentration of BCMA antibody used was 10 μg / ml in PBS / 2% FCS. Bound monoclonal antibody was detected using anti-rat IgG-Fcy-PE (1: 100 in PBS / 2% FCS; Jackson-Immuno-Research # 112-116-071). As a negative control, cells were incubated with PBS / 2% FCS in place of the first antibody. Sample the FACSCanto II instrument (Becton) Measured by flow cytometry in Dickinson) and analyzed by FlowJo software (version 7.6). Transfected CHO cells were analyzed and confirmed for surface expression of human-mouse BCMA chimeras in flow cytometry assays using various anti-BCMA antibodies (Fig. 2).

0345<u style="single">Example D2</u>2.1 Transient expression in HEK293 cells Clone expression plasmids with sequence-validated nucleotide sequences were used for transfection and protein expression in the FreeStyle 293 expression system (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer's protocol. A supernatant containing the expressed protein was obtained, cells were removed by centrifugation and the supernatant was stored at -20 ° C.

03462.2 Stable expression in CHO cells Clone expression plasmids with sequence-verified nucleotide sequences were transfected into DHFR-deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR-deficient CHO cells was performed as described in Kaufman RJ (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing the concentration of methotrexate (MTX) to a final concentration of 20 nM MTX. After two passages in static culture, cells contain nucleoside-free HyQ PF CHO liquid soybean medium (containing 4.0 mM L-glutamine, containing 0.1% Pluronic F-68; HyClone) for 7 days prior to collection. Growing in roller bottles. Cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -20 ° C.

0347<u style="single">Example D3</u>Epitope clustering of mouse scFv fragments Cells transfected with human or mouse BCMA or chimeric BCMA molecules were stained with a crude undiluted periplasmic extract containing scFv that binds to human / macaque BCMA. Bound scFv was detected with 1 μg / ml anti-FLAG antibody (Sigma F1804) and R-PE labeled anti-mouse Fcγ-specific antibody (1: 100; Dianova # 115-116-071). All antibodies were diluted with PBS containing 2% FCS. As a negative control, cells were incubated with PBS / 2% FCS instead of periplasmic extract. Samples were measured by flow cytometry on a FACSCanto II instrument (Becton Dickinson) and analyzed by FlowJo software (version 7.6).

0348<u style="single">Example D4</u>Obtaining different recombinant forms of soluble human and macaque BCMA Code sequences for human and rhesus monkey BCMA (published at GenBank, accession numbers NM_001192 [human], XM_001106892 [rhesus monkey]), human albumin, human Fcγ1 and mouse albumin coding sequences, human and macaque BCMA, respectively, and human albumin, human It was used to construct an artificial cDNA sequence encoding a soluble fusion protein of IgG1 Fc and mouse albumin, respectively, as well as a soluble protein containing only the extracellular domain of BCMA. To generate constructs for the expression of soluble human and macaque BCMA proteins, cDNA fragments were obtained by PCR mutagenesis and molecular cloning of the above full-length BCMA cDNA according to standard protocols. For fusions with human albumin, they first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, respectively. And rhesus monkey BCMA protein coding sequence, then in-frame coding sequence of artificial Ser1-Gly4-Ser1 linker, then in-frame coding sequence of human serum albumin, then in-frame coding sequence of Flag tag, then in-frame Frame-modified histidine tag<img id="000036" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For fusions with mouse IgG1, humans first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, followed by the coding sequence of the human IgG1 hinge and Fcγ moiety in frame, and then the hexahistidine tag code in frame. Modified cDNA fragments were designed to include sequences and stop codons. For fusions with mouse albumin, humans each contain amino acids 1-54 and 1-53, respectively, corresponding to the extracellular domains of human and rhesus BCMA, first the Kozak site for eukaryotic expression of the construct. And the coding sequence of the macaque BCMA protein, followed by the coding sequence of the artificial Ser1-Gly4-Ser1 linker in frame, then the coding sequence of mouse serum albumin in frame, then the coding sequence of the Flag tag in frame, and then in frame. Frame-modified histidine tag<img id="000037" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. For soluble extracellular domain constructs, first contain the Kozak site for eukaryotic expression of the construct, followed by amino acids 1-54 and 1-53 corresponding to the extracellular domains of human and rhesus BCMA, respectively, human and Macaque BCMA protein coding sequence, then in-frame artificial Ser1-Gly1 linker coding sequence, then in-frame Flag tag coding sequence, then in-frame modified histidine tag<img id="000038" he="5" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />The modified cDNA fragment was designed to contain the coding sequence and stop codon of. The cDNA fragment was also designed to introduce restriction sites at the beginning and end of the fragment. The introduced restriction sites, EcoRI at the 5'end and SalI at the 3'end, were used in the cloning procedure below. The cDNA fragment was cloned through EcoRI and SalI into a plasmid called pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). All of the above steps were performed according to standard protocols (Sambrook, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)).

0349<u style="single">Example D5</u>Biacore-based determination of bispecific antibody affinity for human and macaque BCMA and CD3 Biacore analytical experiments were performed with recombinant BCMA fusion proteins containing human serum albumin (ALB) to determine target binding to BCMA. To measure the affinity for CD3, a recombinant fusion protein having the N-terminal 27 amino acids of CD3 epsilon (CD3e) fused to the Fc portion of the human antibody was used. This recombinant protein is available in human CD3e 1-27 and cynomolgus monkey CD3e versions, both of which carry the CD3 conjugate epitope in the bispecific antibody. More specifically, approximately 100-150 RU of each recombinant antigen was immobilized on a CM5 sensor chip (GE Healthcare) using acetate buffer pH 4.5 according to the manufacturer's manual. Bispecific antibody sample, HBS-EP electrophoresis buffer (GE) Five concentrations diluted with Healthcare): filled with 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.13 nM. The flow rate was 30-35 μl / min for 3 minutes, after which HBS-EP electrophoresis buffer was applied again at a flow rate of 30-35 μl / min for 8 minutes. Chip regeneration was performed with 10 mM glycine 0.5 M NaCl pH 2.45. The dataset was analyzed using BiaEval software (see Figure D4). Usually, two independent experiments were performed.

0350<u style="single">Example D6</u>Bispecific binding and cross-reactivity For flow cytometry, 200,000 cells from each cell line were incubated on ice for 30 minutes with 50 μl of purified bispecific molecule at a concentration of 5 μg / ml. Cells were washed twice with 2% FCS-containing PBS and construct binding was detected with mouse PentaHis antibody (Qiagen; 1:20 dilution with 50 μl 2% FCS-containing PBS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS containing 2% FCS.

0351<u style="single">Example D7</u>Scatchard-based determination of bispecific antibody affinity for human and macaque BCMA In Scatchard analysis, saturated binding experiments are performed using a monovalent detection system (anti-His Fab / Alexa 488) developed by Micromet to accurately determine the monovalent binding of bispecific antibodies to each cell line. Each cell line (CHO cell line that recombinantly expresses human BCMA, CHO cell line that recombinantly expresses macaque BCMA) 2 x 10⁴Cells are incubated with 50 μl each of a triple dilution series (1: 2 8-step dilution) of each BCMA bispecific antibody starting at 100 nM, followed by incubation at 4 ° C for 16 hours with stirring, 1 Perform multiple residue cleaning steps. The cells are then incubated with 30 μl of anti-His Fab / Alexa 488 solution (Micromet; 30 μg / ml) for an additional 30 minutes. After one wash step, cells were resuspended in FACS buffer 150μl containing 3.5% formaldehyde, incubator for an additional 15 minutes and Bate, centrifuged, resuspended in FACS buffer and FACS Canto II machine and Analyze using FACS Diva software. Data are obtained from two independent experimental sets. The values are plotted as a combined curve of hyperbolas. Calculations are performed to estimate the maximum binding (Bmax) from each Scatchard analysis. Determine the concentration of bispecific antibody at maximum half-dose binding that reflects each KD. Plot the values of the triple measurements as a hyperbola. The maximum binding is determined using the Scatchard evaluation and each KD is calculated.

0352<u style="single">Example D8</u>Cytotoxic activity 8.1 Chromium release assay with stimulated human T cells CD8⁺T cells enriched and stimulated for T cells were obtained as follows. Petri dishes (145 mm in diameter, Greiner bio-one GmbH, Kremsmunster) were coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) at a final concentration of 1 μg / ml at 37 ° C. for 1 hour. Unbound proteins were removed by a single wash step with PBS. 3-5 x 10 in 120 ml RPMI 1640 containing stabilized glutamine / 10% FCS / IL-2 20 U / ml (Proleukin®, Chiron)⁷Human PBMCs were added to pre-coated Petri dishes and stimulated for 2 days. On day 3, cells were harvested and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U / ml and the cells were again cultured in the same cell culture medium as above for 1 day. CD8⁺Cytotoxic T lymphocytes (CTL) CD4 using Dyna-Beads according to the manufacturer's protocol⁺T cells and CD56⁺Concentrated by depleting NK cells. Macaque or human BCMA-transfected CHO target cells were washed twice with PBS and 11.1 MBq in a final volume of 100 μl RPMI containing 50% FCS.⁵¹Labeled with Cr at 37 ° C for 60 minutes. The labeled target cells were then washed 3 times with 5 ml RPMI, which was then used in the cytotoxicity assay. The assay was performed in 96-well plates at a 10: 1 E: T ratio in supplemented RPMI with a total volume of 200 μl. Purified bispecific antibodies with a starting concentration of 0.01-1 μg / ml and 3-fold dilutions thereof were used. The incubation time in the assay was 18 hours. Cytotoxicity was determined as the relative value of chromium released in the supernatant relative to the difference between maximal lysis (with Triton-X added) and spontaneous lysis (without effector cells). All measurements were made in quadruples. Chromium activity in the supernatant was measured at the Wizard 3''γ counter (Perkin Elmer Life Sciences GmbH, Koln, Germany). Analysis of the results is based on Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, California, USA) was used. EC50 values calculated from the sigmoid dose response curve by the analytical program were used to compare cytotoxic activity (see Figure D5).

03538.2 FACS-based cytotoxic assay with unstimulated human PBMC Isolation of effector cells Human peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient centrifugation from concentrated lymphocyte preparations (soft membranes), a by-product of blood collected by blood banks for transfusion. The buffy coat was provided by a domestic blood bank and PBMCs were prepared on the same day as blood collection. After thorough Ficoll density centrifugation and thorough washing with Dulbecco's PBS (Gibco), the remaining erythrocytes are removed with erythrocyte lysis buffer (155 mM NH).₄Cl, 10mM KHCO₃, 100 μM EDTA) removed from PBMC. Platelets were removed through the supernatant by centrifugation at 100 xg of PBMC. The remaining lymphocytes mainly contain B and T lymphocytes, NK cells and monocytes. PBMC in RPMI medium (Gibco) containing 10% FCS (Gibco) at 37 ° C / 5% CO₂It was maintained by the culture below.

0354CD14⁺And CD56⁺Cell depletion CD14⁺Human CD14 microbeads (Milteny Biotec, MACS, # 130-050-201) were used to deplete cells, and human CD56 microbeads (MACS, # 130-050-401) were used to deplete NK cells. It was used. PBMCs were counted and centrifuged at 300 xg for 10 minutes at room temperature. Discard the supernatant and dispose of the cell pellet in MACS isolation buffer [80 μL / 10⁷Cells; resuspended in PBS (Invitrogen, # 20012-043), 0.5% (v / v) FBS (Gibco, # 10270-106), 2 mM EDTA (Sigma-Aldrich, # E-6511)]. CD14 microbeads and CD56 microbeads (20 μL / 10)⁷Cells) were added and incubated at 4-8 ° C for 15 minutes. MACS isolation buffer (1-2 mL / 10) for cells⁷Washed with cells). After centrifugation (see above), discard the supernatant and isolate cells in MACS isolation buffer (500 μL / 10).⁸Resuspended in cells). Next, CD14 / CD56 negative cells were isolated using an LS column (Miltenyi Biotec, # 130-042-401). PBMC without CD14 + / CD56 + cells in RPMI complete medium, ie, 10% FBS (Biochrom AG, # S0115), 1 x non-essential amino acids (Biochrom AG, # K0293), in an incubator at 37 ° C until required. RPMI1640 (Biochrom AG, # FG1215) supplemented with 10 mM Hepes buffer (Biochrom AG, # L1613), 1 mM sodium pyruvate (Biochrom AG, # L0473) and 100 U / mL penicillin / streptomycin (Biochrom AG, # A2213). Cultured in.

0355Target cell labeling Fluorescent membrane dye DiOC for analysis of cytolysis in flow cytometry assays₁₈(DiO) (Molecular Probes, # V22886) was used to label human BCMA or macaque BCMA-transfected CHO cells as target cells so that they could be distinguished from effector cells. Briefly, cells are collected, washed once with PBS, and 2% (v / v) FBS and membrane dye DiO (5 μL / 10).⁶10 in PBS containing cells)⁶Adjusted to cells / mL. After incubation at 37 ° C for 3 minutes, the cells were washed twice in complete RPMI medium and the cell number was 1.25 x 10⁵Adjusted to cells / mL. Cell viability was determined using 0.5% (v / v) isotonic Eosin G solution (Roth, # 45380).

0356Flow cytometry-based analysis This assay was designed to quantify lysis of macaque or human BCMA-transfected CHO cells in the presence of serial dilutions of BCMA bispecific antibody. Equal amounts of DiO-labeled target cells and effector cells (ie, CD14)⁺Cell-free PBMCs) were mixed to give an E: T cell ratio of 10: 1. 160 μL of this suspension was transferred to each well of a 96-well plate. 40 μL of serial dilutions of BCMA bispecific antibody and negative control bispecific (CD3-based bispecific antibody recognizing unrelated target antigens) or RPMI complete medium as an additional negative control was added. .. Bispecific antibody-mediated cytotoxic response is 7% CO₂It was allowed to proceed for 48 hours in a humidified incubator. The cells were then transferred to a new 96-well plate and the loss of membrane integrity of the target cells was monitored by the addition of propidium iodide (PI) at a final concentration of 1 μg / mL. PI is a membrane-impermeable pigment that is normally eliminated from living cells, but dead cells take it up and become identifiable by fluorescent radiation. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). Target cells were identified as DiO positive cells. PI-negative target cells were classified as living target cells. The percentage of cytotoxicity is calculated by<img id="000039" he="18" wi="128" file="JP6231007B2_D0001.tif" img-format="tif" img-content="drawing" />Calculated according to.

0357GraphPad Prism 5 software (Graph Pad Software, San Diego) was used to plot the percentage of cytotoxicity against the concentration of the corresponding bispecific antibody. Dose response curves were analyzed using a 4-parameter logistic regression model for evaluation of sigmoid dose response curves with a constant hill gradient, and EC50 values were calculated.

0358<u style="single">Example D9</u>Elimination of cross-reactivity with BAFF receptor For flow cytometry, 200,000 cells from each cell line were incubated on ice for 30 minutes with 50 μl of purified bispecific molecule at a concentration of 5 μg / ml. Cells were washed twice with PBS containing 2% FCS and construct binding was detected with mouse PentaHis antibody (Qiagen; diluted 1:20 with 50 μl PBS containing 2% FCS). After washing, bound PentaHis antibody was detected with phycoerythrin-conjugated Fcγ-specific antibody (Dianova) diluted 1: 100 with PBS containing 2% FCS. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). The bispecific conjugate was shown to be non-cross-reactive with the BAFF receptor.

0359<u style="single">Example D10</u>Cytotoxic activity The efficacy of human-like BCMA bispecific antibodies that redirect effector T cells to BCMA-expressing target cells is analyzed in five additional in vitro cytotoxicity assays: 1. The efficacy of BCMA bispecific antibodies that redirect stimulated human effector T cells to BCMA-positive (human) tumor cell lines is measured in a 51 chromium release assay. 2. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs to human BCMA-transfected CHO cells is measured in a FACS-based cytotoxicity assay. 3. The efficacy of BCMA bispecific antibodies that redirect T cells in unstimulated human PBMCs to BCMA-positive (human) tumor cell lines is measured in a FACS-based cytotoxicity assay. 4. FACS-based cytotoxic assay using Makaku T cell lines as effector T cells to confirm that cross-reactive BCMA bispecific antibodies can redirect Makaku T cells to Makaku BCMA-transfected CHO cells. I do. Five. The potency gap between the monomeric and dimeric forms of the BCMA bispecific antibody was determined in a 51 chromium release assay using human BCMA-transfected CHO cells as target cells and human T cells stimulated as effector cells. To do.

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