Multiple target antigen-binding molecules for use in proliferative disorders

Abstract

The present invention provides a bispecific antigen-binding molecule characterized by comprising first and second domains that bind to any of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, or EpCAM, respectively, a third domain that binds to an extracellular epitope of the human and macaque CD3 epsilon chain, and optionally, an Fc-type fourth domain. Furthermore, the present invention provides a polynucleotide encoding this antigen-binding molecule, a vector comprising this polynucleotide, a host cell expressing this construct, and a pharmaceutical composition comprising the same.

Description

[Technical Field]

[0001] This invention relates to biotechnology products and methods, particularly to multi-target antigen-binding molecules, formulations thereof, and uses thereof. [Background technology]

[0002] Compared to small molecule chemical agents, protein-based drugs exhibit high specificity and activity at relatively low concentrations and typically provide therapies for various diseases with significant impact, such as cancers, autoimmune diseases, and metabolic disorders (Roberts, Trends Biotechnol. 2014 Jul;32(7):372-80, Wang, Int J Pharm. 1999 Aug 20;185(2):129-88).

[0003] Such novel protein-based drugs typically include bispecific (monoclonal) antibodies that can simultaneously bind to two different types of antigens. These are known in several structural forms, and their applications in cancer immunotherapy and drug delivery are currently being explored (Fan, Gaowei; Wang, Zujian; Hao, Mingju; Li, Jinming (2015). “Bispecific antibodies and their applications”. Journal of Hematology & Oncology. 8:130).

[0004] Bispecific molecules useful in cancer immunotherapy can be antigen-binding polypeptides such as antibodies, and may be, for example, IgG-like bispecific antibodies (i.e., full-length bispecific antibodies) or non-IgG-like bispecific antibodies that are not full-length antigen-binding molecules. Full-length bispecific antibodies usually retain the normal monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except that the two Fab sites bind to different antigens. Non-full-length bispecific antibodies may completely lack an Fc region. These include chemically linked Fabs consisting only of Fab regions, as well as various types of bivalent and trivalent single-stranded variable fragments (scFv). There are also fusion proteins that mimic the variable domains of two antibodies. An example of such a form is the bispecific T cell engager (BiTE®) (Yang, Fa; Wen, Weihong; Qin, Weijun (2016). “Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies”. International Journal of Molecular Sciences. 18(1):48).

[0005] Exemplary bispecific antibody-derived molecules, such as the BiTE® molecule, are recombinant protein constructs composed of binding domains derived from two flexibly linked antibodies. One binding domain of the BiTE® molecule is specific to a selected tumor-associated surface antigen on target cells, and the second binding domain is specific to CD3, a subunit of the T cell receptor complex on T cells. Due to their special design, BiTE® antigen-binding molecules are uniquely suited to transiently binding T cells to target cells while simultaneously potently activating the intrinsic cytolytic ability of T cells against target cells. Significant further developments of the first-generation BiTE® molecules, deployed in clinics as AMG103 and AMG110 (see International Publication No. 99 / 54440 and International Publication No. 2005 / 040220), provided bispecific antigen-binding molecules that bind to a context-independent epitope at the N-terminus of the CD3 ε chain (see International Publication No. 2008 / 119567). The BiTE® molecule that binds to this selected epitope not only does not exhibit interspecies specificity for human and macaque or marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), or squirrel monkey (Saimiri sciureus) CD3ε chains, but also recognizes this specific epitope (instead of the CD3 conjugate epitopes previously described in bispecific T cell engagement molecules), thus not exhibiting the same degree of nonspecific T cell activation as observed with previous generation T cell engagement antibodies. This reduced T cell activation is associated with less or decreased T cell redistribution in patients, the latter of which has been judged to be a risk of adverse effects, for example, with pasotuximab.

[0006] The antibody-based molecules described in International Publication No. 2008 / 119567 are characterized by rapid clearance from the body; therefore, they can rapidly reach most parts of the body, however their in vivo application may be limited by their short persistence in vivo. On the other hand, their concentrations in the body can be immediately adapted and fine-tuned. Due to the short in vivo half-life of these small single-chain molecules, continuous administration by continuous intravenous infusion is used to achieve therapeutic effects. However, as described in International Publication No. 2017 / 134140, bispecific antigen-binding molecules with more advantageous pharmacokinetic properties, including longer half-lives, are available. Longer half-lives are typically useful in the in vivo application of immunoglobulins, particularly with respect to small antibody fragments or constructs, for example, for patient compliance.

[0007] One of the ongoing challenges in antibody-based cancer immunotherapy is tumor evasion. Such tumor evasion occurs when the immune system fails to adequately eradicate tumors that have accumulated genetic and epigenetic modifications and use several mechanisms to ensure the victory of the immune editing process, even when the immune system is induced or triggered by some antibody-based immunotherapy (Keshavarz-Fathi, Mahsa; Rezaei, Nima (2019) “Vaccines for Cancer Immunotherapy”). Generally, four mechanisms are known to hinder an effective anti-tumor immune response: (1) impaired processing or presentation of tumor antigens, (2) lack of activation mechanisms, (3) suppression mechanisms and immunosuppressive states, and (4) resistant tumor cells. In particular, with respect to the first mechanism, tumor antigens may exist in new forms due to genetic instability, tumor mutations, and immune system evasion. Epitope-negative tumor cells remain hidden and, as a result, exhibit resistance to immune rejection. These are expressed following the removal of epitope-positive tumor cells, similar to Darwin's theory of natural selection. As a result, antibody-based immunotherapy against antigens on tumor cells becomes ineffective when such tumor cells no longer express the respective antigen due to tumor evasion. The aforementioned antigen deficiency is understood herein as the driving force behind tumor evasion and is therefore used interchangeably. Accordingly, there is a need to provide improved antibody-based cancer immunotherapy that addresses the problem of antigen deficiency in order to effectively prevent tumor evasion.

[0008] Furthermore, despite the preclinical and clinical successes achieved so far with antibody-based immunotherapy, significant limitations remain, such as inter-individual and inter-cancer type variability in responses. Not all patients respond to treatment at available safe doses, because dose-limiting toxicity can limit the effectiveness of antibody-based immunotherapy. Therefore, it is also necessary to reduce dose-limiting toxicity in antibody-based immunotherapy to make such therapies available to a wider range of patients suffering from diverse proliferative disorders.

[0009] Another challenge to the widespread use of T cell engagement bispecific molecules in cancer immunotherapy is the availability of appropriate targets (Bacac et al., Clin Cancer Res; 22(13) July 1, 2016). For example, solid tumor targets may be overexpressed on tumor cells, but may be expressed at lower but significant levels on non-malignant primary cells in important tissues. In nature, according to Bacac et al., T cells can distinguish between cells with high antigen expression and those with low antigen expression by T cell receptors (TCRs) that can achieve high affinity binding to target cells that express relatively low but still sufficiently high levels of the target antigen. Therefore, T cell engagement bispecific molecules that can facilitate the same thing and thus maximize the window between the death of cells with high target expression and the death of cells with low target expression are highly desirable. One approach considered in the art is to improve target selectivity compared to normal tissues that express only one or only low levels of both target antigens by using bitargeting of two antigens on the same cell. This effect is thought to depend on the binding activity mediated by the simultaneous binding of bsAb to both antigens on the same cell. Regarding such dual targeting, several multispecific monoclonal antibodies (mAbs) or other immune constructs are known in the art. International Publication No. 2014 / 116846 teaches a multispecific binding protein comprising a first binding site that specifically binds to a target cell antigen, a second binding site that specifically binds to a cell surface receptor on an immune cell, and a third binding site that specifically binds to a cell surface regulator on an immune cell. U.S. Patent Application Publication No. 2017 / 0022274 discloses a trivalent T-cell redirecting complex comprising a bispecific antibody having two binding sites for tumor-associated antigens (TAAs) and one binding site for T cells.In the art, various multispecific antibodies or antibody fragments are known, some of which target T cells, but no multitarget bispecific molecules have been proposed that employ the mechanism of a (preferably single-stranded) bispecific T cell engagement molecule to address the need to overcome antigen deficiency / tumor avoidance and reduce dose-limiting toxicity in antibody-based immunotherapy, while simultaneously addressing the need to effectively redirect T cells in a single stable, ready-to-use therapeutic system. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 99 / 54440 [Patent Document 2] International Publication No. 2005 / 040220 [Patent Document 3] International Publication No. 2008 / 119567 [Patent Document 4] International Publication No. 2017 / 134140 [Patent Document 5] International Publication No. 2014 / 116846 [Patent Document 6] U.S. Patent Application Publication No. 2017 / 0022274 [Non-patent literature]

[0011] [Non-Patent Document 1] Roberts,Trends Biotechnol.2014 Jul;32(7):372-80 [Non-Patent Document 2] Wang,Int J Pharm.1999 Aug 20;185(2):129-88 [Non-Patent Document 3] Fan,Gaowei;Wang,Zujian;Hao,Mingju;Li,Jinming(2015).“Bispecific antibodies and their applications”.Journal of Hematology&Oncology.8:130 [Non-Patent Document 4] Yang, Fa; Wen, Weihong; Qin, Weijun (2016). “Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies”. International Journal of Molecular Sciences. 18(1):48 [Non-Patent Document 5] Keshavarz-Fathi, Mahsa; Rezaei, Nima (2019) “Vaccines for Cancer Immunotherapy” [Non-Patent Document 6] Bacac et al., Clin Cancer Res;22(13)July 1,2016 [Overview of the project] [Problems that the invention aims to solve]

[0012] In consideration of the needs described above, the object of the present invention is to provide a multi-targeted antigen-binding molecule (typically a polypeptide, e.g., a T-cell engagement bispecific molecule) particularly suitable for use in the treatment of a specific condition, by binding to two antigens on target cells associated with the condition and simultaneously to one antigen on effector cells. Accordingly, the present invention provides a multi-targeted bispecific antigen-binding molecule comprising a first domain that binds to a target cell surface antigen (e.g., a first TAA), a second domain that binds to the same or preferably different target cell surface antigen (e.g., a second TAA), a third domain that binds to an extracellular epitope of human and non-human (e.g., macaque) CD3ε chains, and a fourth domain that is preferably a specific Fc mode that modulates the half-life of the molecule. Preferably, these domains are binding domains composed of VH and VL domains in the amino-to-carboxyl direction, respectively, and are mobile, but a short peptide linker links the VL of the first binding domain to the VH of the second binding domain. Remarkably, the activity of the molecule of the present invention against target cells associated with specific diseases can be preserved without steric hindrance between the first and second binding domains, and without the need to provide a long linker that would be unfavorably susceptible to degradation, cleavage, or similar compared to a shorter linker that is immediately available. Furthermore, the present invention provides a polynucleotide encoding an antigen-binding molecule, a vector comprising this polynucleotide, a host cell expressing this construct, and a pharmaceutical composition comprising the same. [Means for solving the problem]

[0013] In a first aspect, the present invention relates to a multispecific antigen-binding molecule comprising at least three binding domains, (i.) The first binding domain contains a paratope that binds immunospecifically to a first target cell surface antigen (e.g., TAA1), (ii) The second binding domain comprises a paratope that immunospecifically binds to a second target cell surface antigen (e.g., TAA2), and (iii) The third binding domain contains a paratope that immunospecifically binds to the extracellular epitope of the human and / or macaque CD3ε chain. The first, second, and third binding domains are arranged in the order of amino to carboxyl, and the first and second binding domains are linked by a peptide linker having a length of 5 to 24 amino acids, preferably 5 to 18 amino acids, thereby providing a multispecific antigen-binding molecule.

[0014] In the above embodiments, it is also conceivable that the present invention provides a multispecific antigen-binding molecule comprising a fourth domain containing two polypeptide monomers, each containing a hinge, a CH2, and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker.

[0015] In the above embodiment, a multispecific antigen-binding molecule relating to the present invention, wherein the fourth domain is arranged in the order of amino to carboxyl, Hinge-CH2-CH3-Linker-Hinge-CH2-CH3 It is also conceivable that this could provide a multispecific antigen-binding molecule.

[0016] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which each of the polypeptide monomers in the fourth domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 24, and preferably, each of the polypeptide monomers has an amino acid sequence selected from SEQ ID NOs: 17 to 24.

[0017] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which the CH2 domain includes an intradomain cysteine ​​disulfide crosslink.

[0018] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which the first, second, third and optionally fourth binding domains are arranged in the order of amino to carboxyl.

[0019] In the above embodiments, it is also conceivable that, in connection with the present invention, a multispecific antigen-binding molecule is provided, which is a single-chain antigen-binding molecule, preferably a multispecific scFv antigen-binding molecule.

[0020] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which the first, second, and third binding domains each contain a VH domain and a VL domain in the order of amino to carboxyl.

[0021] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which the peptide linker between the first binding domain VL and the second binding domain VH is selected from having a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids, preferably 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, more preferably 6 amino acids.

[0022] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which the peptide linker between the first binding domain VL and the second binding domain VH is a mobile linker and comprises serine and glycine as amino acid components, preferably a mobile linker comprising only serine (Ser, S) and glycine (Gly, G).

[0023] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which the peptide linker between the first binding domain and the second binding domain is preferably rich in small and / or hydrophilic amine acids, and preferably selected from the group consisting of S(G4S)n, (G4S)n, (G4)n and (G5)n (where n is equal to 1, 2, 3 or 4, more preferably n is equal to 1 or 2), more preferably selected from SG4S.

[0024] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which either the first target cell surface antigen or the second target cell surface antigen is selected from the group consisting of CS1, BCMA, FLT3, CD123, CD20, CD22, EpCAM, MSLN, CDH3, and CLL1.

[0025] In the above embodiments, it is also conceivable that, in connection with the present invention, a multispecific antigen-binding molecule is provided in which the first target cell surface antigen and the second cell surface antigen are not the same.

[0026] In the above embodiments, it is also conceivable that the present invention provides a multispecific antigen-binding molecule in which a first binding domain has the ability to bind to a first target cell surface antigen, and a second binding domain has the ability to simultaneously bind to a second target cell surface antigen, preferably the first target cell surface antigen and the second target cell surface antigen are located on the same target cell.

[0027] In the above embodiments, it is also conceivable to provide a multispecific antigen-binding molecule in which the first target cell surface antigen and the second target cell surface antigen are respectively selected from the group consisting of CS1 and BCMA, BCMA and CS1, FLT3 and CD123, CD123 and FLT3, CD20 and CD22, CD22 and CD20, EpCAM and MSLN, MSLN and EpCAM, CDH3 and MSLN, MSLN and CDH3, FLT3 and CLL1, and CLL1 and FLT3.

[0028] In the above embodiment, the multispecific antigen-binding molecule is (i) The first and second domains each contain two antibody-variable domains, and the third domain also contains two antibody-variable domains; (ii) The first and second domains each contain one antibody variable domain, and the third domain contains two antibody variable domains; (iii) The first and second domains contain two antibody-variable domains, and the third domain contains one antibody-variable domain; or (iv) The first domain contains one antibody variable domain, and the second domain contains one antibody variable domain. It is also conceivable that it could be characterized by the following.

[0029] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule in which the first, second, and third domains, each fused by a peptide linker, are fused to a fourth domain via a peptide linker.

[0030] In the above embodiment, in relation to the present invention, a multispecific antigen-binding molecule is provided, wherein the order from amino to carboxyl is: (a) The first domain; (b) Peptide linkers having amino acid sequences preferably selected from the group consisting of SEQ ID NOs: 1-4 and 9-12, preferably from SEQ ID NO: 11; (c) Second domain; (d) Peptide linkers having an amino acid sequence preferably selected from the group consisting of SEQ ID NOs: 1 to 3; and (e) Third domain It is also conceivable that a multispecific antigen-binding molecule containing such a molecule may be provided.

[0031] In the above embodiment, in relation to the present invention, a multispecific antigen-binding molecule is provided, wherein the order from amino to carboxyl is: (f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11 and 12. (g) The first polypeptide monomer of the fourth domain; (h) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs. 5, 6, 7, and 8; and (i) Second polypeptide monomer of the fourth domain It is also conceivable to provide a multispecific antigen-binding molecule that further contains the above.

[0032] In the above embodiment, relating to the present invention, an antigen-binding molecule wherein the first and second binding domains are sequence numbers 33-35, 44-46, 55-57, 66-68, 77-79, 88-90, 99-101, 110-112, 121-123, 132-134, 143-145, 154-156, 165-167, 176-178, 187-189, 198-200, 209-211, 220-222, 231-233, 242-244, 253-255, 264-266, 275-277, 286-288, 297-299, 308-310, 319-321, 330~332, 341~343, 352~354, 363~365, 374~376, 385~387, 396~398, 407~409, 418~420, 429~431, 440~442, 451~453, 462~464, 473~475, 484~486, 495~ 497, 506~508, 517~519, 528~530, 539~541, 550~552, 561~563, 572~574, 583~585, 594~596, 605~607, 616~618, 627~629, 638~640, 649~651, 660~662, 8 96-898, 907-909, 918-920, 929-931, 940-942, 951-953, 962-964, 973-975, 984-986, 995-997, 1006-1008, 1017-1019, 1028-1030, 1039-1041, 1050- 1052, 1061-1063, 1072-1074, 1083-1085, 1094-1096, 1105-1107, 1116-1118, 1127-1129, 1138-1140, 1149-1151, 1160-1162, 1171-1173, 1182-1184, 1193~1195, 1204~1206, 1215~1217, 1226~1228, 1237~1239, 1248~1250, 1259~1261, 1270~1272, 1281~1283, 1292~1294, 1303~1305, 1314~1316, 1325~ 1327, 1336-1338, 1347-1349, 1358-1360, 1369-1371, 1380-1382, 1391-1393, 1402-1404, 1413-1415, 1424-1426, 1489-1491, 1500-1502, 1511-1513,1522-1524, 1533-1535, 1544-1546, 1555-1557, 1566-1568, 1577-1579, 1588-1590, 1599-1601, 1610-1612, 1621-1623, 1632-1634, 1643-1645, 1654-1656, 1827-1829, 1840-1842, 1853-1855, 1866-1868, 1879-1881, 1892-1894, 190 5-1907, 1922-1924, 1935-1937, 1948-1950, 1961-1963, 1974-1976, 1987-1989, 2000-2002, 2013-2015, 2026-2028, 2039-2041, 2052-2054, 2065-2067, 2078-2080, 2091-2093, 2104-2106, 2117-2119, 2130-2132, 2143-2145, 2156-2 158, 2169~2171, 2182~2184, 2195~2197, 2208~2210, 2221~2223, 2234~2236, 2247~2249, 3346~3348, 3357~3359, 3368~3370, 3379~3381, 3390~3392, 3401~3403, 3412~3414, 3423~3425, 3434~3436, 3445~3447, 3456~3458, 3467~3469 It is also conceivable to provide antigen-binding molecules containing a VH region including CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of 3478-3480, 3489-3491, 3500-3502, 3511-3513, 3522-3524, 3533-3535, 3544-3546, 3555-3557, 3566-3568, 3679-3681, 3690-3692, 3712-3714, and 3723-3725.

[0033] In the above embodiment, a multispecific antigen-binding molecule relating to the present invention, wherein the first and second binding domains are SEQ ID NOs: 36-38, 47-49, 58-60, 69-71, 80-82, 91-93, 102-104, 113-115, 124-126, 135-137, 146-148, 157-159, 168-170, 179-181, 190-192, 201-203, 212-214, 223-225, 234-236, 245-247, 256-258, 267-269, 278-280, 289-291, 300-302, 311-313, 3 22-324, 333-335, 344-346, 355-357, 366-368, 377-379, 388-390, 399-401, 410-412, 421-423, 432-434, 443-445, 454-456, 465-467, 476-478, 487-4 89, 498~500, 509~511, 520~522, 531~533, 542~544, 553~555, 564~566, 575~577, 586~588, 597~599, 608~610, 619~621, 630~632, 641~643, 652~654, 66 3-665, 899-901, 910-912, 921-923, 932-934, 943-945, 954-956, 965-967, 976-978, 987-989, 998-1000, 1009-1011, 1020-1022, 1031-1033, 1042-10 43, 1053~1055, 1064~1066, 1075~1077, 1086~1088, 1097~1099, 1108~1110, 1119~1121, 1130~1132, 1141~1143, 1152~1154, 1163~1165, 1174~1176, 1 185~1187, 1196~1198, 1207~1209, 1218~1220, 1229~1231, 1240~1242, 1251~1253, 1262~1264, 1273~1275, 1284~1286, 1295~1297, 1306~1308, 1317~ 1319, 1328-1330, 1339-1341, 1350-1352, 1361-1363, 1372-1374, 1383-1385, 1394-1396, 1405-1407, 1416-1418, 1427-1429, 1492-1494, 1503-1505,1514-1516, 1525-1527, 1536-1538, 1547-1549, 1558-1560, 1569-1571, 1580-1582, 1591-1593, 1602-1604, 1613-1615, 1624-1626, 1635-1637, 1646-1648, 1657-1659, 1830-1832, 1843-1845, 1856-1858, 1869-1871, 1882-1884, 1895-18 97, 1908-1910, 1925-1927, 1938-1940, 1951-1953, 1964-1966, 1977-1979, 1990-1992, 2003-2005, 2016-2018, 2029-2031, 2042-2044, 2055-2057, 2068-2070, 2081-2083, 2094-2096, 2107-2109, 2120-2122, 2133-2135, 2146-2148, 2159 ~2131, 2172~2174, 2085~2187, 2198~2200, 2211~2213, 2224~2226, 2237~2239, 2250~2252, 3349~3351, 3360~3362, 3371~3373, 3382~3384, 3393~3392, 3404~3406, 3415~3417, 3426~3428, 3437~3439, 3448~3450, 3459~3461, 3470~3472, 3 It is also conceivable to provide a multispecific antigen-binding molecule containing a VL region including CDR-L1, CDR-L2, and CDR-L3 selected from the group consisting of 481-3483, 3492-3494, 3503-3505, 3514-3516, 3525-3527, 3536-3538, 3547-3549, 3558-3560, 3569-3571, 3682-3684, 3693-3695, 3715-3717, and 3726-3728.

[0034] In the above embodiment, a multispecific antigen-binding molecule relating to the present invention, wherein the first and second binding domains are SEQ ID NOs: 39, 50, 61, 72, 83, 94, 105, 116, 127, 138, 149, 160, 171, 182, 193, 204, 215, 226, 237, 248, 259, 270, 281, 292, 303, 314, 325, 336, 347, 358, 369, 380, 391, 402, 413, 424, 435, 446, 457, 468, 479, 490, 501, 512, 523, 534, 545 ,556,567,578,589,600,611,622,633,644,655,666,902,913,924,935,946,957,968,979,990,1001,1012,1023,1034,1045,1056,1067,1078,1089,1100,1111,1122,1133,1144,1155,1166,1177,1188,1199,1210,1221,1232,1243,1254,1265,1276,1287,1298,1309,1320,1 331, 1342, 1353, 1364, 1375, 1386, 1397, 1408, 1419, 1430, 1495, 1506, 1517, 1528, 1539, 1550, 1561, 1572, 1583, 1594, 1605, 1616, 1627, 1638, 1649, 1660, 1833, 1846, 1859, 1872, 1885, 1898, 1911, 1928, 1941, 1954, 1967, 1980, 1993, 2006, 2019, 2032, 2045, 2058, 2071, 2084, 209 It is also conceivable to provide a multispecific antigen-binding molecule containing a VH region selected from the group consisting of 7, 2110, 2123, 2136, 2149, 2162, 2175, 2188, 2201, 2214, 2227, 2240, 2253, 3352, 3363, 3374, 3385, 3396, 3407, 3418, 3429, 3440, 3451, 3462, 3473, 3484, 3495, 3506, 3517, 3528, 3539, 3550, 3561, 3572, 3686, 3696, 3718, and 3729.

[0035] In the above embodiment, a multispecific antigen-binding molecule relating to the present invention, wherein the first and second binding domains are SEQ ID NOs: 40, 51, 62, 73, 84, 95, 106, 117, 128, 139, 150, 161, 172, 183, 194, 205, 216, 227, 238, 249, 260, 271, 282, 293, 304, 315, 326, 337, 348, 359, 370, 381, 392, 403, 414, 425, 436, 447, 458, 469, 480, 491, 502, 513, 524, 535, 546 ,557,568,579,590,601,612,623,634,645,656,667,903,914,925,936,947,958,969,980,991,1002,1013,1024,1035,1046,1057,1068,1079,1090,1101,1112,1123,1134,1145,1156,1167,1178,1189,1200,1211,1222,1233,1244,1255,1266,1277,1288,1299,1310,1321,1 332, 1343, 1354, 1365, 1376, 1387, 1398, 1409, 1420, 1431, 1496, 1507, 1518, 1529, 1540, 1551, 1562, 1573, 1584, 1595, 1606, 1617, 1628, 1639, 1650, 1661, 1834, 1847, 1860, 1873, 1886, 1899, 1912, 1929, 1942, 1955, 1968, 1981, 1994, 2007, 2020, 2033, 2046, 2059, 2072, 2085, 209 It is also conceivable to provide a multispecific antigen-binding molecule that includes a VL region selected from the group consisting of 8, 2111, 2124, 2137, 2150, 2163, 2176, 2189, 2202, 2215, 2228, 2241, 2254, 3353, 3364, 3375, 3386, 3397, 3408, 3419, 3430, 3441, 3452, 3463, 3474, 3485, 3496, 3507, 3518, 3529, 3540, 3551, 3562, 3573, 3685, 3697, 3719, and 3730.

[0036] In the above embodiment, a multispecific antigen-binding molecule relating to the present invention, wherein the first and second binding domains are SEQ ID NOs: 41, 52, 63, 74, 85, 96, 107, 118, 129, 140, 151, 162, 173, 184, 195, 206, 217, 228, 239, 250, 261, 272, 283, 294, 305, 316, 327, 338, 349, 360, 371, 382, ​​393, 404, 415, 426, 437, 448, 459, 470, 481, 492, 503, 514, 525, 536, 547, 558, 569, 580, 591, 602, 613, 624, 635, 646, 657, 668, 671, 674, 677, 680, 683, 686, 689, 692, 695, 698, 701, 704, 707, 710, 713, 716, 719, 722, 725, 728, 731, 734, 737, 740, 7 43, 746, 749, 752, 755, 758, 761, 764, 767, 770, 773, 776, 779, 782, 785, 788, 791, 794, 797, 800, 803, 806, 809, 812, 815, 818, 821, 824, 827, 830, 833, 83 6, 839, 842, 845, 848, 851, 854, 857, 860, 863, 866, 869, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 904, 915, 926, 937, 948, 959, 970, 981 ,992,1003,1014,1025,1036,1047,1058,1069,1080,1091,1102,1113,1124,1135,1146,1157,1168,1179,1190,1201,1212,1223,1234,1245,1256, 1267, 1278, 1289, 1300, 1311, 1322, 1333, 1344, 1355, 1366, 1377, 1388, 1399, 1410, 1421, 1432, 1435, 1438, 1441, 1444, 1447, 1450, 1453, 1456, 1459, 1462, 1465, 1468, 1471, 1474, 1477, 1480, 1483, 1486, 1497, 1508, 1519, 1530, 1541, 1552, 1563, 1574, 1585, 1596, 1607, 1618, 1629, 1640, 1651, 1662,1665、1668、1671、1674、1677、1680、1683、1686、1689、1692、1695、1698、1701、1704、1707、1710、1713、1716、1719、1722、1725、1728、1731、1734、1737、1740、1743、1746、1749、1752、1755、1758、1761、1764、1767、1770、1773、1776、1779、1782、1785、1788、1791、1794、1797、1800、1803、1806、1809、1812、1815、1818、1821、1824、1835、1848、1861、1874、1887、1900、1913、1930、1943、1956、1969、1982、1995、2008、2021、2034、2047、2060、2073、2086、2099、2112、2125、2138、2151、2164、2177、2190、2203、2216、2229、2242、2255、2264、2265、2274、2275、2284、2285、2294、2295、2304、2305、2314、2315、2324、2325、2334、2335、2344、2345、2354、2355、2364、2365、2374、2375、2384、2385、2394、2395、2404、2405、2414、2415、2424、2425、2434、2435、2444、2445、2454、2455、2464、2465、2474、2475、2484、2485、2494、2495、2504、2505、2514、2515、2524、2525、2534、2535、2544、2545、2554、2555、2564、2565、2574、2575、2584、2585、2594、2595、2604、2605、2614、2615、2624、2625、2634、2635、2644、2645、2654、2655、2664、2665、2674、2675、2684、2685、2694、2695、2704、2705、2714、2715、2724、2725、2734、2735、2744、2745、2754、2755、2764、2765、2774、2775、2784、2785、2794、2795、2804、2805、2814、2815、2824、2825, 2834, 2835, 2844, 2845, 2854, 2855, 2864, 2865, 2874, 2875, 2884, 2885, 2894, 2895, 2904, 2905, 2914, 2915, 2924, 2925, 2934, 2935, 2944, 2945, 2954, 2955, 2964, 2965, 2974, 2975, 2984, 2985, 2994, 2995, 3004, 3005, 3014, 3015, 3024, 3025, 3034, 3035, 3044, 3045, 3 054, 3055, 3064, 3065, 3074, 3075, 3084, 3085, 3094, 3095, 3104, 3105, 3114, 3115, 3124, 3125, 3134, 3135, 3144, 3145, 3154, 3155, 3164, 3165, 3174, 3175, 3184, 3185, 3194, 3195, 3204, 3205, 3214, 3215, 3224, 3225, 3234, 3235, 3244, 3245, 3254, 3255, 3264, 3265, 3274, 327 5, 3284, 3285, 3294, 3295, 3304, 3305, 3314, 3315, 3324, 3325, 3334, 3335, 3354, 3365, 3376, 3387, 3398, 3409, 3420, 3431, 3442, 3453, 3464, 3475, 3486, 3497, 3508, 3519, 3530, 3541, 3552, 3563, 3574, 3577, 3580, 3583, 3586, 3589, 3592, 3595, 3598, 3601, 3604, 3607, 3610, It is also conceivable to provide a multispecific antigen-binding molecule containing an scFv sequence selected from the group consisting of 3613, 3616, 3619, 3622, 3625, 3628, 3631, 3634, 3637, 3640, 3643, 3646, 3649, 3652, 3655, 3658, 3661, 3664, 3667, 3670, 3673, 3676, 3687, 3698, 3701, 3706, 3720, 3731, 3734, 3737, 3740, 3749, and 3750, preferably 1399 or 1435.

[0037] In the above embodiment, a multispecific antigen-binding molecule relating to the present invention comprises a first and / or second target-binding domain together with a third effector-binding domain, where two or three of these binding domains are sequence numbers 42, 53, 64, 75, 86, 97, 108, 119, 130, 141, 152, 163, 174, 185, 196, 207, 218, 229, 240, 251, 262, 273, 284, 295, 306, 317, 328, 339, 350, 361, 372, 383, 394, 405, 416, 427, 438, 449, 460, 471 ,482,493,504,515,526,537,548,559,570,581,592,603,614,625,636,647,658,669,672,675,678,681,684,687,690,693,696,699,702,705,708, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 759, 762, 765, 768, 771, 774, 777, 780, 783, 786, 789, 792, 795, 798, 801, 8 04, 807, 810, 813, 816, 819, 822, 825, 828, 831, 834, 837, 840, 843, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 875, 877, 879, 881, 883, 885, 887, 88 9, 891, 893, 895, 905, 916, 927, 938, 949, 960, 971, 982, 993, 1004, 1015, 1026, 1037, 1048, 1059, 1070, 1081, 1092, 1103, 1114, 1125, 1136, 1147, 1158, 1169, 1180, 1191, 1202, 1213, 1224, 1235, 1246, 1257, 1268, 1279, 1290, 1301, 1312, 1323, 1334, 1345, 1356, 1367, 1378, 1389, 1400, 1411, 1422, 1433, 1436, 1439, 1442, 1445, 1448, 1451, 1454, 1457, 1460, 1463, 1466, 1469, 1472, 1475, 1478, 1481, 1484, 1487, 1498, 1509, 1520, 1531, 1542, 1553, 1564,1575、1586、1597、1608、1619、1630、1641、1652、1663、1666、1669、1672、1675、1678、1681、1684、1687、1690、1693、1696、1699、1702、1705、1708、1711、1714、1717、1720、1723、1726、1729、1732、1735、1738、1741、1744、1747、1750、1753、1756、1759、1762、1765、1768、1771、1774、1777、1780、1783、1786、1789、1792、1795、1798、1801、1804、1807、1810、1813、1816、1819、1822、1825、1836、1849、1862、1875、1888、1901、1914、1931、1944、1957、1970、1983、1996、2009、2022、2035、2048、2061、2074、2087、2300、2113、2126、2139、2152、2165、2178、2191、2204、2217、2230、2243、2256、2260、2266、2267、2276、2277、2286、2287、2296、2297、2306、2307、2316、2317、2326、2327、2336、2337、2346、2347、2356、2357、2366、2367、2376、2377、2386、2387、2396、2397、2406、2407、2416、2417、2426、2427、2436、2437、2446、2447、2456、2457、2466、2467、2476、2477、2486、2487、2496、2497、2506、2507、2516、2517、2526、2527、2536、2537、2546、2547、2556、2557、2566、2567、2576、2577、2586、2587、2596、2597、2606、2607、2616、2617、2626、2627、2636、2637、2646、2647、2656、2657、2666、2667、2676、2677、2686、2687、2696、2697、2706、2707、2716、2717、2726、2727、2736、2737、2746、2747、2756、2757、2766、2767、2776、2777, 2786, 2787, 2796, 2797, 2806, 2807, 2816, 2817, 2826, 2827, 2836, 2837, 2846, 2847, 2856, 2857, 2866, 2867, 2876, 2877, 2886, 2887, 2896, 2897, 2906, 2907, 2916, 2917, 2926, 2927, 2936, 2937, 2946, 2947, 2956, 2957, 2966, 2967, 2976, 2977, 2986, 2987, 2996, 2997, 3006, 3007, 3016, 3017, 3026, 3027, 3036, 3037, 3046, 3047, 3056, 3057, 3066, 3067, 3076, 3077, 3086, 3087, 3096, 3097, 3106, 3107, 3116, 3117, 3126, 3127, 3136, 3137, 3146, 3147, 3156, 3157, 3166, 3167, 3176, 3177, 3186, 3187, 3196, 3197, 3206, 3207, 3216, 3217, 3226, 3227, 3236, 3237, 3246, 3247, 3256, 3 257, 3266, 3267, 3276, 3277, 3286, 3287, 3296, 3297, 3306, 3307, 3316, 3317, 3326, 3327, 3336, 3337, 3355, 3366, 3377, 3388, 3399, 3410, 3421, 3432, 3443, 3454, 3465, 3476, 3487, 3498, 3509, 3520, 3531, 3542, 3553, 3564, 3575, 3578, 3581, 3584, 3587, 3590, 3593, 3596, 3599, 3602, 3605, 3 It is also conceivable to provide a multispecific antigen-binding molecule having sequences selected from the group consisting of 608, 3611, 3614, 3617, 3620, 3623, 3626, 3629, 3632, 3635, 3638, 3641, 3644, 3647, 3650, 3653, 3656, 3659, 3662, 3665, 3668, 3671, 3674, 3677, 3688, 3699, 3702, 3703, 3707, 3721, 3732, 3735, 3738, 3741, 3751, and 3752, preferably 1400 or 1436.

[0038] In the above embodiments, it is also conceivable that the present invention may provide a multispecific antigen-binding molecule comprising first and second target bindings, a third domain, and optionally a fourth domain that provides an extended half-life, as disclosed herein.

[0039] In the above embodiment, the antigen-binding molecule, in addition to (a) to (d), is arranged in the order from amino to carboxyl. (e) A first polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17-24; (f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs. 5, 6, 7, and 8; and (g) A second polypeptide monomer of a third domain having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17-24. It is also conceivable to provide antigen-binding molecules that further contain [the specified element].

[0040] In the above embodiment, the antigen-binding molecule relating to the present invention is sequence numbers 41, 52, 63, 74, 85, 96, 107, 118, 129, 140, 151, 162, 173, 184, 195, 206, 217, 228, 239, 250, 261, 272, 283, 294, 305, 316, 327, 338, 349, 360, 371, 382, ​​393, 404, 415, 426, 437, 448, 459, 470, 481, 492, 503, 514, 525, 536, 547, 558, 569, 580, 591, 602, 613, 624, 635, 646, 657, 668, 671, 674, 677, 680, 683, 686, 689, 692, 695, 698, 701, 704, 707, 710, 713, 716, 719, 722, 725, 728, 731, 734, 737, 740, 743, 746, 749, 752, 755, 7 58, 761, 764, 767, 770, 773, 776, 779, 782, 785, 788, 791, 794, 797, 800, 803, 806, 809, 812, 815, 818, 821, 824, 827, 830, 833, 836, 839, 842, 845, 848, 85 1, 854, 857, 860, 863, 866, 869, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 904, 915, 926, 937, 948, 959, 970, 981, 992, 1003, 1014, 1025, 1036, 1047, 1058, 1069, 1080, 1091, 1102, 1113, 1124, 1135, 1146, 1157, 1168, 1179, 1190, 1201, 1212, 1223, 1234, 1245, 1256, 1267, 1278, 1289, 1300, 1311, 1322, 1333, 1344, 1355, 1366, 1377, 1388, 1399, 1410, 1421, 1432, 1435, 1438, 1441, 1444, 1447, 1450, 1453, 1456, 1459, 1462, 1465, 1468, 1471, 1474, 1477, 1480, 1483, 1486, 1497, 1508, 1519, 1530, 1541, 1552, 1563, 1574, 1585, 1596, 1607, 1618, 1629, 1640, 1651, 1662, 1665, 1668, 1671, 1674,1677, 1680, 1683, 1686, 1689, 1692, 1695, 1698, 1701, 1704, 1707, 1710, 1713, 1716, 1719, 1722, 1725, 1728, 1731, 1734, 1737, 1740, 1743, 1746, 1749 , 1752, 1755, 1758, 1761, 1764, 1767, 1770, 1773, 1776, 1779, 1782, 1785, 1788, 1791, 1794, 1797, 1800, 1803, 1806, 1809, 1812, 1815, 1818, 1821 and 182 A group consisting of 4, preferably 1399 or 1435, preferably sequence numbers 42, 53, 64, 75, 86, 97, 108, 119, 130, 141, 152, 163, 174, 185, 196, 207, 218, 229, 240, 251, 262, 273, 284, 295, 306, 317, 328, 339, 350, 361, 372, 383, 394, 405, 416, 427, 438, 449, 460, 471, 482, 493, 504, 515, 526, 537, 548, 559, 570, 581, 592, 603, 614, 625, 636, 647, 658, 669, 672, 675, 678, 681, 684, 687, 690, 693, 696, 699, 702, 705, 708, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 7 59, 762, 765, 768, 771, 774, 777, 780, 783, 786, 789, 792, 795, 798, 801, 804, 807, 810, 813, 816, 819, 822, 825, 828, 831, 834, 837, 840, 843, 846, 849, 85 2, 855, 858, 861, 864, 867, 870, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 905, 916, 927, 938, 949, 960, 971, 982, 993, 1004, 1015, 1026, 1037, 1048, 1059, 1070, 1081, 1092, 1103, 1114, 1125, 1136, 1147, 1158, 1169, 1180, 1191, 1202, 1213, 1224, 1235, 1246, 1257, 1268, 1279, 1290, 1301,1312, 1323, 1334, 1345, 1356, 1367, 1378, 1389, 1400, 1411, 1422, 1433, 1436, 1439, 1442, 1445, 1448, 1451, 1454, 1457, 1460, 1463, 1466, 1469, 1472 , 1475, 1478, 1481, 1484, 1487, 1498, 1509, 1520, 1531, 1542, 1553, 1564, 1575, 1586, 1597, 1608, 1619, 1630, 1641, 1652, 1663, 1666, 1669, 1672, 1675 , 1678, 1681, 1684, 1687, 1690, 1693, 1696, 1699, 1702, 1705, 1708, 1711, 1714, 1717, 1720, 1723, 1726, 1729, 1732, 1735, 1738, 1741, 1744, 1747, 1750 , 1753, 1756, 1759, 1762, 1765, 1768, 1771, 1774, 1777, 1780, 1783, 1786, 1789, 1792, 1795, 1798, 1801, 1804, 1807, 1810, 1813, 1816, 1819, 1822 and 182 A group consisting of 5, preferably 1400 or 1436, more preferably 43, 54, 65, 76, 87, 98, 109, 120, 131, 142, 153, 164, 175, 186, 197, 208, 219, 230, 241, 252, 263, 274, 285, 296, 307, 318, 329, 340, 351, 362, 373, 384, 395, 406, 417, 428, 439, 450, 461, 472, 483, 494, 505, 516, 527, 538, 549, 560, 571, 582, 593, 604, 615, 626, 637, 648, 65 9, 670, 673, 676, 679, 682, 685, 688, 691, 694, 697, 700, 703, 706, 709, 712, 715, 718, 721, 724, 727, 730, 733, 736, 739, 742, 745, 748, 751, 754, 757, 760 ,763,766,769,772,775,778,781,784,787,790,793,796,799,802,805,808,811,814,817,820,823,826,829,832,835,838,841,844,847,850,853,856、859、862、865、868、871、906、917、928、939、950、961、972、983、994、1005、1016、1027、1038、1049、1060、1071、1082、1093、1104、1115、1126、1137、1148、1159、1170、1181、1192、1203、1214、1225、1236、1247、1258、1269、1280、1291、1302、1313、1324、1335、1346、1357、1368、1379、1390、1401、1412、1423、1434、1437、1440、1443、1446、1449、1452、1455、1458、1461、1464、1467、1470、1473、1476、1479、1482、1485、1488、1499、1510、1521、1532、1543、1554、1565、1576、1587、1598、1609、1620、1631、1642、1653、1664、1667、1670、1673、1676、1679、1682、1685、1688、1691、1694、1697、1700、1703、1706、1709、1712、1715、1718、1721、1724、1727、1730、1733、1736、1739、1742、1745、1748、1751、1754、1757、1760、1763、1766、1769、1772、1775、1778、1781、1784、1787、1790、1793、1796、1799、1802、1805、1808、1811、1814、1817、1820、1823、1826、1838、1851、1864、1877、1890、1903、1916、1933、1946、1959、1972、1985、1998、2011、2024、2037、2050、2063、2076、2089、2102、2115、2128、2141、2154、2167、2180、2194、2206、2219、2232、2245、2258、2262、2270、2271、2280、2281、2290、2291、2300、2301、2310、2311、2320、2321、2330、2331、2340、2341、2350、2351、2360、2361、2370、2371、2380、2381、2390、2391、2400、2401、2410、2411、2420、2421、2430、2431、2440、2441、2450、2451、2460、2461、2470、2471、2480、2481、2490、2491、2500、2501、2510、2511、2520、2521、2530、2531、2540、2541、2550、2551、2560、2561、2570、2571、2580、2581、2590、2591、2600、2601、2610、2611、2620、2621、2630、2631、2640、2641、2650、2651、2660、2661、2670、2671、2680、2681、2690、2691、2700、2701、2710、2711、2720、2721、2730、2731、2740、2741、2750、2751、2760、2761、2770、2771、2780、2781、2790、2791、2800、2801、2810、2811、2820、2821、2830、2831、2840、2841、2850、2851、2860、2861、2870、2871、2880、2881、2890、2891、2900、2901、2910、2911、2920、2921、2930、2931、2940、2941、2950、2951、2960、2961、2970、2971、2980、2981、2990、2991、3000、3001、3010、3011、3020、3021、3030、3031、3040、3041、3050、3051、3060、3061、3070、3071、3080、3081、3090、3091、3100、3101、3110、3111、3120、3121、3130、3131、3140、3141、3150、3151、3160、3161、3170、3171、3180、3181、3190、3191、3200、3201、3210、3211、3220、3221、3231、3240、3241、3250、3251、3260、3261、3270、3271、3280、3281、3290、3291、3300、3301、3310、3311、3320、3321、3330、3331、3340、3341、3344、3345、3356、3367、3378、3389、3400、3411、3422、3433、3444、3455, 3466, 3477, 3488, 3499, 3510, 3521, 3532, 3543, 3554, 3565, 3576, 3579, 3582, 3585, 3588, 3591, 3594, 3597, 3600, 3603, 3606, 3609, 3612, 361, It is also conceivable to provide antigen-binding molecules having an amino acid sequence selected from the group consisting of 5, 3618, 3621, 3624, 3627, 3630, 3633, 3636, 3639, 3642, 3645, 3648, 3651, 3654, 3657, 3660, 3663, 3666, 3669, 3672, 3675, 3678, 3689, 3700, 3704, 3705, 3708, 3709, 3710, 3711, 3722, 3733, 3736, 3739, 3744, 3747, 3748, 3756, 3757, 3761 and 3762, preferably 1401 or 1437.

[0041] In the above embodiment, an antigen-binding molecule having a CD3 conjugate with optimized stability, relating to the present invention, is provided for SEQ ID NOs: 1839, 1852, 1865, 1878, 1891, 1904, 1917, 1919, 1921, 1934, 1947, 1960, 1973, 1986, 1999, 2012, 2025, 2038, 2051, 2064, 2077, 2090, 2103, 2116, 2029, 2142, 2155, 2168, 2181, 2193, 2207, 2220, 2233, 2246, 2259, 2263, 2272, 2273, 2282, 2283 ,2292,2293,2302,2303,2312,2313,2322,2323,2332,2333,2342,2343,2352,2353,2362,2363,2372,2373,2382,2383,2392,2393,2402,2403,2412 ,2413,2422,2423,2432,3433,2442,2443,2452,2453,2462,2463,2472,2473,2482,2483,2492,2493,2502,2503,2512,2513,2522,2523,2532,2533 ,2542,2543,2552,2553,2562,2563,2572,2573,2582,2583,2592,2593,2602,2603,2612,2613,2622,2623,2632,2633,2642,2643,2652,2653,2662 ,2663,2672,2673,2682,2683,2692,2693,2702,2703,2712,2713,2722,2723,2732,2733,2742,2743,2752,2753,2762,2763,2772,2773,2782,2783 ,2792,2793,2802,2803,2812,2813,2822,2823,2832,2833,2842,2843,2852,2853,2862,2863,2872,2873,2882,2883,2892,2893,2902,2903,2912 ,2913,2922,2923,2932,2933,2942,2943,2952,2953,2962,2963,2972,2973,2982,2983,2992,2993,3002,3003,3012,3013,3022,3023,3032,3033,3042, 3043, 3052, 3053, 3062, 3063, 3072, 3073, 3082, 3083, 3092, 3093, 3112, 3113, 3122, 3123, 3132, 3133, 3142, 3143, 3152, 3153, 3162, 3163, 3172, 3173, 3182, 3183, 3192, 3193, 3202, 3203, 3212, 3213, 3222, 3223, 3 It is also conceivable to provide molecules having an amino acid sequence selected from the group consisting of 232, 3233, 3242, 3243, 3252, 3253, 3262, 3263, 3272, 3273, 3282, 3283, 3292, 3293, 3302, 3303, 3312, 3313, 3322, 3323, 3332, 3333, 3342, 3343, 3745, 3746, 3758, 3759, and 3760.

[0042] In a second aspect, it is conceivable that, in connection with the present invention, polynucleotides encoding the antigen-binding molecule of the present invention are provided.

[0043] In a third aspect, it is also conceivable that a vector containing the polynucleotide of the present invention may be provided in connection with the present invention.

[0044] In a fourth aspect, it is conceivable that, in connection with the present invention, host cells transformed or transfected with the polynucleotide or vector of the present invention may be provided.

[0045] In a fifth aspect of the present invention, it is also conceivable to provide a process for generating the antigen-binding molecule of the present invention, comprising the steps of culturing host cells of the present invention under conditions that enable the expression of the antigen-binding molecule, and recovering the generated antigen-binding molecule from the culture.

[0046] In a sixth aspect, it is conceivable that, in connection with the present invention, a pharmaceutical composition comprising an antigen-binding molecule of the present invention or an antigen-binding molecule produced according to the process of the present invention is provided.

[0047] In the above embodiments, it is also assumed that the pharmaceutical composition is stable at approximately -20°C for at least 4 weeks in connection with the present invention.

[0048] In connection with the present invention, it is further envisioned to provide antigen-binding molecules of the present invention or antigen-binding molecules produced according to the process of the present invention for use in the prevention, treatment, or remission of diseases selected from proliferative disorders, neoplastic disorders, cancer, or immunodeficiencies.

[0049] In the above embodiments, the diseases in relation to the present invention are preferably multiple myeloma (MM), acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC), and preferably the CS1×BCMA or BCMA×CS1 multispecific antigen-binding molecule is for use in the treatment of multiple myeloma, preferably the FLT3×CD123 or CD123×FLT3 multispecific antigen-binding molecule is for use in the treatment of acute myeloid leukemia, and preferably the CD20×CD22 or CD22×CD20 multispecific antigen-binding molecule is for use in the treatment of non-Hodgkin lymphoma.

[0050] In a seventh aspect, it is further envisioned that the present invention provides a method for the treatment or remission of a proliferative disorder, neoplastic disorder, cancer or immunodeficiency, comprising the step of administering an antigen-binding molecule of the present invention or an antigen-binding molecule produced according to a process of the present invention to a subject in need thereof, wherein the disorder is preferably multiple myeloma, acute myeloid leukemia, non-Hodgkin lymphoma, non-small cell lung cancer and / or colorectal cancer, preferably the CS1×BCMA or BCMA×CS1 multispecific antigen-binding molecule for the treatment of multiple myeloma, preferably the FLT3×CD123 or CD123×FLT3 multispecific antigen-binding molecule for the treatment of acute myeloid leukemia, and preferably the CD20×CD22 or CD22×CD20 multispecific antigen-binding molecule for the treatment of non-Hodgkin lymphoma.

[0051] In the eighth aspect, it is also conceivable that a kit comprising the antigen-binding molecule of the present invention or an antigen-binding molecule produced according to the process of the present invention, the polynucleotide of the present invention, the vector of the present invention, and / or the host cell of the present invention may be provided in connection with the present invention. [Brief explanation of the drawing]

[0052] [Figure 1] The overall structure of the multispecific antigen-binding molecule of the present invention is shown. [Figure 2] A schematic model outlines the reasons why the multispecific antigen-binding molecule according to the present invention, which has a short (6 aa) peptide linker between the first and second binding domains, is not expected to be able to bind to TAA2 (A), but is expected to be able to bind to TAA2 if it has a longer peptide linker with 30 aa (B). For mobility reasons, a third binding domain and an optional fourth domain are not shown. [Figure 3-1] This shows an overview of three different modeled three-dimensional structures of the second binding domain relative to the first binding domain of the exemplary multispecific antigen-binding molecule MSLN×EpCAM HLE BiTE® antigen-binding molecule: three-dimensional structure 2 (B) which allows for maximum space for TAA2 of 10-30 Å, three-dimensional structure 1 (C) which allows for maximum space for TAA2 of 3-8 Å, and three-dimensional structure 3 (D) which does not leave space for TAA2 (i.e., causes steric collision between binding domains). [Figure 3-2] This shows an overview of three different modeled three-dimensional structures of the second binding domain relative to the first binding domain of the exemplary multispecific antigen-binding molecule MSLN×EpCAM HLE BiTE® antigen-binding molecule: three-dimensional structure 2 (B) which allows for maximum space for TAA2 of 10-30 Å, three-dimensional structure 1 (C) which allows for maximum space for TAA2 of 3-8 Å, and three-dimensional structure 3 (D) which does not leave space for TAA2 (i.e., causes steric collision between binding domains). [Figure 4-1]The modeled space between the first and second binding domains of an exemplary multispecific antigen-binding molecule, the MSLN×EpCAM HLE BiTE® antigen-binding molecule, is shown depending on the linker length, and the mobility (SGn)x linker (where n is 2–4 and x is 3–6) is investigated. The maximum available space is 39–50 Å for an exemplary linker (A) with a length of 12 aa, 54–60 Å for an exemplary linker (B) with a length of 18 aa, and 84–94 Å for an exemplary linker (C) with a length of 30 aa. In the case of the CD20×CD22 HLE BiTE® molecule, the available space is modeled by linkers of length 5 aa (D), linkers of length 30 aa (E), and linkers of length 50 aa (F). In the case of the CS1×BCMA HLE BiTE® molecule, the available space is modeled by linkers of length 5 aa (G), linkers of length 30 aa (H), and linkers of length 50 aa (I). [Figure 4-2] The modeled space between the first and second binding domains of an exemplary multispecific antigen-binding molecule, the MSLN×EpCAM HLE BiTE® antigen-binding molecule, is shown depending on the linker length, and the mobility (SGn)x linker (where n is 2–4 and x is 3–6) is investigated. The maximum available space is 39–50 Å for an exemplary linker (A) with a length of 12 aa, 54–60 Å for an exemplary linker (B) with a length of 18 aa, and 84–94 Å for an exemplary linker (C) with a length of 30 aa. In the case of the CD20×CD22 HLE BiTE® molecule, the available space is modeled by linkers of length 5 aa (D), linkers of length 30 aa (E), and linkers of length 50 aa (F). In the case of the CS1×BCMA HLE BiTE® molecule, the available space is modeled by linkers of length 5 aa (G), linkers of length 30 aa (H), and linkers of length 50 aa (I). [Figure 4-3]The modeled space between the first and second binding domains of an exemplary multispecific antigen-binding molecule, the MSLN×EpCAM HLE BiTE® antigen-binding molecule, is shown depending on the linker length, and the mobility (SGn)x linker (where n is 2–4 and x is 3–6) is investigated. The maximum available space is 39–50 Å for an exemplary linker (A) with a length of 12 aa, 54–60 Å for an exemplary linker (B) with a length of 18 aa, and 84–94 Å for an exemplary linker (C) with a length of 30 aa. In the case of the CD20×CD22 HLE BiTE® molecule, the available space is modeled by linkers of length 5 aa (D), linkers of length 30 aa (E), and linkers of length 50 aa (F). In the case of the CS1×BCMA HLE BiTE® molecule, the available space is modeled by linkers of length 5 aa (G), linkers of length 30 aa (H), and linkers of length 50 aa (I). [Figure 5-1]Binding experiments using electrically switchable nanolever technology are shown. The association and dissociation of exemplary MSLN×EpCAM HLE BiTE® antigen-binding molecules measured at three concentrations (12.5 nM, 25 nM, and 50 nM) are shown (A, B, C, D). The biochips to be tested were coated with a 1:1 mix of antigen EpCAM (A, B) and antigen MSLN (C, D). The quenching of the green fluorophore binding to antigen EpCAM is shown (A, B). The quenching of the red fluorophore binding to antigen MSLN is shown (C, D). Binding experiments using switchSENSE® (Planegg, Germany) technology are further shown at E, F, G, H, I, J, K, L, M, N, O, and P. The association and dissociation of dual-targeted CLL1×FLT3 HLE BiTE® antigen-binding molecules at three concentrations (12.5 nM, 25 nM, and 50 nM) are shown. The biochips to be tested were coated with either the antigen CLL1 (E, F), the antigen FLT3 (G, H), or a 1:1 mix of both antigens CLL1 and FLT3 (I, J). The association and dissociation of the dual-targeted CS1×BCMA HLE BiTE® antigen-binding molecule were shown at three concentrations (12.5 nM, 25 nM, and 50 nM). The biochips to be tested were also coated with either the antigen CS1 (K, L), the antigen BCMA (M, N), or a 1:1 mix of both antigens CS1 and BCMA (O, P). [Figure 5-2]Binding experiments using electrically switchable nanolever technology are shown. The association and dissociation of exemplary MSLN×EpCAM HLE BiTE® antigen-binding molecules measured at three concentrations (12.5 nM, 25 nM, and 50 nM) are shown (A, B, C, D). The biochips to be tested were coated with a 1:1 mix of antigen EpCAM (A, B) and antigen MSLN (C, D). The quenching of the green fluorophore binding to antigen EpCAM is shown (A, B). The quenching of the red fluorophore binding to antigen MSLN is shown (C, D). Binding experiments using switchSENSE® (Planegg, Germany) technology are further shown at E, F, G, H, I, J, K, L, M, N, O, and P. The association and dissociation of dual-targeted CLL1×FLT3 HLE BiTE® antigen-binding molecules at three concentrations (12.5 nM, 25 nM, and 50 nM) are shown. The biochips to be tested were coated with either the antigen CLL1 (E, F), the antigen FLT3 (G, H), or a 1:1 mix of both antigens CLL1 and FLT3 (I, J). The association and dissociation of the dual-targeted CS1×BCMA HLE BiTE® antigen-binding molecule were shown at three concentrations (12.5 nM, 25 nM, and 50 nM). The biochips to be tested were also coated with either the antigen CS1 (K, L), the antigen BCMA (M, N), or a 1:1 mix of both antigens CS1 and BCMA (O, P). [Figure 5-3]Binding experiments using electrically switchable nanolever technology are shown. The association and dissociation of exemplary MSLN×EpCAM HLE BiTE® antigen-binding molecules measured at three concentrations (12.5 nM, 25 nM, and 50 nM) are shown (A, B, C, D). The biochips to be tested were coated with a 1:1 mix of antigen EpCAM (A, B) and antigen MSLN (C, D). The quenching of the green fluorophore binding to antigen EpCAM is shown (A, B). The quenching of the red fluorophore binding to antigen MSLN is shown (C, D). Binding experiments using switchSENSE® (Planegg, Germany) technology are further shown at E, F, G, H, I, J, K, L, M, N, O, and P. The association and dissociation of dual-targeted CLL1×FLT3 HLE BiTE® antigen-binding molecules at three concentrations (12.5 nM, 25 nM, and 50 nM) are shown. The biochips to be tested were coated with either the antigen CLL1 (E, F), the antigen FLT3 (G, H), or a 1:1 mix of both antigens CLL1 and FLT3 (I, J). The association and dissociation of the dual-targeted CS1×BCMA HLE BiTE® antigen-binding molecule were shown at three concentrations (12.5 nM, 25 nM, and 50 nM). The biochips to be tested were also coated with either the antigen CS1 (K, L), the antigen BCMA (M, N), or a 1:1 mix of both antigens CS1 and BCMA (O, P). [Figure 6] Surface expression of CS1 and BCMA in human multiple myeloma cell lines and additional human hematological cancer cell lines is shown. C1 was detected using antibody clone 162.1 (●), and BCMA was detected using antibody clone 19F2 (■). [Figure 7] This shows redirected T cell lysis (A), T cell activation (B), and cytokine induction (C) by a multi-targeted (dual) CS1×BCMA BiTE® antigen-binding molecule using ARH-77 target cells. [Figure 8]This shows redirected T cell lysis (A), T cell activation (B), and cytokine induction using T cell subpopulations (C, D) by a multi-targeted (dual) CS1×BCMA BiTE® antigen-binding molecule using MM.1R-targeted cells. [Figure 9] This shows redirected T cell lysis using multi-targeted CS1×BCMA(dual)BiTE® antigen-binding molecules, CS1(single)BiTE® antigen-binding molecules, and BCMA(single)BiTE® antigen-binding molecules, using ARH-77 target cells (A), MM.1R target cells (B), OPM-2 target cells (C), and U266B1 target cells (D). [Figure 10-1] This shows redirected T cell lysis by multitargeted CS1×BCMA Dual BiTE® antigen-binding molecules, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules in the presence of soluble CS1 and / or soluble BCMA at various concentrations of soluble CS1 and / or soluble BCMA, respectively, from 0 ng / ml (A) to a maximum of 80 ng / ml (G), using U266B1 target cells. [Figure 10-2] This shows redirected T cell lysis by multitargeted CS1×BCMA Dual BiTE® antigen-binding molecules, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules in the presence of soluble CS1 and / or soluble BCMA at various concentrations of soluble CS1 and / or soluble BCMA, respectively, from 0 ng / ml (A) to a maximum of 80 ng / ml (G), using U266B1 target cells. [Figure 10-3]This shows redirected T cell lysis by multitargeted CS1×BCMA Dual BiTE® antigen-binding molecules, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules in the presence of soluble CS1 and / or soluble BCMA at various concentrations of soluble CS1 and / or soluble BCMA, respectively, from 0 ng / ml (A) to a maximum of 80 ng / ml (G), using U266B1 target cells. [Figure 11-1] This shows redirected T cell lysis using OPM-2 target cells, mediated by multispecific CS1×BCMA Dual BiTE®, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules, in the presence of soluble CS1 or soluble BCMA at various concentrations of soluble BCMA(I) from 0 ng / ml(A) to a maximum of 250 ng / ml, respectively. [Figure 11-2] This shows redirected T cell lysis using OPM-2 target cells, mediated by multispecific CS1×BCMA Dual BiTE®, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules, in the presence of soluble CS1 or soluble BCMA at various concentrations of soluble BCMA(I) from 0 ng / ml(A) to a maximum of 250 ng / ml, respectively. [Figure 11-3] This shows redirected T cell lysis using OPM-2 target cells, mediated by multispecific CS1×BCMA Dual BiTE®, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules, in the presence of soluble CS1 or soluble BCMA at various concentrations of soluble BCMA(I) from 0 ng / ml(A) to a maximum of 250 ng / ml, respectively. [Figure 12-1]This shows redirected T cell lysis in the presence of soluble BCMA, using U266B1 target cell lines (A-C), NCI-H929 target cell lines (D-F), and OPM-2 target cell lines (G-I), mediated by multispecific CS1×BCMA dual BiTE® antigen-binding molecules, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules. [Figure 12-2] This shows redirected T cell lysis in the presence of soluble BCMA, using U266B1 target cell lines (A-C), NCI-H929 target cell lines (D-F), and OPM-2 target cell lines (G-I), mediated by multispecific CS1×BCMA dual BiTE® antigen-binding molecules, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules. [Figure 12-3] This shows redirected T cell lysis in the presence of soluble BCMA, using U266B1 target cell lines (A-C), NCI-H929 target cell lines (D-F), and OPM-2 target cell lines (G-I), mediated by multispecific CS1×BCMA dual BiTE® antigen-binding molecules, CS1 Mono BiTE® antigen-binding molecules, and BCMA Mono BiTE® antigen-binding molecules. [Figure 13-1] This shows redirected T cell lysis using U266B1 target cells with CS1×BCMA Dual BiTE® antigen-binding molecule, CS1 Mono BiTE® antigen-binding molecule, and BCMA Mono BiTE® antigen-binding molecule, using T cell subpopulations Pan T cells (A), CD4+ T cells (B), and CD8+ T cells. [Figure 13-2] This shows redirected T cell lysis using U266B1 target cells with CS1×BCMA Dual BiTE® antigen-binding molecule, CS1 Mono BiTE® antigen-binding molecule, and BCMA Mono BiTE® antigen-binding molecule, using T cell subpopulations Pan T cells (A), CD4+ T cells (B), and CD8+ T cells. [Figure 14] This demonstrates redirected T cell lysis using a T cell subpopulation with the CS1×BCMA Dual BiTE® antigen-binding molecule, using U266B1 target cells. [Figure 15] This shows the J-chain mRNA levels plotted against BiTE® concentration for CS1 mono BiTE® antigen-binding molecule (CS1 HLE BiTE®), BCMA mono BiTE® antigen-binding molecule, and multi-targeted CS1×BCMA BiTE® antigen-binding molecule (dBiTE). [Figure 16] This shows the lysis of target cells in Ramos wt cells by two representative CD20-CD22 T cell engager molecules compared to an equimolar mixture of CD20 and CD22 T cell engager molecules. [Figure 17] BCMA-CS1 T cell molecule 1, which has a 6-amino acid linker, showed an EC50 value [pM] against OPM-2 cells equivalent to engager molecules 2 and 3, which have longer linker variants (Figure 17A). EpCAM-MSLN T cell engager molecules 2 and 3, which have longer linker variants, showed an EC50 value [pM] against HCT-116 cells equivalent to EpCAM-MSLN T cell engager molecule 1, which has the original 6-amino acid linker (Figure 17B). CD123-FLT3 T cell engager molecules 2 and 3, which have longer linker variants, showed an EC50 value [pM] against OPM-2 cells equivalent to CD123-FLT3 T cell engager molecule 1, which has the original 6-amino acid linker (Figure 17C). [Figure 18] The tested CD22 T cell engager molecule 1 showed an EC50 value [pM] (Figure 18A) for Raji cells equivalent to that of CD20-CD22 T cell engager molecule 1 (Figure 18B) for Raji CRISPR CD20 cells. [Figure 19-1]Alignment of human and mouse CD20×CLL1 protein sequences with epitope sections is shown. The extracellular loop 1 (ECL1) of the CD20 protein was named E1, and the extracellular loop 2 (ECL2) was named E2. For more precise epitope mapping, the extracellular loop 1 (ECL1) was further divided into sub-parts E1A and E1B, and the extracellular loop 2 (ECL2) was further divided into sub-parts E2A, E2B, E2C, or E2D. [Figure 19-2] Alignment of human and mouse CD20×CLL1 protein sequences with epitope sections is shown. The extracellular loop 1 (ECL1) of the CD20 protein was named E1, and the extracellular loop 2 (ECL2) was named E2. For more precise epitope mapping, the extracellular loop 1 (ECL1) was further divided into sub-parts E1A and E1B, and the extracellular loop 2 (ECL2) was further divided into sub-parts E2A, E2B, E2C, or E2D. [Figure 20] Flow cytometry-bound analysis of a known CD20 antibody (A) and a T cell engager molecule (B) disclosed herein against transfected CHO cells expressing a full-length human or mouse CD20 protein construct or a human / mouse chimeric CD20 protein construct. [Figure 21-1]Sequence alignment of human and mouse CD22. Top sequence: human CD22, bottom sequence: mouse CD22. Sequence alignment of CD22 proteins shows which mouse sequence portions (V+C2-1, C2-1+C2-2, C2-2+C2-3, C2-3+C2-4, C2-4+C2-5, C2-5+C2-6) are replaced with the corresponding human sequences, and which amino acids differ between the two species. To ensure structural integrity of the transition region between domains, two adjacent domains of the mouse CD22 protein were swapped with the corresponding sequences of the human CD22 protein. To obtain single-domain specific results, epitope clustering constructs were designed to overlap each other. The swapped sequence regions include two CD22 domains, an amino acid stretch between the swapped domains, and an amino acid stretch between the second swapped domain and the subsequent CD22 domain. [Figure 21-2] Sequence alignment of human and mouse CD22. Top sequence: human CD22, bottom sequence: mouse CD22. Sequence alignment of CD22 proteins shows which mouse sequence portions (V+C2-1, C2-1+C2-2, C2-2+C2-3, C2-3+C2-4, C2-4+C2-5, C2-5+C2-6) are replaced with the corresponding human sequences, and which amino acids differ between the two species. To ensure structural integrity of the transition region between domains, two adjacent domains of the mouse CD22 protein were swapped with the corresponding sequences of the human CD22 protein. To obtain single-domain specific results, epitope clustering constructs were designed to overlap each other. The swapped sequence regions include two CD22 domains, an amino acid stretch between the swapped domains, and an amino acid stretch between the second swapped domain and the subsequent CD22 domain. [Figure 21-3]Sequence alignment of human and mouse CD22. Top sequence: human CD22, bottom sequence: mouse CD22. Sequence alignment of CD22 proteins shows which mouse sequence portions (V+C2-1, C2-1+C2-2, C2-2+C2-3, C2-3+C2-4, C2-4+C2-5, C2-5+C2-6) are replaced with the corresponding human sequences, and which amino acids differ between the two species. To ensure structural integrity of the transition region between domains, two adjacent domains of the mouse CD22 protein were swapped with the corresponding sequences of the human CD22 protein. To obtain single-domain specific results, epitope clustering constructs were designed to overlap each other. The swapped sequence regions include two CD22 domains, an amino acid stretch between the swapped domains, and an amino acid stretch between the second swapped domain and the subsequent CD22 domain. [Figure 22-1] Flow cytometry-bound analysis of CD22 antibodies and T cell engager molecules against HEK cell membrane fragments: truncated CD22 protein - control (i.e., known antibody) (A), mouse / human chimeric CD22 protein - control (i.e., known antibody) (B), truncated CD22 protein - CD22 T cell engager molecule disclosed herein (C). [Figure 22-2] Flow cytometry-bound analysis of CD22 antibodies and T cell engager molecules against HEK cell membrane fragments: truncated CD22 protein - control (i.e., known antibody) (A), mouse / human chimeric CD22 protein - control (i.e., known antibody) (B), truncated CD22 protein - CD22 T cell engager molecule disclosed herein (C). [Figure 23-1]FACS-based cytotoxicity assay for testing the CD20×CD22 antigen-binding molecules U4U, Z3L, G3P, Y3N, B5K, and C8V disclosed herein against target cells CHO ff / Luc pCMV / hu orl CD22 v1 pEFDHFR without effector cell stimulation. PBMC #263 (E:T ratio 50.000:5.000 S100μl) in RPMI plus+10% FCS at an initial concentration of 160nM and a 1:6 dilution on an F-bottom plate (A) against target cells CHO huCD20 pEFDHFR / ffLuc pCMV without effector cell stimulation. A LUC-based cytotoxicity assay to test the CD20×CD22 antigen-binding molecules Y3N and CD20 antigen-binding molecules T9J and S3 disclosed herein against unstimulated target cells huCHO CD22+ effector cells with PBMC #263 (E:T ratio 50.000:5.000 S100 μl) in RPMI plus+10% FCS at an initial concentration of 160 nM and a 1:6 dilution on a 384-wellPBMC #773 (E:T ratio 25.000:2.500 S50 μl) in RPMI plus+10% FCS at an initial concentration of 160 nM and a 1:6 dilution. PBMC #773 in RPMI plus + 10% FCS at an initial concentration of 160 nM and a 1:6 dilution on a 384-well F-bottom plate (D) (E:T ratio 25.000:2.500 S 50 μl). [Figure 23-2]FACS-based cytotoxicity assay for testing the CD20×CD22 antigen-binding molecules U4U, Z3L, G3P, Y3N, B5K, and C8V disclosed herein against target cells CHO ff / Luc pCMV / hu orl CD22 v1 pEFDHFR without effector cell stimulation. PBMC #263 (E:T ratio 50.000:5.000 S100μl) in RPMI plus+10% FCS at an initial concentration of 160nM and a 1:6 dilution on an F-bottom plate (A) against target cells CHO huCD20 pEFDHFR / ffLuc pCMV without effector cell stimulation. A LUC-based cytotoxicity assay to test the CD20×CD22 antigen-binding molecules Y3N and CD20 antigen-binding molecules T9J and S3 disclosed herein against unstimulated target cells huCHO CD22+ effector cells with PBMC #263 (E:T ratio 50.000:5.000 S100 μl) in RPMI plus+10% FCS at an initial concentration of 160 nM and a 1:6 dilution on a 384-wellPBMC #773 (E:T ratio 25.000:2.500 S50 μl) in RPMI plus+10% FCS at an initial concentration of 160 nM and a 1:6 dilution. PBMC #773 in RPMI plus + 10% FCS at an initial concentration of 160 nM and a 1:6 dilution on a 384-well F-bottom plate (D) (E:T ratio 25.000:2.500 S 50 μl). [Figure 24] Three dual-targeting CLL1×FLT3 antigen-binding molecules with three different configurations of the domains described below were tested for cytotoxic activity against huCHO FLT3-positive cells, huCHO CLL1-positive cells, and dual-positive cells (DT): Form 1 1CL1 9-G4 CC×I2C0×scFc×FL 4-E9 CC(A), Form 2 CL1 9-G4 CC×FL 4-E9 CC×I2C0×ScFc(B), and Form 3 CL1 9-G4 CC×ScFc×4-E9 CC×I2C0(C). [Modes for carrying out the invention]

[0053] In connection with the present invention, a multitarget antigen-binding molecule is provided, comprising at least three binding domains, wherein the first and second binding domains in the amino-to-carboxyl direction can preferably simultaneously target two target cell surface antigens associated with malignant tumors, and the third binding domain binds to an extracellular epitope of human and / or macaque CD3ε chain on effector cells, which are T cells.

[0054] In relation to the present invention, it is a remarkable finding that the T cell engagement multispecific antigen-binding molecule according to the present invention is preferably suitable for targeting two (different) antigens on one target cell (e.g., cancer cell), and conversely, targets fewer non-cancer cells. By being able to address two target antigens simultaneously, (a) the possibility of targeting target cells such as cancer cells increases significantly when such target cells undergo antigen deficiency, and therefore, since one effective antigen remains as a target on cells that have undergone antigen evasion, tumors tend to evade effective antitumor therapy, and (b) the possibility of targeting disease-related target cells instead of physiological cells increases significantly when two TAAs typically associated with disease-related target cells are selected instead of physiological cells. In this regard, the Specified Prototype of multitarget antigen-binding molecule is envisioned that not only prevents antigen evasion in the tumor environment, for example, but also further broadens the range of treatment by addressing cells having a pattern of two antigens typically associated with a particular disease, for example. Therefore, biological tissues in which cells express only one of the two targets are not addressed by the immediate dual-target antigen-binding molecule. In particular, the selective gap can be achieved by a dual-targeting molecule of the form described herein, for example, having a dual-specificity entity comprising, for example, a target-binding domain (or conjugate, as used synonymously throughout this disclosure) and a CD3 conjugate, further target conjugates and optionally a half-life extension domain (e.g., an scFc domain). The dual-targeting antigen-binding molecules described herein typically feature an EC50 of less than 100 pM for cells positive for both targets, preferably less than 50 pM, more preferably less than 30 pM, and even more preferably less than or equal to about 10 pM, although such dual-targeting molecules typically exhibit significantly higher EC50 values ​​(e.g., at least 50 pM, 100 pM, 250 pM, and even more than 500 pM) when used with single-targeting cells.This finding suggests that the multi-targeting molecules of the present invention may be advantageously used to specifically target pathogenic target cells that express both targets and to which the molecules can simultaneously bind in order to induce T cell-mediated cytotoxicity, exhibiting a selective gap in activity of at least 10-fold, preferably at least 20-fold, and even more 30-fold. This may reduce off-target toxicity and associated side effects, and, based on the concepts immediately described, may provide safer treatment. Thus, the T cell engagement multi-targeting antigen-binding molecules of the present invention, which are typically single-stranded, offer improvements in efficacy and safety compared to existing bispecific antibodies or antigen-binding molecules that bind to T cells. The advantageous properties are preferably achieved by the fact that the first and second binding domains of this multi-targeting antigen-binding molecule can maintain their biological activity independently of each other, i.e., each can bind to its respective target without being sterically hindered by the target to which the other binding domain and / or each other target conjugate is bound. Preservation of biological activity is preferably achieved by (a) a VH-VL configuration of both binding domains in the amino-to-carboxyl direction, and / or (b) careful selection of a linker connecting the first and second binding domains. The linker needs to have a length that ensures both the biological activity of both binding domains and sufficient (chemical) stability of this construct. Surprisingly relatively short peptide linkers with a length of about 5 to 24 amino acids, preferably 5 to 18, more preferably 6 or 12 amino acids, satisfy both requirements. Preferably, such linkers are rich in small or hydrophilic amino acids such as Gly and Ser, because such a composition preferably provides mobility. As a result, such mobility preferably allows interaction of each binding domain independently of other binding domains of the multi-targeted antigen-binding molecule according to the present invention. At the same time, it is remarkable that even such short, preferably mobile, peptide linkers typically provide sufficient spatial separation between the first and second binding domains so that both domains retain the bioactivity required to have a therapeutically useful molecule in relation to the present invention.A further advantage of such short linkers disclosed in connection with the present invention is that inter-chain mispairing is preferably prevented compared to longer linkers.

[0055] The specific findings described above, which form the basis of this invention, are surprising considering the teachings of the prior art. For example, Liu et al. showed that the longer the interpeptide linker, the more the independent folding and biological activity of the two molecules are conserved (Liu ZG, Lin JB, Du W, et al. Anti-proteolysis study of recombinant IIn-UK fusion protein in CHO cell. Prog Biochem Biophys 2005;32:544-50). An excessively short linker between binding domains (preferably scFv binding domains) negatively affects protein folding due to spatial occupancy, while an excessively long linker enhances the antigenicity of scFv antibodies and also affects the functionality and activity of scFv antibodies. Xu et al. teach that sufficient length and certain sequence characteristics are important factors for two halves to have enough space to perform their functions, and that avoiding the formation of α-helices and β-sheets is important for stability (Xue F, Gu Z, Feng JA. LINKER: a web server to generate peptide sequences with extended conformation. Nucleic Acids Res 2004;32:W562-5). Therefore, those skilled in the art who aim to maintain the distance between binding domains will typically intend to employ a helical structure or a proline-rich rigid linker. However, the length of the rigid linker also has a significant impact on the biological activity of the protein. McCormick et al. investigated the rigid peptide linker (Ala-Pro)n (10-34 aa) applied by the interferon-γ-gp120 fusion protein (McCormick A, Thomas M, Heath A. Immunization with an interferon-gamma-gp120 fusion protein induces enhanced immune responses to human immunodeficiency virus gp120. J Infect Dis. 2001;184:1423-1430).Due to the short linker of 10 aa residues, this fusion protein exhibited relatively low biological activity of interferon-γ. By lengthening this linker, the biological activity of the fusion protein gradually improved, peaking at 88% of the activity of free interferon-γ with the longest linker of 34 residues. Furthermore, in some cases, even with the insertion of mobile or rigid linkers, the reduction in biological activity due to steric hindrance between domains could not be overcome (Bai Y, Ann DK, Shen WC. Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent. Proc Natl Acad Sci US A. 2005;102:7292-7296).

[0056] Given the known hindrances in the art, those skilled in the art are urged to avoid short, mobile, or even rigid linkers and turn to longer, rigid linkers, where “long” can be understood from the art as approximately 30 amino acids, preferably containing proline. Based on this information, those skilled in the art will preferably use state-of-the-art modeling techniques to model the first and second binding domains linked by the peptide linker and determine which linker length should be taken and which should be avoided. If the linker is a mobile linker rich in Ger and Ser, a linker length of 30 amino acids typically results in a fairly large space between the first and second binding domains (typically at least 70 Å, more typically at least 80 Å), which those skilled in the art will consider a safe size for accommodating a second target cell surface antigen (e.g., TAA2) to facilitate binding by the second binding domain of a multitarget antigen-binding molecule. In relation to the present invention, it is important to note that the first binding domain (i.e., the N-terminal binding domain) is relatively easily activated because it has only one adjacent binding domain which may cause steric hindrance during binding to the target, while the second binding domain is connected to the first binding domain in the N direction.

[0057] Typically, when the SGGGGS linker is modeled between two target-binding domains that are scFv (e.g., MSLN × EpCAM), when three (GGGGS) linkers are modeled between VH and VL within the binding domain, when the first binding domain (e.g., anti-MSLN binding domain) is fixed, and when three possible expected three-dimensional structures (linkers oscillating in different orthogonals) are applied (linker three-dimensional structures 1, 2, and 3, respectively), a complete collision is observed in the case of linker position 3, while no collision is observed in positions 1 and 2. However, this space is still insufficient to accommodate TAA2 EpCAM, based on the location where the CDR is preferably located within the second binding domain of the multi-targeted antigen-binding molecule according to the present invention. Thus, this result strongly indicates the need for a longer linker between the two target-binding domains. Those skilled in the art will predict that, using the size of the target EpCAM as a guide, a better linker would preferably have at least about 30 residues, and less preferably at least 20 residues (i.e., a preferred distance of 70 Å divided by 3.8 per aa). Therefore, due to the lack of space, short linker solutions such as the SGGGS linker and its short multiplicity (e.g., S(G4S)2 and S(G4S)2 between the two target-binding domains according to the present invention) are undesirable and thus a non-trivial choice for this structure of the target conjugate in multi-targeted antigen-binding molecules (particularly the bi-targeted BiTE® molecule). The same applies to a 12 aa linker, which typically grants a small maximum usable space of about 35 Å, which can be up to about 50 Å depending on the context, and therefore a typical target to be bound, which is at least about 45, 50, 55, 60, 65, 70, 75, 80, or 85 Å in size, would not be able to safely accommodate.Similarly, a linker with 18 aa lengths (e.g., SGGGGSGGGGSGGGGSGG) where the maximum available space between binding domains in the structures disclosed herein is 60 Å or less, typically 55 Å or less, for example 54–60 Å, may make binding to a second TAA2 of exemplary size 45–70 Å impossible. In contrast, a linker with 30 aa lengths typically provides a maximum space of 84–94 Å, thus enabling the target conjugate to securely bind to an exemplary target EpCAM of approximately 45–70 Å. Therefore, those skilled in the art will likely select a linker with at least 18 aa lengths to ensure binding to a second TAA2 in MSLN×EpCAM HLE dual BiTE®, for example, as a multi-targeted antigen-binding molecule according to the present invention. It should be noted that the above considerations are based on mobile linkers with high Ser and / or Gly content. Those skilled in the art will anticipate that, in order to maintain the biological function of the target-binding domain, a less mobile linker may require more amino acids to ensure a length sufficient to maintain the distance between two adjacent target-binding domains according to the present invention.

[0058] The bispecific antigen-binding molecule according to the present invention is expected to have cross-reactivity with, for example, cynomolgus monkey tumor antigens (e.g., CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, and EpCAM). In connection with the present invention, two targets may be addressed simultaneously by a single multi-targeting antigen-binding molecule, and this bi-targeting, as described throughout herein, for example, (i) the presence of a soluble target (e.g., soluble BCMA) that would normally "mask" the target on target cells by binding to an antibody-based drug without enabling any therapeutic effect, and (ii) the low risk of cell surface BCMA expression in the process of antigen deficiency as a driving factor for tumor avoidance.

[0059] For example, the multi-targeted antigen-binding molecule according to the present invention (e.g., constructs for CS1 as TAA1 and BCMA as TAA2) is suitable for use in the treatment of multiple myeloma (MM). This multi-targeted antigen-binding molecule is particularly suitable for achieving effective exposure despite the presence of a soluble target (e.g., BCMA), where antigen deficiency may lead to resistance. BCMA and CS1 are good partners in combination due to their broad MM expression and the fact that their normal expression is restricted compared to other MM antigens (e.g., CD38). The BCMA × CS1 HLE (extended half-life) dual BiTE® antigen-binding molecule disclosed herein as an example is manufacturable, has an acceptable safety profile, and exhibits increased efficacy because the multi-targeted BiTE® antigen-binding molecule induces lysis of CS-expressing cells and / or BCMA-expressing cells and is active in the presence of high concentrations of sBCMA and / or sCS1.

[0060] In relation to the present invention, it is particularly assumed that a multi-targeted antigen-binding molecule, preferably one that addresses two different target cell surface antigens, is thereby highly specific to its target cells and therefore preferably safe in the therapeutic use of this molecule. This has been demonstrated in cynomolgus monkey toxicity studies. Exemplary CS1×BCMA HLE dual BiTE® antigen-binding molecules typically exhibit good tolerability and, despite peripheral CS1 target expression, typically do not cause cytokine release syndrome (CRS, a typical but serious side effect of T cell redirection therapy). Peripheral CS1-expressing NK and T cells are preferably unaffected. The histopathological findings of the multi-targeted BiTE® antigen-binding molecule are comparable to those of the BiTE® antigen-binding molecule having only a binding domain for TAA BCMA. Furthermore, typically, depletion of plasma cells from the periphery and BM mediated by the multi-targeted BiTE® antigen-binding molecule has been observed and correlates with exposure. Furthermore, when J-chain mRNA levels are plotted against the BiTE® concentration of the CS1 HLE BiTE® antigen-binding molecule versus the BCMA HLE BiTE® antigen-binding molecule versus the monospecific CS1×BCMA HLE BiTE® antigen-binding molecule, the data preferably show that the exemplary CS1×BCMA HLE BiTE® antigen-binding molecule induces deeper target cell depletion compared to either the CS1 HLE BiTE® antigen-binding molecule or BCMA HLE BiTE®, indicating a better clinical effect of the multi-targeted antigen-binding molecule according to the present invention. Mesoserine, also known as MSLN, is a 40 kDa protein expressed in mesothelial cells. Mesoserine is a tumor differentiation antigen normally present on mesothelial cells lining the pleura, peritoneum, and pericardium. Since MSLN is overexpressed in several cancers and is immunogenic, this protein may be used as a tumor marker or an antigenic target for therapeutic cancer vaccines.

[0061] Preferred target cell surface antigens related to the present invention are CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, and EpCAM. Typically, target cell surface antigens related to the present invention are tumor-associated antigens (TAAs). [ka] It is a member of the CD2 subset of the immunoglobulin superfamily (IgSF) expressed on NK, T, and stimulated B cells. It is also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17) and is a B cell maturation antigen. [ka] BCM (or B lymphocyte antigen) is a protein expressed in mature B lymphocytes. [ka] It is expressed on the surface of all B cells, starting in the pre-B phase (CD45R+, CD117+) and gradually increasing in concentration until maturation. [ka] Alternatively, surface antigen classification 22 is a molecule belonging to the SIGLEC family of lectins. This molecule is found on the surface of mature B cells and to a relatively low degree on some immature B cells. Fms-like tyrosine kinase 3 [ka] It is also known as surface antigen classification 135 (CD135), receptor tyrosine-protein kinase FLT3, or fetal liver kinase-2 (Flk2). FLT3 is a cytokine receptor belonging to receptor tyrosine kinase class III. CD135 is the receptor for the cytokine FLT3 ligand (FLT3L). The FLT3 gene is frequently mutated in acute myeloid leukemia (AML). Interleukin-3 receptor [ka] This molecule is found on cells and plays a role in the signal transduction of interleukin-3, an important soluble cytokine in the immune system. It is a type C lectin-like receptor. [ka] It is also known as CLEC12A or MICL. It contains an ITIM motif in the cytoplasmic tail that may be associated with the signaling phosphatases SHP-1 and SHP-2. Human MICL is primarily expressed as a monomer on myeloid cells (e.g., granulocytes, monocytes, macrophages, and dendritic cells) and is associated with AML. Mesoserine [ka] Cadherin-3 is a 40 kDa protein expressed in mesothelial cells and overexpressed in several human tumors. It is also known as P-cadherin. [ka] It is a calcium-dependent cell-to-cell adhesion glycoprotein composed of five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail. It is associated with several types of tumors. Epithelial cell adhesion molecule [ka] EpCAM is a transmembrane glycoprotein that mediates Ca2+-independent isoplastic cell-cell adhesion in epithelium. EpCAM is oncogenic and is thought to play a role in tumorigenesis and metastasis of carcinomas.

[0062] Furthermore, optionally but advantageously in relation to the present invention, the multi-targeted antigen-binding molecule is envisioned to possess a fourth domain (typically an scFc domain, i.e., HLE), enabling intravenous administration at a frequency of as little as once a week, once every two weeks, once every three weeks, or even once every four weeks or less.

[0063] For example, mapping was performed as described herein to determine the epitopes of preferred multi-targeted antigen-binding molecules according to the present invention directed toward the CD20 epitope. The extracellular region of the human CD20 protein was divided into the following two parts: (1) Extracellular loop 1 called E1 (ECL1, amino acids 72-84, see reference in Example 17) and Extracellular loop 2 called E2 (ECL2). Extracellular loop 1 (E1) was further divided into two subparts called E1A (aa72-79) and E1B (aa80-84). Extracellular loop 2 (E2, aa142-188) was further divided into four subparts called E2A (aa142-161), E2B (aa162-166), E2C (aa167-175), and E2D (aa176-188). Surprisingly, we found that the CD20 antigen-binding molecule exhibits preferably higher cytotoxic activity when bound to (i) E1A, E2B, and E2C epitopes, or (ii) E2A and E2B epitopes, in both single-target and dual-target configurations. Similarly, to characterize the epitope, the extracellular region of the human CD22 protein was divided into the following seven parts: V (aa20~142 as defined by Uniprot P20273+RPFP), C2-1 (aa143~241 as defined by Uniprot P20273+LNVKHT), C2-2 (aa242~330 as defined by Uniprot P20273+VQYA), C2-3 (aa331~418 as defined by Uniprot P20273+YP), C2-4 (aa419~504 as defined by Uniprot P20273+VQYA), C2-5 (aa505~592 as defined by Uniprot P20273+KAWTLEVLYA), and C2-6 (Uniprot (as defined by P20273+VYYSPETIGRR, aa593~687). Surprisingly, we found that the CD22 antigen-binding molecule exhibits preferably higher cytotoxic activity when bound to the C2-1 epitope, in both single-targeting and dual-targeting configurations.

[0064] It is particularly remarkable that the multispecific antigen-binding molecule according to the present invention can preferably bind to two different targets simultaneously, despite the short linker between the target-binding domains. Simultaneous binding has been demonstrated herein for several targets. However, this is remarkable considering the typical distance between the targets. For example, CD20 contains two small extracellular domains with only 13 aa(E1) and 47 aa(E2). In contrast, CD22 contains an extracellular domain the length of a 7Ig domain with 676 aa. However, despite the significant differences in extracellular size and composition, the multispecific antigen-binding molecule according to this invention can successfully target both TAA CD20 and CD22 simultaneously, for the advantages of high efficacy and low toxicity. This is preferably achieved.

[0065] The antigen-binding molecules of the present invention are preferably multi-targeting and bispecific. However, monospecific (for one target) and bispecific antigen-binding molecules comprising (i) a first binding domain for one cell surface target antigen and a second binding domain which is preferably an effector binding domain that binds to CD3e, or (ii) a first and second binding domain for the same cell surface target and a third binding domain which is preferably an effector binding domain that binds to CD3e, are also included in the present invention, and the cell surface target antigens are selected from CS1, CD20, CD22, CD123, and CLL1. For example, a CS1×CD3 bispecific antigen-binding molecule (i.e., a molecule that directs CS1 only to the target cell surface antigen after the effector CD3) has undergone long-term clinical evaluation and assessment with BCMA×CD3 antibodies and EC 50 They demonstrate comparable efficacy in terms of EC50 values. Similarly, for example, the CD20 antigen-binding molecules described herein are characterized by high activity against CD20-containing targets in terms of EC50 values, particularly when addressing the specific epitopes disclosed herein.

[0066] In relation to the present invention, preferred multispecific antigen-binding molecules are expected to exhibit not only a favorable ratio of cytotoxicity and affinity, but also sufficient stability properties to facilitate practical handling when formulating, storing, and administering the construct. Sufficient stability is characterized by a high monomer content (i.e., undenatured molecules that are not aggregated and / or associated) after standard preparation, such as at least 65%, more preferably at least 70%, and even more preferably at least 75%, as determined, for example, by preparative size exclusion chromatography (SEC). Furthermore, the turbidity measured at 340 nm as light absorption at a concentration of 2.5 mg / ml should preferably be 0.025 or less, more preferably 0.020 or less, to conclude, for example, the essential absence of undesirable aggregates. Advantageously, the high monomer content is maintained after incubation under stress conditions such as freeze / thaw or incubation at 37 or 40°C. Furthermore, the multispecific antigen-binding molecules according to the present invention have at least equivalent or even higher thermal stability compared to bispecific antigen-binding molecules that typically have only one target-binding domain but otherwise include a CD3-binding domain and optionally a half-life-extending scFc domain (i.e., are structurally less complex). Those skilled in the art would expect that more structurally complex protein-based molecules would be less susceptible to thermal degradation and other degradation (i.e., have lower thermal stability). Surprisingly, however, the opposite is true; for example, the CS1×BCMA or BCMA×CS1 multispecific antigen-binding molecules according to the present invention exhibit higher thermal stability, lower monomer loss after storage, higher monomer percentage after three freeze-thaw cycles, and higher protein homogeneity compared to the CS1 or BCMA bispecific single-target antigen-binding molecules disclosed herein. The same is true for the CD123×FLT3 multispecific antigen-binding molecule disclosed herein, with respect to the FTL3 bispecific antigen-binding molecule disclosed herein.

[0067] Therefore, the present invention relates to a multispecific antigen-binding molecule, (i) A first binding domain that specifically binds to a first target cell surface antigen (e.g., TAA1), (ii) A second binding domain that specifically binds to a second target cell surface antigen (e.g., TAA2), and (iii) A third binding domain that binds to an extracellular epitope of the human and / or macaque CD3ε chain, wherein the first, second and third binding domains are arranged in the order of amino to carboxyl, and the first and second binding domains are linked by a peptide linker having a length of 5 to 25 (preferably 5 to 18 or 6 to 16) amino acids, and optionally, (iv) A fourth domain comprising two polypeptide monomers, each containing a hinge, a CH2, and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker. This invention provides a multispecific antigen-binding molecule containing the following:

[0068] In one embodiment, the present invention provides a multispecific antigen-binding molecule comprising all four such domains. In a preferred embodiment, domains (i), (ii), (iii), and (iv) are arranged in the N-to-C direction (Form 2). However, instead, this multispecific antigen-binding molecule may have domains arranged in the order (ii), (iii), (iv), and (i) (Form 1) or (ii), (iv), (i), and (iii) (Form 3) in the N-to-C direction. Surprisingly, all arrangements (a) confer significant efficacy with respect to target cytotoxicity and (b) can be manufactured with acceptable product quality. As a general requirement for the multitargeted bispecific antigen-binding molecule of the present invention, one target antigen domain must be positioned adjacent to the N-terminus of the effector CD3-binding domain in order to act as a bispecific entity and thereby form a cytolytic synapse between the target cell and the effector T cell (preferably bi-positive).

[0069] In this specification, the term "polypeptide" is understood to mean an organic polymer comprising at least one continuous, unbranched amino acid chain. Polypeptides comprising multiple amino acid chains are also envisioned in connection with the present invention. The amino acid chains of a polypeptide typically contain at least 50 amino acids, preferably at least 100, 200, 300, 400, or 500 amino acids. Furthermore, in connection with the present invention, the amino acid chains of a polymer may also be linked to entities not composed of amino acids.

[0070] The term “antigen-binding polypeptide” in the present invention is preferably a polypeptide that immunospecifically binds to a target or antigen of the polypeptide. This polypeptide typically includes or comprises domains derived from the heavy chain variable region (VH) and / or light chain variable region (VL) of an antibody. The polypeptide according to the present invention includes the minimum structural requirements of an antibody that enable immunospecific target binding. These minimum requirements can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region), and preferably by the presence of all six CDRs. Thus, a T-cell engagement polypeptide may be characterized by the presence of three or six CDRs within either one or both binding domains, and the location (and order) of these CDRs within the binding domain is known to those skilled in the art. Typically, “antigen-binding molecule” is understood to mean “antigen-binding polypeptide” as relating to the present invention.

[0071] Instead, in relation to the present invention, an antigen-binding polypeptide corresponds to an “antibody construct,” which typically refers to a molecule whose structure and / or function are based on the structure / function of an antibody (e.g., a full-length immunoglobulin molecule or a whole immunoglobulin molecule). Thus, an antigen-binding molecule can bind to its specific target or antigen and / or is derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of the antibody or its fragment. Furthermore, a domain that binds to a binding partner according to the present invention is understood herein as the binding domain of the antigen-binding molecule according to the present invention. Typically, the binding domain according to the present invention includes the minimum structural requirements of the antibody that enable target binding. These minimum requirements can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region), preferably all six CDRs. Alternative methods for defining the minimum structural requirements of an antibody include defining the structure of a specific target, defining the antibody's epitope within the protein domains (epitope clusters) of the target protein that constitute the epitope region, or referencing a specific antibody that competes with the defined antibody's epitope. Antibodies on which the construct according to the present invention is based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0072] The binding domain of the antigen-binding molecule according to the present invention may include, for example, the CDRs of the group referenced above. Preferably, these CDRs are contained within the framework of the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), but do not necessarily contain both. An Fd fragment, for example, has two VH regions and often retains the antigen-binding function of a portion of the intact antigen-binding domain. Further examples of antibody fragments, antibody variants, or binding domains include (1) Fab fragments, which are monovalent fragments having VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, which are bivalent fragments having two Fab fragments linked by disulfide crosslinks in the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains in one arm of the antibody; (5) dAb fragments having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity-determining regions (CDRs); and (7) single-stranded Fv (scFv), the latter of which is preferred (e.g., those derived from scFV libraries). Examples of embodiments of the antigen-binding molecule according to the present invention are described, for example, in International Publication No. 00 / 006605, International Publication No. 2005 / 040220, International Publication No. 2008 / 119567, International Publication No. 2010 / 037838, International Publication No. 2013 / 026837, International Publication No. 2013 / 026833, U.S. Patent Application Publication No. 2014 / 0308285, U.S. Patent Application Publication No. 2014 / 0302037, International Publication No. 2014 / 144722, International Publication No. 2014 / 151910, and International Publication No. 2015 / 048272.

[0073] The definition of “binding domain” or “domain that binds to ~” also includes fragments of full-length antibodies such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or “rIgG” (“half-antibody”). Antigen-binding molecules according to the present invention may also include modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabody, single-stranded diabody, tandem diabody (Tandab's), tandem di-scFv, tandem tri-scFv, “multibody,” such as triabody or tetrabody, and single-domain antibodies, such as nanobody, or single variable-domain antibodies containing only one variable domain, which may be VHH, VH, or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0074] As used herein, the terms “single-chain Fv,” “single-chain antibody,” or “scFv” refer to a single polypeptide chain antibody fragment that contains variable regions derived from both the heavy and light chains but lacks a constant region. Typically, single-chain antibodies further include a polypeptide linker between the VH and VL domains, enabling the formation of a desired structure that allows binding to an antigen. Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods for generating single-chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; and Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, single-chain antibodies may be bispecific, multispecific, human and / or humanized and / or synthetic.

[0075] Furthermore, the definition of the term "antigen-binding molecule" preferably includes a polyvalent / multivalent construct, and therefore includes a bispecific molecule (bispecificity means that it specifically binds to two cell types containing different antigenic structures (i.e., target cells and effector cells)). Since the antigen-binding molecule of the present invention is preferably multitargeted, it is typically a polyvalent / multivalent molecule that specifically binds to three or more (preferably three) antigenic structures via different binding domains related to the present invention, which are two target-binding domains and one CD3-binding domain. Furthermore, the definition of the term "antigen-binding molecule" includes molecules consisting of only one peptide chain and molecules consisting of multiple polypeptide chains (these chains may be identical (homodimers, homotrimers, or homoolibomers) or different (heterodimers, heterotrimers, or heterooligomers)). Such molecules, which contain multiple polypeptide chains (i.e., typically two chains), have these chains typically linked to each other as heterodimers by charge pair bonds within a hetero-Fc entity, for example, at the C-terminal position of the CD3 conjugate described herein, and functioning as a half-life extension portion. Examples of antigen-binding molecules (e.g., antibody-based molecules) identified above are described in particular in Harlow and Lane, Antibodies: A Laboratory Manual, CSHL Press (1988) and Using Antibodies: A Laboratory Manual, CSHL Press (1999), Kontermann and Duebel, Antibody Engineering, Springer, 2nd ed. 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0076] The term “bispecificity,” as used herein, refers to an antigen-binding molecule that is “at least bispecific,” i.e., the antigen-binding molecule addresses two different cell types, i.e., targets effector cells, and comprises at least a first binding domain and a second binding domain, where at least one of the binding domains preferably binds to an antigen or target selected from CS1, BCMA, CD20, CD22, FLT3, CD123, MSLN, CLL1, and EpCAM, and the other binding domain of the same molecule binds to another antigen or target (CD3 in this specification). Thus, the antigen-binding molecule according to the present invention comprises specificity for at least two different antigens or targets. For example, one domain preferably does not bind to one or more extracellular epitopes of CD3e species described herein.

[0077] The term "target cell surface antigen" refers to an antigenic structure expressed by a cell and present on the cell surface in a manner accessible to the antigen-binding molecules described herein. A preferred target cell surface antigen in relation to the present invention is a tumor-associated antigen (TAA). This surface antigen may be a protein (preferably the extracellular portion of a protein) or a carbohydrate structure (preferably a protein-carbohydrate structure such as a glycoprotein). This surface antigen is preferably a tumor antigen. The term "bispecific antigen-binding molecule" in the present invention also includes triple-specific antigen-binding molecules containing three binding domains or multispecific antigen-binding molecules such as constructs having more than three (e.g., four, five...) specificities.

[0078] Preferred in relation to the present invention are molecules that are "multispecific," as understood herein, "at least bispecific." In this regard, multispecific molecules, such as antigen-binding molecules, are specific to effectors such as CD3 (more preferably CD3e) and at least two target cell surface antigens. The specificity is conferred by each binding domain as defined herein. Typically, "multispecific" refers to a molecule that is specific to two different target cell surface effectors, and therefore, multispecificity confers a preferred property of the multispecific antigen-binding molecule according to the present invention (i.e., reduction of antigen deficiency and increased therapeutic range or good high tolerability).

[0079] If the antigen-binding molecule according to the present invention is (at least) bispecific, it does not exist in nature and is significantly different from naturally occurring products. Therefore, a "bispecific" antigen-binding molecule or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sites with different specificities. Bispecific antigen-binding molecules can be generated by various methods, such as hybridoma fusion or Fa' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

[0080] At least three binding domains and variable domains (VH / VL) of the antigen-binding molecule of the present invention typically include a peptide linker (spacer peptide). According to the present invention, the term "peptide linker" includes an amino acid sequence that links the amino acid sequences of one (variable and / or binding) domain and the other (variable and / or binding) domain of the antigen-binding molecule of the present invention to each other. The peptide linker between the first and second binding domains, which can simultaneously bind to two targets, preferably different targets (e.g., TAA1 and TAA2), is preferably mobile and of limited length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids). The peptide linker may also be used to fuse the third domain to other domains of the antigen-binding molecule of the present invention. An essential technical feature of such a peptide linker is that it does not contain any polymerization activity. Some suitable peptide linkers are described in U.S. Patent Nos. 4,751,180 and 4,935,233 or in International Publication No. 88 / 09344. Peptide linkers can also be used to attach other domains, modules, or regions (e.g., half-life extension domains) to the antigen-binding molecule of the present invention. However, typically, the linker between the first and second target-binding domains differs from the in-conjugate linker that links the VH and VL within the target-binding domain. The difference is that the linker between the first and second binding domains has one more amino acid than the in-conjugate linker (e.g., six and five amino acids, such as SGGGGS versus GGGGS). Surprisingly, this simultaneously confers mobility and stability in the specific antigen-binding molecule formats described herein.

[0081] The antigen-binding molecule of the present invention is preferably an "in vitro-generated antigen-binding molecule." This term refers to an antigen-binding molecule as defined above, in which all or part of the variable region (e.g., at least one CDR) is generated by any other method that allows for the selection of non-immune cells, e.g., in vitro phage display, protein chip, or testing of candidate sequences with respect to antigen-binding ability. Accordingly, this term preferably excludes sequences that are generated solely by genomic rearrangement in animal immune cells. A "recombinant antibody" is an antibody produced by the use of recombinant DNA technology or genetic engineering.

[0082] As used herein, the term “monoclonal antibody” (mAb) or “monoclonal antigen-binding molecule” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., an individual antibody that is identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and are induced to a single antigenic site or determinant on an antigen, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies induced to different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are therefore free from contamination by other immunoglobulins. The modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0083] Any technique that yields antibodies produced by continuous cell line culture can be used to prepare monoclonal antibodies. For example, the monoclonal antibodies used may be produced by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Further examples of techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0084] Next, hybridomas can be screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis, e.g., Biacore®, to identify one or more hybridomas that produce antibodies that specifically bind to a specified antigen. Any form of the relevant antigen can be used as an immunogen, for example, recombinant antigen, a naturally occurring form, any variant or fragment thereof, and its antigenic peptide. Surface plasmon resonance, as employed in the Biacore system, can be used to enhance the efficiency of phage antibody binding to the epitope of the target cell surface antigen (Schier, Human Antibodies Hybridomas 7(1996), 97-105; Malmborg, J.Immunol.Methods 183(1995), 7-13).

[0085] Another exemplary method for generating monoclonal antibodies involves screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al., Nature 352:624-628 (1991); and Marks et al., J.Mol.Biol. 222:581-597 (1991).

[0086] In addition to using display libraries, non-human animals, such as rodents (mice, hamsters, rabbits, or rats), can be immunized using relevant antigens. In one embodiment, the non-human animal contains at least a portion of the human immunoglobulin gene. For example, a mouse strain lacking mouse antibody production can be modified using a large fragment of the human Ig (immunoglobulin) locus. Using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with desired specificity can be generated and selected. See, for example, XENOMOUSE®, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, International Publication No. 96 / 34096 and International Publication No. 96 / 33735.

[0087] Monoclonal antibodies, after being obtained from non-human animals, can be modified using recombinant DNA techniques known in the art, such as humanization, deimmunization, and chimerization. Examples of modified antigen-binding molecules include humanized variants of non-human antibodies, "affinity-mature" antibodies (see, e.g., Hawkins et al. J.Mol.Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants with altered effector function (see, e.g., U.S. Patent No. 5,648,260, Kontermann and Duebel (2010) and Little (2009) cited above).

[0088] In immunology, affinity maturation is the process by which B cells produce antibodies with increased affinity to an antigen during an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with continuously increasing affinity. Similar to natural prototypes, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation is used without issue to optimize antibodies, antigen-binding molecules, and antibody fragments. Random mutations within the CDR are introduced using radiation, chemical mutagens, or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinity in the low nanomolar range.

[0089] A preferred type of amino acid substitution variant of an antigen-binding molecule involves substitution of one or more hypervariable region residues of the parent antibody (e.g., a humanized antibody or a human antibody). Generally, variants selected for further development will have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus generated are presented in a monovalent form from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystalline structure of the antigen-antibody complex to identify contact sites between the binding domain and, for example, human CS1, BCMA, CD20, CD22, FLT3, CD123, MSLN, CLL1, or EpCAM. Such contact residues and adjacent residues are candidates for substitution by the techniques detailed herein. After generating such variants, a panel of variants may be screened as described herein, and antibodies exhibiting superior properties in one or more relevant assays may be selected for further development.

[0090] The monoclonal antibodies and antigen-binding molecules of the present invention include, in particular, “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain originates from a particular species or is identical or homologous to a corresponding sequence in an antibody belonging to a particular class or subclass of antibodies, while the remainder of the chain originates from another species or is identical or homologous to a corresponding sequence in an antibody belonging to another class or subclass of antibodies and in a fragment of such an antibody, insofar as it exhibits the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The chimeric antibodies of interest as used herein include “primatized” antibodies that contain a variable domain antigen-binding sequence and a human constant region sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.). Various methods for generating chimeric antibodies are described. For example, see Morrison et al., Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent No. 0171496; European Patent No. 0173494; and British Patent No. 2177096.

[0091] Antibodies, antigen-binding molecules, antibody fragments, or antibody variants can also be modified by specific deletion of human T cell epitopes (a method called "deimmunization") by the methods disclosed in the examples of International Publication No. 98 / 52976 or International Publication No. 00 / 34317. Briefly, for peptides that bind to MHC class II, the heavy and light chain variable domains of antibodies can be analyzed. These peptides correspond to potential T cell epitopes (as defined in International Publication No. 98 / 52976 and International Publication No. 00 / 34317). For the detection of potential T cell epitopes, a computer modeling technique called "peptide threading" can be applied, as described in International Publication No. 98 / 52976 and International Publication No. 00 / 34317, and in addition, motifs present in VH and VL sequences can be searched in a database of human MHC class II-binding peptides. These motifs bind to any of the 18 major MHC class II DR allotypes and thus become potential T cell epitopes. 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, though not all, amino acids common to the positions in the antibody sequences of human germline cells can be used. Human germline cells are disclosed, for example, in 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 catalog provides a comprehensive overview of human immunoglobulin variable region sequences (edited by Tomlinson, LA. et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for framework regions and CDRs.For example, the consensus human framework area described in U.S. Patent No. 6,300,064 can also be used.

[0092] A “humanized” antibody, antigen-binding molecule, variant, or fragment thereof (Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) is an antibody or immunoglobulin that is predominantly human, containing minimal sequences derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient’s hypervariable region (also known as the CDR) are replaced with residues from the hypervariable region of a non-human species (e.g., rodents) such as mouse, rat, hamster, or rabbit (donor antibody) that have the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, as used herein, “humanized antibody” 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. A humanized antibody may also contain the immunoglobulin constant region (Fc), usually at least a portion of the constant region of human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0093] Humanized antibodies or fragments thereof can be produced by substituting sequences of Fv variable domains not directly involved in antigen binding with equivalent sequences derived from human Fv variable domains. Exemplary methods for producing humanized antibodies or fragments thereof are provided in Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214 and U.S. Patents 5,585,089, 5,693,761, 5,693,762, 5,859,205 and 6,407,213. These methods involve isolating, manipulating and expressing nucleic acid sequences encoding all or part of an immunoglobulin Fv variable domain derived from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas and other sources that produce antibodies against a given target as described above. Next, recombinant DNA encoding a humanized antibody molecule can be cloned into a suitable expression vector.

[0094] Humanized antibodies can also be produced using transgenic animals, such as mice that express human heavy and light chain genes but cannot express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes exemplary CDR transplantation methods that can be used for the preparation of humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of non-human CDRs, or only a portion of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for the humanized antibody to bind to a given antigen.

[0095] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or reverse mutations. Such modified immunoglobulin molecules can be generated by any of several techniques known in the art (e.g., 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 European Patent No. 239400).

[0096] The terms “human antibody,” “human antigen-binding molecule,” and “human binding domain” include antibodies, antigen-binding molecules, and binding domains having antibody regions such as variable and constant regions or domains that substantially correspond to human germline immunoglobulin sequences known in the art, including those described by Kabat et al. (1991) (cited above). The human antibody, antigen-binding molecule, or binding domain of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis or in vivo somatic mutation) for example, CDR, particularly CDR3. The human antibody, antigen-binding molecule, or binding domain may have at least one, two, three, four, five, or more positions substituted with amino acid residues not encoded by human germline immunoglobulin sequences. As used herein, the definitions of human antibody, antigen-binding molecule, and binding domain also refer to fully human antibodies that contain only the human sequence of an antibody that has not been artificially and / or genetically modified, which can be obtained by using technologies or systems such as Xenomouse. Preferably, a “fully human antibody” does not contain amino acid residues not encoded by human germline immunoglobulins.

[0097] In some embodiments, the antigen-binding molecules of the present invention are “isolated” or “substantially pure” antigen-binding molecules. When used to describe the antigen-binding molecules disclosed herein, “isolated” or “substantially pure” means antigen-binding molecules identified, separated, and / or recovered from components of their production environment. Preferably, the antigen-binding molecules have no or substantially no association with all other components from their production environment. Contaminants in their production environment, such as components arising from recombinant transfected cells, are typically materials that interfere with diagnostic or therapeutic applications of polypeptides, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Antigen-binding molecules may constitute, for example, at least about 5% by weight or at least about 50% by weight of the total protein in a given sample. Isolated proteins are understood to constitute 5% to 99.9% by weight of the total protein content, depending on the environment. Polypeptides can be produced at significantly high concentrations by the use of inducible promoters or high-expression promoters, so that polypeptides are produced at high concentration levels. This definition includes the production of antigen-binding molecules in a variety of organisms and / or host cells known in the art. In preferred embodiments, the antigen-binding molecule is purified (1) using a spinning cup sequencer to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (2) to a degree of homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. However, typically, the isolated antigen-binding molecule is prepared by at least one purification step.

[0098] In relation to the present invention, the term "binding domain" is characterized by a domain that (specifically) binds to / interacts with / recognizes a given target epitope or target site on a target molecule (antigen), such as CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM, respectively, and CD3. The structure and function of the first and / or second binding domain (which recognizes CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM), and preferably the structure and / or function of the effector binding domain (typically a third binding domain that recognizes CD3), are based on the structure and / or function of the antibody, for example, the full-length or full immunoglobulin molecule, and / or derived from the variable heavy chain (VH) and / or variable light chain (VL) domains of the antibody or its fragments. Preferably, the target cell surface antigen-binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The effector (typically CD3) binding domain preferably also includes the minimum structural requirements of the antibody that enable target binding. More preferably, the second binding domain includes at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The first and / or second binding domains are expected to be generated or obtained by phage display or library screening methods other than transplanting CDR sequences derived from existing (monoclonal) antibodies onto a scaffold.

[0099] According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may comprise a proteinaceous portion and a non-proteinaceous portion (e.g., a chemical linker or a chemical crosslinking agent such as glutaraldehyde). A protein (including its fragments, preferably biologically active fragments and peptides having typically fewer than 30 amino acids) comprises two or more amino acids linked to each other through covalent peptide bonds (resulting in a chain of amino acids).

[0100] As used herein, the term “polypeptide” typically refers to a group of molecules consisting of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers, and higher-order oligomers, i.e., they may consist of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc., may be identical or different. The corresponding higher-order structures of such multimers are therefore referred to as homodimers or heterodimers, homodimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule in its natural form, consisting of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms “peptide,” “polypeptide,” and “protein” also refer to naturally modified peptides / polypeptides / proteins that have undergone post-translational modifications such as glycosylation, acetylation, and phosphorylation. As used herein, “peptide,” “polypeptide,” or “protein” may also be chemically modified, such as pegylation. Such modifications are well known in the art and are described below herein.

[0101] Preferably, the binding domain that binds to any of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, and EpCAM, and / or the binding domain that binds to CD3ε, is a human binding domain. Antibodies and antigen-binding molecules containing at least one human binding domain avoid some of the problems associated with antibodies or antigen-binding molecules having non-human variable and / or constant regions, such as those from rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins may lead to rapid clearance of the antibody or antigen-binding molecule or to trigger an immune response by the patient to the antibody or antigen-binding molecule. To avoid the use of rodent-derived antibodies or antigen-binding molecules, human or fully human antibody / antigen-binding molecules can be generated by introducing human antibody function into rodents so that the rodents produce fully human antibodies.

[0102] The ability to clone and reconstruct megabase-sized human loci in yeast artificial chromosomes (YACs) and to introduce them into mouse germline cells provides a powerful method for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human diseases. Furthermore, using such techniques to replace mouse loci with their human equivalents could provide unique insights into the expression and regulation of human gene products during development, their transduction to other systems, and their involvement in disease induction and progression.

[0103] A key practical application of such strategies is the "humanization" of the mouse humoral immune system. Introducing human Ig loci into mice with inactivated endogenous immunoglobulin (Ig) genes provides an opportunity to study the mechanisms underlying programmed antibody expression and construction, as well as their roles in B cell development. Furthermore, such strategies could provide an ideal source for the production of fully human monoclonal antibodies (mAbs), a crucial milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies or antigen-binding molecules are expected to minimize the immunogenicity and allergic reactions inherent in mouse mAbs or mouse-derived mAbs, thereby increasing the efficacy and safety of the administered antibody / antigen-binding molecules. The use of fully human antibodies or antigen-binding molecules is expected to offer significant advantages in the treatment of chronic and recurrent human diseases requiring repeated administration of compounds, such as inflammation, autoimmunity, and cancer.

[0104] One approach to achieving this goal involves manipulating mouse strains lacking mouse antibody production using large fragments of the human Ig locus. This is based on the prediction that such mice would produce a broad repertoire of human antibodies without producing mouse antibodies. Large human Ig fragments are thought to retain broad diversity in variable genes and appropriate regulation of antibody production and expression. By utilizing mouse mechanisms for antibody diversification and selection, and for the lack of immune tolerance to human proteins, the human antibody repertoire reproduced in this mouse strain should produce high-affinity antibodies against any target antigen, including human antigens. Using hybridoma technology, antigen-specific human mAbs with desired specificity can be easily generated and selected. This general strategy was demonstrated in connection with the creation of the first XenoMouse mouse strain (see Green et al. Nature Genetics 7:13-21 (1994)). This XenoMouse strain was engineered using YACs containing germline-arranged fragments of 245kb and 190kb sizes, respectively, of the human heavy chain locus and kappa light chain locus, containing core sequences of the variable and constant regions. These human Ig-containing YACs proved compatible with the mouse strain in terms of both antibody rearrangement and expression, and were capable of replacing inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development to produce an adult-like human repertoire of fully human antibodies and antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing numerous V genes, additional regulatory elements, and the human Ig constant region, could reproduce a substantially complete repertoire characterized by the human humoral response to infection and immunization. More recently, building upon the work of Green et al., the introduction of megabase-sized germline-arranged YAC fragments of the human heavy chain locus and kappa light chain locus resulted in the introduction of over 80% of the human antibody repertoire. See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. Patent Application Publication No. 08 / 759,620.

[0105] The creation of the XenoMouse animal is described in U.S. Patent Publication Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, 08 / 463,191, 08 / 462,837, and 08 / 4 This is further discussed and detailed in Patent No. 86,853, Patent No. 08 / 486,857, Patent No. 08 / 486,859, Patent No. 08 / 462,513, Patent No. 08 / 724,752 and Patent No. 08 / 759,620; and U.S. Patent No. 6,162,963, Patent No. 6,150,584, Patent No. 6,114,598, Patent No. 6,075,181 and Patent No. 5,939,598; and Japanese Patent No. 3068180B2, Japanese Patent No. 3068506B2 and Japanese Patent No. 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), European Patent No. 0463151B1, International Publication Nos. 94 / 02602, 96 / 34096, 98 / 24893, 00 / 76310, and 03 / 47336.

[0106] Another approach, employed by companies including GenPharm International, Inc., utilizes the "mini-locus" method. In this mini-locus method, an exogenous Ig locus is mimicked by including fragments (individual genes) derived from this Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a muon constant region, and a second constant region (preferably a gamma constant region) form a construct that is inserted into the animal. This method applies to U.S. Patent No. 5,545,807 to Surani et al., and to U.S. Patent Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, and 5,877,397 to Lonberg and Kay, respectively. Detailed documents; Specification No. 5,874,299; and Specification No. 6,255,458; U.S. Patent Nos. 5,591,669 and 6,023,010 against Krimpenfort and Berns; U.S. Patent No. 5,612,205; No. 5,721,367; and 5,789,215 against Berns et al.; and U.S. Patent No. 5,643,763 against Choi and Dunn; and GenPharm This is described in U.S. Patent Publication Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 of International.See also European Patent No. 0546073B1, International Publication No. 92 / 03918, International Publication No. 92 / 22645, International Publication No. 92 / 22647, International Publication No. 92 / 22670, International Publication No. 93 / 12227, International Publication No. 94 / 00569, International Publication No. 94 / 25585, International Publication No. 96 / 14436, International Publication No. 97 / 13852, and International Publication No. 98 / 24884, as well as U.S. Patent No. 5,981,175. Furthermore, see Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), Tuaillon et al. (1995), and Fishwild et al. (1996).

[0107] Kirin has also demonstrated the production of human antibodies from mice in which large chromosome fragments or entire chromosomes are introduced by microcell fusion. See European Patent Publications No. 773288 and No. 843961. Xenerex Biosciences is developing a promising human antibody production technology in which SCID mice are reconstituted with human lymphocytes, such as B cells and / or T cells. The mice can then be immunized with an antigen and generate an immune response to that antigen. See U.S. Patents No. 5,476,996; No. 5,698,767; and No. 5,958,765.

[0108] Human anti-mouse antibody (HAMA) reactions have led to the industry producing chimeric or otherwise humanized antibodies. However, certain human anti-chimeric antibody (HACA) reactions are expected to be observed, particularly in chronic or multi-dose antibody use. Therefore, it is desirable to provide antigen-binding molecules containing human-binding domains for CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM, and human-binding domains for CD3ε, in order to eliminate concerns and / or effects of HAMA or HACA reactions.

[0109] The terms “(specifically) or (immunospecifically) bind,” “(specifically) recognize,” “(specifically) induce,” and “(specifically) react,” according to the present invention, mean that the binding domain, preferably by its paratope, interacts with or specifically interacts with a given epitope or a given target site on a target molecule (antigen, preferably CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, or EpCAM and CD3ε, respectively).

[0110] In relation to the present invention, a paratope is understood as an antigenic site that is part of a polypeptide described herein and recognizes and binds to an antigen. A paratope is typically a small region of at least about five amino acids. Paratopes as understood herein typically include a portion of the heavy chain (VH) and light chain (VL) sequences derived from an antibody. Each binding domain of the polypeptide according to the present invention comprises a paratope that includes a set of six complementarity-determining regions (CDR loops), three of which are contained within the antibody-derived VH and VL sequences.

[0111] In relation to the present invention, the antigen-binding molecule (i.e., preferably a polypeptide) of the present invention binds to each target structure in a specific manner. Preferably, the polypeptide according to the present invention contains one paratope per binding domain that "specifically or immunospecifically binds to" or "recognizes" (specifically or immunospecifically) or "reacts" (specifically or immunospecifically) with each target structure. This means that, according to the present invention, the polypeptide or its binding domain interacts with or (immunologically)-specifically with the target molecule (antigen) and a given epitope on CD3, respectively. This interaction or association occurs more frequently, more rapidly, more persistently, more favorably, or in some combination of these parameters with respect to the epitope on a particular target compared to alternative substances (non-target molecules). However, due to sequence similarities between homologous proteins in various species, an antibody construct or binding domain that immunospecifically binds to a target (e.g., a human target) may cross-react with homologous target molecules from different species (e.g., non-human primates). Therefore, the term “specific / immunospecific binding” may include the binding of an antibody construct or binding domain to epitopes in multiple species and / or structurally related epitopes. The term “(immuno)selective binding” excludes binding to structurally related epitopes.

[0112] The term "epitope" refers to a site on an antigen to which a binding domain of an antibody or immunoglobulin, or a derivative, fragment, or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and therefore, the term epitope may also be referred to herein as an "antigenic structure" or "antigenic determinant." Thus, the binding domain is an "antigen interaction site." This binding / interaction is understood to also define "specific recognition."

[0113] An "epitope" can be formed by both consecutive amino acids or discontinuous amino acids that are paralleled by the three-dimensional folding of a protein. A "linear epitope" is an epitope that contains an epitope in which the primary amino acid sequence is recognized. Linear epitopes typically contain at least three or at least four, and more commonly at least five, at least six, or at least seven, for example, about eight to about ten amino acids, within a characteristic sequence.

[0114] A "structural epitope," in contrast to a linear epitope, is an epitope in which the primary sequence of amino acids containing the epitope is not the sole element defining the recognized epitope (for example, an epitope in which the primary sequence of amino acids is not necessarily recognized by the binding domain). Generally, structural epitopes contain a larger number of amino acids compared to linear epitopes. In relation to the recognition of structural epitopes, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein, or a fragment thereof (in relation to the present invention, the antigenic structure for one of the binding domains is contained within the target cell surface antigen protein). For example, when a protein molecule folds to form a three-dimensional structure, specific amino acids and / or polypeptide backbone that form the structural epitope are arranged in parallel, thereby enabling the antibody to recognize that epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, site-specific spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

[0115] The epitope mapping method is described below: When a region (adjacent amino acid stretch) in a human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM protein is exchanged or substituted with a corresponding region in a non-human or non-primate CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (for example, mouse CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM, but other species such as chicken, rat, hamster, or rabbit are also possible), a reduction in binding affinity of the binding domain is expected to occur unless the binding domain is cross-reactive to the non-human or non-primate CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, or EpCAM used. The reduction is preferably at least 10%, 20%, 30%, 40%, or 50%, more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even further 100%, compared to the binding to each region in the human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM proteins, with the binding to each region in the human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM proteins, with the binding to each region in the human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM proteins being 100%. The above human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM / non-human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM chimeras are expected to be expressed in CHO cells. Furthermore, it is conceivable that chimeras of human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM / non-human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM may fuse with the transmembrane and / or cytoplasmic domains of different membrane-bound proteins such as EpCAM.

[0116] An alternative or additional method to epitope mapping may generate several truncated forms of the extracellular domains of human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM to determine specific regions recognized by binding domains. In these truncated forms, various extracellular CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM domains / subdomains or regions are deleted stepwise, starting from the N-terminus. It is hypothesized that truncated CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM may be expressed in CHO cells. It is also hypothesized that truncated CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM may fuse with the transmembrane and / or cytoplasmic domains of different membrane-bound proteins such as EpCAM. Furthermore, shortened versions of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM may contain a signal peptide domain at their N-terminus, and it is conceivable that they may contain a signal peptide derived from, for example, mouse IgG heavy chain signal peptide. In addition, shortened versions of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM may contain a v5 domain at the N-terminus (following the signal peptide) that enables accurate confirmation of their expression on the cell surface. For shortened versions of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM that no longer contain the region recognized by the binding domain, a reduction or loss of binding is expected. The reduction in binding is preferably at least 10%, 20%, 30%, 40%, or 50%, more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even 100%, when the binding to the entire human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM protein (or its extracellular region or domain) is set to 100%.

[0117] Additional methods for determining the contribution of specific residues of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN or EpCAM to recognition by an antigen-binding molecule or binding domain involve alanine scanning, which substitutes each residue to be analyzed with alanine, for example, by site-directed mutagenesis (see, for example, Morrison KL & Weiss GA. Cur Opin Chem Biol. 2001 Jun;5(3):302-7). Alanine is used because it is not bulky, is chemically inert, and yet has a methyl functional group that mimics the secondary structure criteria of many other amino acids. If it is desired to preserve the size of the mutated residue, bulkier amino acids such as valine or leucine may sometimes be used. Alanine scanning is a well-established technique that has been used for a long time.

[0118] The interaction between a binding domain and an epitope or region containing an epitope means that the binding domain exhibits a measurable affinity for an epitope / region containing an epitope on a particular protein or antigen (herein CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3 or EpCAM and CD3, respectively), and generally does not exhibit significant reactivity against proteins or antigens other than CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3 or EpCAM or CD3. "Measurable affinity" includes bindings having an affinity of about 10 -6 M (KD) or stronger. Preferably, the binding affinity is 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 -9If M is present, the binding is considered specific. Whether the binding domain specifically reacts with or binds to a target can be easily tested, in particular, by comparing the reaction of the binding domain to a target protein or antigen with the reaction of the binding domain to proteins or antigens other than CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, EpCAM, or CD3. Preferably, the binding domain of the present invention does not essentially or substantially bind to proteins or antigens other than CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, EpCAM, or CD3 (i.e., the first binding domain cannot bind to proteins other than CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, or EpCAM, and the second binding domain cannot bind to proteins other than CD3). Having superior affinity properties compared to other HLE forms is a presumed feature of the antigen-binding molecule according to the present invention. Such superior affinity consequently suggests an extended half-life in vivo. The longer half-life of the antigen-binding molecule according to the present invention may reduce the duration and frequency of administration, which typically contributes to improved patient compliance. This is particularly important because the antigen-binding molecule of the present invention is especially beneficial for cancer patients who are severely debilitated or even more multi-disease-ridden.

[0119] The terms "essentially / substantially non-binding" or "unable to bind" mean that the binding domain of the present invention does not bind to proteins or antigens other than CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, EpCAM, or CD3. In other words, if binding to each of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, EpCAM, or CD3 is considered 100%, then the domain does not exhibit reactivity of more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6%, or 5% to proteins or antigens other than CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, CDH3, EpCAM, or CD3.

[0120] Specific binding is thought to be brought about by specific motifs within the binding domain and the amino acid sequence of the antigen. Therefore, binding occurs as a result of their primary, secondary, and / or tertiary structures, as well as as a result of secondary modifications of said structures. Specific interaction between the antigen interaction site and the specific antigen can lead to simple binding of the site to the antigen. Furthermore, specific interaction between the antigen interaction site and the specific antigen can alternatively or additionally initiate a signal, for example, by inducing conformational changes in the antigen or oligomerization of the antigen.

[0121] The term "variable" refers to a portion of an antibody or immunoglobulin domain (i.e., a "variable domain") that exhibits variability within its sequence and is involved in determining the specificity and binding affinity of a particular antibody. A pair of variable heavy chains (VH) and variable light chains (VL) together form a single antigen-binding site.

[0122] The variability is not uniformly distributed throughout the antibody's variable domain, but rather concentrated in the respective subdomains of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs or FRs), which provide a scaffold for the six CDRs in the three-dimensional space that forms the antigen-binding surface. The naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3, and FR4), which mostly take a β-sheet configuration. These are connected by three hypervariable regions that form loop connections and, in some cases, form part of the β-sheet structure. The hypervariable regions of each chain are held collectively in close proximity by the FRMs and, together with the hypervariable regions of other chains, contribute to the formation of the antigen-binding site (see Kabat et al. cited above).

[0123] The term "CDR" and its plural form "CDRs" refer to complementarity-determining regions, three of which constitute the binding properties of the light chain variable region (CDR-L1, CDR-L2, and CDR-L3), and three which constitute the binding properties of the heavy chain variable region (CDR-H1, CDR-H2, and CDR-H3). CDRs contain the majority of the residues responsible for the specific interaction between antibodies and antigens, and therefore contribute to the functional activity of antibody molecules. In other words, they are the main determinants of antigen specificity.

[0124] The precise definition of CDR boundaries and length follows various classification and numbering schemes. Therefore, CDRs may be represented by Kabat, Chothia, Contact, or any other boundary definition, including the numbering schemes described herein. Even with different boundaries, each of these schemes has some overlap in the portions constituting the so-called "hypervariable regions" within the variable sequence. Consequently, the definitions of CDRs by these schemes may differ in terms of length and the boundary regions with respect to adjacent framework regions. See, for example, Kabat (a method based on interspecies sequence variability), Chothia (a method based on crystallographic studies of antigen-antibody complexes), and / or MacCallum (see Kabat et al., op. cit.; Chothia et al., J.Mol.Biol, 1987, 196:901-917; and MacCallum et al., J.Mol.Biol, 1996, 262:732). Another standard for characterizing antigen-binding sites is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Two residue identification techniques can be combined to define overlapping regions rather than identical regions to define hybrid CDRs. However, numbering following the so-called Kabat system is preferred.

[0125] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the three-dimensional structure of the main chain adopted by the antigen-binding (CDR) loop. Comparative structural studies have revealed that five of the six antigen-binding loops have only a limited repertoire of available three-dimensional structures. Each canonical structure can be characterized by the twist angle of the polypeptide backbone. Therefore, corresponding loops between antibodies can have very similar three-dimensional structures, despite high amino acid sequence variability being observed in most of the loops (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). Furthermore, there is a correlation between the adopted loop structure and the surrounding amino acid sequence. The three-dimensional structure 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 (i.e., outside the loop). Therefore, assignment to a particular canonical class can be based on the presence of these key amino acid residues.

[0126] The term "canonical structure" may also include considerations regarding the linear sequence of an antibody, as classified, for example, by Kabat (Kabat et al., cited above). Kabat's numbering scheme is a widely adopted standard for numbering amino acid residues of antibody variable domains in a consistent manner and is a preferred scheme for application in the present invention, as mentioned elsewhere in this specification. Further structural considerations may also be used to determine the canonical structure of an antibody. For example, differences not adequately reflected by Kabat's numbering scheme can be described by the numbering scheme of Chothia et al., and / or revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence may be classified, among other things, into a canonical class from which appropriate chassis sequences can be identified (for example, based on the requirement to include various canonical structures in a library). The significance of Kabat numbering for antibody amino acid sequences, the structural considerations described by Chothia et al. (cited above), and their implications for interpreting the canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional arrangements of various classes of immunoglobulins are well known in this field. For an overview of antibody structures, please refer to Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0127] Light chain CDR3, and especially heavy chain CDR3, can be the most important determinants of antigen binding within the light and heavy chain variable regions. In some antigen-binding molecules, heavy chain CDR3 appears to be the primary contact region between the antigen and the antibody. In vitro selection schemes that alter only the CDR3 can be used to change the binding properties of an antibody or to determine which residues contribute to antigen binding. Therefore, CDR3 is usually the greatest source of molecular diversity within antibody binding sites. For example, H3 can be as short as two amino acid residues or more than 26 amino acids.

[0128] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to the heavy (H) chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The CH domain closest to the VH is usually referred to as CH1. The constant ("C") domain does not directly participate in antigen binding but exhibits various effector functions, such as antibody-dependent, cell-mediated cytotoxicity, and complement activation. The Fc region of the antibody is contained within the heavy chain constant domain and can interact with Fc receptors located, for example, on the cell surface.

[0129] The sequences of antibody genes after construction and somatic mutation are extremely diverse, and these diversified genes are 10 10 It is presumed to encode different antibody molecules (Immunoglobulin Genes, 2 nd (eds. Jonio et al., Academic Press, San Diego, CA, 1995). Thus, the immune system provides a repertoire of immunoglobulins. The term “repertoire” refers to at least one nucleotide sequence that is derived in whole or in part from at least one sequence encoding at least one immunoglobulin. This sequence may be generated by in vivo rearrangement of the V, DJ segments of the heavy chain and the V and J segments of the light chain. Alternatively, this sequence may be generated from cells in response to a stimulus that causes rearrangement, for example, in vitro. Alternatively, some or all of this sequence may be obtained by DNA splicing, nucleotide synthesis, mutagenesis and other methods (see, for example, U.S. Patent No. 5,565,332). A repertoire may consist of only one sequence or multiple sequences that are part of a genetically diverse collection.

[0130] In relation to the present invention, the term "Fc moiety" or "Fc monomer" means a polypeptide comprising at least one domain having the function of the CH2 domain and at least one domain having the function of the CH3 domain of an immunoglobulin molecule. As is clear from the term "Fc monomer," polypeptides containing these CH domains are "polypeptide monomers." An Fc monomer may be a polypeptide comprising a fragment of the constant region of an immunoglobulin, excluding at least the first constant region immunoglobulin domain (CH1) of the heavy chain, but maintaining the functional portion of at least one CH2 domain and the functional portion of one CH3 domain, with the CH2 domain located on the amino-terminal side of the CH3 domain. In a preferred embodiment of this definition, an Fc monomer may be a polypeptide constant region comprising a portion of the Ig-Fc hinge region, a CH2 region and a CH3 region, with the hinge region located on the amino-terminal side of the CH2 domain. The hinge region of the present invention is intended to promote dimerization. Such Fc polypeptide molecules can be obtained, for example, by papain digestion of an immunoglobulin region (which naturally produces a dimer of two Fc polypeptides), but are not limited thereto. In another embodiment of this definition, the Fc monomer may be a polypeptide region containing parts of the CH2 and CH3 regions. Such Fc polypeptide molecules can be obtained, for example, by pepsin digestion of an immunoglobulin molecule, but are not limited thereto. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the Fc polypeptide sequences of the IgG1Fc, IgG2Fc, IgG3Fc, IgG4Fc, IgMFc, IgAFc, IgDFc, and IgEFc regions (see, for example, Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because certain variants exist among immunoglobulins, and for the sake of clarity, the Fc monomer refers to the two final heavy chain constant-region immunoglobulin domains of IgA, IgD, and IgG, and the three final heavy chain constant-region immunoglobulin domains of IgE and IgM. As mentioned, the Fc monomer may also include a mobile hinge on the N-terminal side of these domains. In the case of IgA and IgM, the Fc monomer may include a J chain.In the case of IgG, the Fc portion includes the immunoglobulin domains CH2 and CH3, as well as the hinge between the first two domains and CH2. The boundaries of the Fc portion may differ, but an example of a human IgG heavy chain Fc portion containing the functional hinge, CH2 and CH3 domains can be defined, for example, as including residues D231 (corresponding to D234 in Table 1 below) to P476 and L476 (in the case of IgG4), respectively, in numbering according to Kabat. Two Fc portions or Fc monomers fused to each other via a peptide linker define a third domain of the antigen-binding molecule of the present invention, which may also be defined as the scFc domain.

[0131] In one embodiment of the present invention, the scFc domains disclosed herein and the Fc monomers to which they are fused are assumed to be contained only in the third domain of the antigen-binding molecule.

[0132] In accordance with the present invention, the IgG hinge region can be identified by similarity using Kabat numbering as described in Table 1. In accordance with the above, the minimum requirement for the hinge domain / region of the present invention is assumed to be the inclusion of amino acid residues corresponding to a sequence of IgG1 sequences D231 D234~P243 by Kabat numbering. It is also assumed that the hinge domain / region of the present invention contains or consists of the IgG1 hinge sequence DKTHTCPPCP(SEQ ID NO) (corresponding to a sequence of D234~P243 shown in Table 1 below - variations of the sequence are also assumed, insofar as the hinge region still promotes dimerization). In a preferred embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain in the third domain of the antigen-binding molecule is removed by an N314X substitution, where X is any amino acid other than Q. The substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat) V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0133] The third domain of the antigen-binding molecule of the present invention may include or consist of DKTHTCPPCP(SEQ ID NO:)(i.e., hinge)-CH2-CH3-linker-DKTHTCPPCP(SEQ ID NO:)(i.e., hinge)-CH2-CH3 in the order of amino to carboxyl. The peptide linker of the antigen-binding molecule is, in a preferred embodiment, characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser(SEQ ID NO: 1) or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer of 5 or greater (e.g., 5, 6, 7, 8, etc. or greater), with 6 being preferred ((Gly4Ser)6). The construct may further include the above substitution: N314X, preferably N314G, and / or further substitutions V321C and R309C. In preferred embodiments of the antigen-binding molecule of the present invention as defined above herein, the second domain is envisioned to bind to an extracellular epitope of the human and / or macaque CD3ε chain. Table 1: Kabat numbering of amino acid residues in the hinge region.

[0134] [Table 1]

[0135] In further embodiments of the present invention, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP(sequence ID), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP(sequence ID) or ELKTPLGDTTHTCPRCP(sequence ID) and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP(sequence ID). The IgG1 subtype hinge sequence may be the following sequence EPKSCDKTHTCPPCP (shown in Table 1 and sequence ID). Accordingly, these core hinge regions are also conceivable in connection with the present invention.

[0136] The locations and sequences of the IgG CH2 and IgG CD3 domains can be identified by similarity using the Kabat numbering described in Table 2.

[0137] [Table 2]

[0138] In one embodiment of the present invention, the amino acid residues highlighted in bold within the CH3 domain of the first or both Fc monomers are deleted.

[0139] The peptide linker into which the polypeptide monomers of the third domain ("Fc moieties" or "Fc monomers") are fused to each other preferably contains at least 25 amino acid residues (e.g., 25, 26, 27, 28, 29, 30). More preferably, the peptide linker contains at least 30 amino acid residues (e.g., 30, 31, 32, 33, 34, 35). It is also preferable that the linker contains up to 40 amino acid residues, more preferably up to 35 amino acid residues, and most preferably just 30 amino acid residues. A preferred embodiment of such a peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 1), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer greater than 5 (e.g., 6, 7, or 8). Preferably, the integer is 6 or 7, and more preferably, the integer is 6.

[0140] When a linker is used to fuse a first domain with a second domain, or to fuse a first or second domain with a third domain, the linker is preferably of sufficient length and sequence to ensure that each of the first and second domains independently maintains their different binding specificities. In the case of a peptide linker connecting at least two binding domains (or two variable domains) within the antigen-binding molecule of the present invention, the peptide linkers are preferably those containing only a few amino acid residues, for example, 12 amino acid residues or less. Therefore, peptide linkers of 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues are preferred. Of the assumed peptide linkers having fewer than 5 amino acids, linkers containing 4, 3, 2, or 1 amino acid and rich in Gly are preferred. A preferred embodiment of the peptide linker for fusion of the first and second domains is shown in SEQ ID NO: 1. A preferred linker embodiment of the peptide linker for fusion of the second and third domains is a (Gly)4-linker, also known as a G4-linker.

[0141] In relation to one of the above-mentioned "peptide linkers," a particularly preferred "single" amino acid is Gly. Thus, the above-mentioned peptide linker may consist of a single amino acid Gly. In a preferred embodiment of the present invention, the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 1) or a polymer thereof, i.e., (Gly4Ser)x, where x is 1 or a greater integer (e.g., 2 or 3). Preferred linkers are shown in SEQ ID NOs: 1 to 12. Features of the peptide linkers, including not promoting secondary structures, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Furthermore, peptide linkers that do not promote any secondary structures are preferred. The interconnection of the above domains can be provided, for example, by the genetic manipulations described in the examples. Methods for preparing fused and operably linked bispecific single-stranded constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., International Publication No. 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0142] In an antigen-binding molecule or, in a preferred embodiment of the present invention, the first and second domains form an antigen-binding molecule of a form selected from the group consisting of (scFv)2, scFv-single-domain mAb, diabody, and any oligomer of these forms.

[0143] In particular, according to preferred embodiments and as described in the appended examples, the first and second domains of the antigen-binding molecule of the present invention are a “bispecific single-chain antigen-binding molecule,” more preferably a bispecific “single-chain Fv” (scFv). The two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be linked by a synthetic linker as described herein, which allows them to be produced as a single protein chain paired together so that the VL and VH regions form a monovalent molecule using recombination; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. These antibody fragments can be obtained using prior art known to those skilled in the art, and the fragments can be evaluated for function in the same manner as complete or full-length antibodies. Thus, a single-chain variable fragment (scFv) is typically a fusion protein of the variable region (VH) of the heavy chain and the variable region (VL) of the light chain of an immunoglobulin, linked by a short linker peptide as described herein. The linker is typically rich in glycine for mobility and serine or threonine for solubility, and may ligate the N-terminus of VH to the C-terminus of VL or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker.

[0144] Bispecific single-chain antigen-binding molecules are known in the art and are described in International Publication No. 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loeffler, Blood, (2000), 95, 6, 2098-2103, Bruehl, Immunol., (2001), 166, 2420-2426, and Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. The techniques described for the production of single-chain antibodies (see, in particular, U.S. Patent No. 4,946,778, Kontermann and Duebel (2010), cited above, and Little (2009), cited above) can be adapted to produce single-chain antigen-binding molecules that specifically recognize selected targets.

[0145] Bivalent (also called divalent) or bispecific single-stranded variable fragments (bi-scFv or di-scFv having the form (scFv)2) can be produced by linking two scFv molecules (for example, using the linkers described above herein). If these two scFv molecules have the same binding specificity, the resulting (scFv)2 molecule is preferably called bivalent (i.e., it has a valency of 2 for the same target epitope). If these two scFv molecules have different binding specificities, the resulting (scFv)2 molecule is preferably called bispecific. Linking can be done by generating a single peptide chain having two VH regions and two VL regions to produce a tandem scFv (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to generate an scFv molecule using a linker peptide that is too short (e.g., about 5 amino acids) to fold the two variable regions together, and then dimerize the scFv. This form is known as a diabody (see, for example, Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0146] In accordance with the present invention, the first domain, the second domain, or either the first or second domain may each comprise a single-domain antibody, a variable domain of a single-domain antibody, or at least a CDR of a single-domain antibody. A single-domain antibody comprises only one (monomer) antibody variable domain that can selectively bind to a specific antigen independently of other V regions or domains. The initial single-domain antibodies were produced from heavy-chain antibodies found in camels, and these are V H This is called an H fragment. Cartilaginous fish also have heavy chain antibodies (IgNAR), and from there, V NARSingle-domain antibodies, known as fragments, can be obtained. An alternative method involves, for example, splitting a dimeric variable domain derived from a common immunoglobulin of human or rodent origin into monomers, thereby obtaining a single-domain Ab (VH) or VL (VL). While most research on single-domain antibodies currently relies on heavy-chain variable domains, light-chain-derived nanobodies have also been shown to specifically bind to target epitopes. Examples of single-domain antibodies are called sdAbs, nanobodies, or single-variable-domain antibodies.

[0147] Therefore, (single-domain mAb)2 is V H , V L , V H H and V NAR It is a monoclonal antigen-binding molecule composed of at least two single-domain monoclonal antibodies individually selected from the group including the above. The linker is preferably in the form of a peptide linker. Similarly, "scFv-single-domain mAb" is a monoclonal antigen-binding molecule composed of at least one of the above single-domain antibodies and one of the above scFv molecules. In this case as well, the linker is preferably in the form of a peptide linker.

[0148] Whether an antigen-binding molecule competes to bind to another given antigen-binding molecule can be measured by a competitive assay, such as a competitive ELISA or a cell-based competitive assay. Avidin-conjugated microparticles (beads) can also be used. Similar to avidin-coated ELISA plates, each of these beads can be used as a substrate when reacting with biotinylated proteins, and assays can be performed on them. The antigen is coated onto the beads, then pre-coated with a primary antibody. A secondary antibody is added to confirm any further binding. Flow cytometry is a possible method for reading the results.

[0149] T cells, or T lymphocytes, are a type of lymphocyte (a type of white blood cell) that plays a central role in cellular immunity. Several subsets of T cells exist, each with different functions. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a T cell receptor (TCR) on their cell surface. The TCR is involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules and is composed of two distinct protein chains. In 95% of T cells, the TCR consists of an alpha (α) chain and a beta (β) chain. When the TCR binds to antigen peptides and MHC (peptide / MHC complexes), the T lymphocyte is activated through a series of biochemical events mediated by related enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors.

[0150] The CD3 receptor complex is a protein complex composed of four chains. In mammals, this complex contains a CD3γ (gamma) chain, a CD3δ (delta) chain, and two CD3ε (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the T cell receptor CD3 complex, which generates activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are cell surface proteins of the highly related immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif, known as the immunoreceptor activation tyrosine motif, or ITAM, which is essential for TCR signaling. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on human chromosome 11. The most preferred CD3 epsilon epitopes are those in the range of amino acid residues 1-27 of the human CD3 epsilon extracellular domain. The antigen-binding molecules according to the present invention are expected to exhibit only minimal nonspecific T-cell activation, which is typically and advantageously undesirable in specific immunotherapy. This results in a low risk of side effects.

[0151] Lysis of redirected target cells via T cell recruitment by multispecific, or at least bispecific, antigen-binding molecules is accompanied by the formation of cytolytic synapses and the delivery of perforin and granzyme. The bound T cells are capable of sequential target cell lysis and are unaffected by immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation (see, for example, International Publication No. 2007 / 042261).

[0152] Cytotoxicity mediated by the antigen-binding molecule of the present invention can be measured by various methods. Effector cells may be, for example, stimulated enriched (human) CD8-positive cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). If the target cells are of macaque origin or have been transfected with macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM to which the first domain binds, the effector cells should also be of macaque origin, such as a macaque T cell line (e.g., 4119LnPx). Target cells should express CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (at least its extracellular domain), for example, human or macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM. The target cells may be cell lines (e.g., CHO) that have been stably or transiently transfected with CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (e.g., human or macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM). Typically, EC 50 The values ​​are expected to be lower in target cell lines expressing higher levels of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM on the cell surface. The effector-to-target cell (E:T) ratio is usually about 10:1, but can vary. The cytotoxic activity of the bispecific antigen-binding molecules CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM is... 51It can be measured by a Cr release assay (approximately 18 hours incubation time) or a FACS-based cytotoxicity assay (approximately 48 hours incubation time). The incubation time (cytotoxicity response) of the assay can also be modified. Other methods for measuring cytotoxicity are known to those skilled in the art and include MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, clonality assays, and ECIS technology.

[0153] The cytotoxic activity mediated by the CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM×CD3 bispecific antigen-binding molecules of the present invention is preferably measured by a cytotoxic assay using cells. 51 It can also be measured by a Cr release assay. Cytotoxic activity is EC 50 This is expressed by a value, which corresponds to the half-effect concentration (the concentration of the antigen-binding molecule that induces an intermediate cytotoxic response between baseline and maximum). Preferably, the EC of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM×CD3 bispecific antigen-binding molecules. 50 The values ​​are ≤5000pM or ≤4000pM, more preferably ≤3000pM or ≤2000pM, even more preferably ≤1000pM or ≤500pM, even more preferably ≤400pM or ≤300pM, even more preferably ≤200pM, even more preferably ≤100pM, even more preferably ≤50pM, even more preferably ≤20pM or ≤10pM, and most preferably ≤5pM.

[0154] In various assays, the given EC above 50 The value can be measured. When stimulated / enriched CD8+ T cells are used as effector cells, the EC is compared to unstimulated PBMCs. 50 Those skilled in the art are aware that a low value can be expected. Furthermore, EC 50The values ​​may be expected to be lower compared to rats with low target expression when target cells express a large number of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM. For example, when stimulated / enriched human CD8+ T cells are used as effector cells (and cells transfected with CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (e.g., CHO cells) or CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM-positive human cells are used as target cells), the EC of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM bispecific antigen-binding molecules 50 The value is preferably ≤1000pM, more preferably ≤500pM, even more preferably ≤250pM, even more preferably ≤100pM, even more preferably ≤50pM, even more preferably ≤10pM, and most preferably ≤5pM. When human PBMCs are used as effector cells, the EC of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM×CD3 bispecific antigen-binding molecules is used. 50The value is preferably ≤5000pM or ≤4000pM (especially when the target cells are CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM-positive cell lines), more preferably ≤2000pM (especially when the target cells are cells transfected with CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (e.g., CHO cells)), more preferably ≤1000pM or ≤500pM, even more preferably ≤200pM, even more preferably ≤150pM, even more preferably ≤100pM, and most preferably ≤50pM or lower. When macaque T cell lines such as LnPx4119 are used as effector cells, and cell lines transfected with macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (e.g., CHO cells) are used as target cell lines, the EC of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM × CD3 bispecific antigen-binding molecules 50 The value is preferably ≤2000pM or ≤1500pM, more preferably ≤1000pM or ≤500pM, even more preferably ≤300pM or ≤250pM, even more preferably ≤100pM, and most preferably ≤50pM.

[0155] Preferably, the CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM × CD3 bispecific antigen-binding molecules of the present invention do not induce / mediate the lysis of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM-negative cells such as CHO cells, or are essentially derivative / non-mediating. The terms “does not induce lysis,” “does not essentially induce lysis,” “does not mediate lysis,” or “does not essentially mediate lysis” mean that, with lysis of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM-positive human cell lines being defined as 100%, the antigen-binding molecules of the present invention do not induce or mediate the lysis of more than 30% of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM-negative cells, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6%, or 5%. This typically applies to antigen-binding molecule concentrations up to 500 nM. Those skilled in the art will know how to measure cell lysis without further effort. Furthermore, this specification provides specific instructions for methods of measuring cell lysis.

[0156] The difference in cytotoxic activity between the monomeric isoform and dimeric isoform of individual CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM×CD3 bispecific antigen-binding molecules is called the "potency gap." This potency gap is, for example, the EC of the monomeric form of this molecule. 50 Value and dimeric form of EC 50 It can be calculated as a ratio to the value. The resistance gap of the CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM×CD3 bispecific antigen-binding molecules of the present invention is preferably ≤5, more preferably ≤4, even more preferably ≤3, even more preferably ≤2, and most preferably ≤1.

[0157] The first and / or second (or any further) binding domains of the antigen-binding molecule of the present invention are preferably interspecies-specific to members of the order Mammalia of primates. Interspecies-specific CD3-binding domains are described, for example, in International Publication No. 2008 / 119567. According to one embodiment, the first and / or second binding domains bind to human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM and human CD3, as well as to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM / CD3 of primates, including (but not limited to) New World primates (e.g., marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus Oedipus), or squirrel monkeys (Saimiri sciureus)), Old World primates (e.g., baboons and macaques), gibbons, and non-homininae primates.

[0158] In one embodiment of the antigen-binding molecule of the present invention, the first domain binds to human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM, and further binds to macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (for example, CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM of cynomolgus monkey (Macaca fascicularis)), and more preferably further binds to macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM expressed on the surface of cells such as CHO cells or 293 cells. The affinity of the first domain to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (preferably human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM) is preferably ≤100 nM or ≤50 nM, more preferably ≤25 nM or ≤20 nM, more preferably ≤15 nM or ≤10 nM, even more preferably ≤5 nM, even more preferably ≤2.5 nM or ≤2 nM, even more preferably ≤1 nM, even more preferably ≤0.6 nM, even more preferably ≤0.5 nM, and most preferably ≤0.4 nM. The affinity can be measured, for example, by a BIAcore assay or a Scatchard assay. Other methods for determining affinity are also known to those skilled in the art. The affinity of the first domain to macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM is preferably ≤15 nM, more preferably ≤10 nM, even more preferably ≤5 nM, even more preferably ≤1 nM, even more preferably ≤0.5 nM, even more preferably ≤0.1 nM, and most preferably ≤0.05 nM or even ≤0.01 nM.

[0159] Preferably, the affinity gap of the antigen-binding molecule according to the present invention with respect to binding to macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM (as determined, for example, by BiaCore or Scatchar analysis) is <100, preferably <20, more preferably <15, even more preferably <10, even more preferably <8, even more preferably <6, and most preferably <2. The preferred range for the affinity gap of the antigen-binding molecule according to the present invention with respect to binding to macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM against human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM is 0.1 to 20, more preferably 0.2 to 10, even more preferably 0.3 to 6, even more preferably 0.5 to 3 or 0.5 to 2.5, and most preferably 0.5 to 2 or 0.6 to 2.

[0160] The third binding domain of the antigen-binding molecule of the present invention binds to human CD3 epsilon and / or macaque CD3 epsilon. In preferred embodiments, the second domain further binds to CD3 epsilon of marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus Oedipus), or squirrel monkey (Saimiri sciureus). Both marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus) are New World primates belonging to the family Callitrichidae, while squirrel monkeys (Saimiri sciureus) are New World primates belonging to the family Cebidae. The binding domain may preferably be referred to as "I2C" or "I2C0" in Table 5.

[0161] With respect to the antigen-binding molecule of the present invention, the third binding domain that binds to the extracellular epitope of the human and / or macaque CD3 epsilon chain is (a) CDR-L1 as shown in Sequence ID 27 of International Publication No. 2008 / 119567, CDR-L2 as shown in Sequence ID 28 of International Publication No. 2008 / 119567, and CDR-L3 as shown in Sequence ID 29 of International Publication No. 2008 / 119567; (b) CDR-L1 as shown in Sequence ID 117 of International Publication No. 2008 / 119567, CDR-L2 as shown in Sequence ID 118 of International Publication No. 2008 / 119567, and CDR-L3 as shown in Sequence ID 119 of International Publication No. 2008 / 119567; and (c) CDR-L1 as shown in Sequence ID 153 of International Publication No. 2008 / 119567, CDR-L2 as shown in Sequence ID 154 of International Publication No. 2008 / 119567, and CDR-L3 as shown in Sequence ID 155 of International Publication No. 2008 / 119567 It is preferable to include a VL region that includes CDR-L1, CDR-L2, and CDR-L3 selected from the above.

[0162] In a more preferred embodiment of the antigen-binding molecule of the present invention, the third binding domain that binds to the extracellular epitope of the human and / or macaque CD3 epsilon chain is (a) CDR-H1 as shown in Sequence ID 12 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 13 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 14 of International Publication No. 2008 / 119567; (b) CDR-H1 as shown in Sequence ID 30 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 31 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 32 of International Publication No. 2008 / 119567; (c) CDR-H1 as shown in Sequence ID 48 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 49 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 50 of International Publication No. 2008 / 119567; (d) CDR-H1 as shown in Sequence ID 66 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 67 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 68 of International Publication No. 2008 / 119567; (e) CDR-H1 as shown in Sequence ID 84 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 85 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 86 of International Publication No. 2008 / 119567; (f) CDR-H1 as shown in Sequence ID 102 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 103 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 104 of International Publication No. 2008 / 119567; (g) CDR-H1 as shown in Sequence ID 120 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 121 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 122 of International Publication No. 2008 / 119567; (h) CDR-H1 as shown in Sequence ID 138 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 139 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 140 of International Publication No. 2008 / 119567; (i) CDR-H1 as shown in Sequence ID 156 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 157 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 158 of International Publication No. 2008 / 119567; and (j) CDR-H1 as shown in Sequence ID 174 of International Publication No. 2008 / 119567, CDR-H2 as shown in Sequence ID 175 of International Publication No. 2008 / 119567, and CDR-H3 as shown in Sequence ID 176 of International Publication No. 2008 / 119567 Includes a VH region containing CDR-H1, CDR-H2, and CDR-H3 selected from the above.

[0163] In a preferred embodiment of the antigen-binding molecule of the present invention, the three groups of VL CDRs described above are combined with the ten groups of VH CDRs described above within a third binding domain to form a (30) group comprising CDR-L1~3 and CDR-H1~3, respectively.

[0164] With respect to the antigen-binding molecule of the present invention, the third domain that binds to CD3 preferably includes a VL region selected from the group consisting of those shown in SEQ ID NOs. 17, 21, 35, 39, 53, 57, 71, 75, 89, 93, 107, 111, 125, 129, 143, 147, 161, 165, 179, or 183 of International Publication No. 2008 / 119567, or those shown in SEQ ID NO. 13 according to the present invention.

[0165] The third domain bound to CD3 may also preferably include a VH region selected from the group consisting of sequence numbers 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177, or 181 in International Publication No. 2008 / 119567, or sequence number 14.

[0166] More preferably, the antigen-binding molecule of the present invention is (a) the VL area shown in Sequence ID No. 17 or 21 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID No. 15 or 19 of International Publication No. 2008 / 119567; (b) The VL area shown in Sequence ID 35 or 39 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 33 or 37 of International Publication No. 2008 / 119567; (c) VL area as shown in Sequence ID 53 or 57 of International Publication No. 2008 / 119567 and VH area as shown in Sequence ID 51 or 55 of International Publication No. 2008 / 119567; (d) The VL area shown in Sequence ID 71 or 75 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 69 or 73 of International Publication No. 2008 / 119567; (e) The VL area shown in Sequence ID 89 or 93 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 87 or 91 of International Publication No. 2008 / 119567; (f) The VL area shown in Sequence ID 107 or 111 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 105 or 109 of International Publication No. 2008 / 119567; (g) the VL area shown in Sequence ID 125 or 129 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID 123 or 127 of International Publication No. 2008 / 119567; (h) The VL area as shown in Sequence ID 143 or 147 of International Publication No. 2008 / 119567 and the VH area as shown in Sequence ID 141 or 145 of International Publication No. 2008 / 119567; (i) the VL area shown in Sequence ID No. 161 or 165 of International Publication No. 2008 / 119567 and the VH area shown in Sequence ID No. 159 or 163 of International Publication No. 2008 / 119567; and (j) VL area as shown in Sequence ID 179 or 183 of International Publication No. 2008 / 119567 and VH area as shown in Sequence ID 177 or 181 of International Publication No. 2008 / 119567 It is characterized by a third domain that binds to DC3, which includes a VL region and a VH region selected from the group consisting of the above.

[0167] Furthermore, in relation to the antigen-binding molecule of the present invention, a preferred third domain is one that binds to CD3, including the VL region shown in SEQ ID NO: 13 and the VH region shown in SEQ ID NO: 14.

[0168] According to a preferred embodiment of the antigen-binding molecule of the present invention, the first and / or third domains have the following configuration: 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 VH-VL or VL-VH. Preferably, the VH region is located at the N-terminus of the linker sequence and the VL region is located at the C-terminus of the linker sequence.

[0169] A preferred embodiment of the antigen-binding molecule of the present invention is characterized by a third domain that binds to CD3, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, or 187 in International Publication No. 2008 / 119567, or the amino acid sequence shown in SEQ ID NO: 15.

[0170] The present invention relates to Sequence IDs 673, 676, 679, 682, 685, 688, 691, 694, 697, 700, 703, 706, 709, 712, 715, 718, 721, 724, 727, 730, 733, 736, 739, 742, 745, 748, 751, 754, 757, 760, 763, 766, 769, 772, 775, 778, 781, 784, 787, 790, 793, 796, 799, 802, 805, 808, 811, 814, 817, 820, 823, 826, 829, 832, 835, 838, 841, 844, 847, 850, 853, 856, 859, 862, 865, 868, 871, 1437, 1440, 1443, 1446, 1449, 1452, 1455, 1458, 1461, 1464, 1467, 1470, 1473, 1476, 1479, 1482, 1485, 1488, 1499, 1 667, 1670, 1673, 1676, 1679, 1682, 1685, 1688, 1691, 1694, 1697, 1700, 1703, 1706, 1709, 1712, 1715, 1718, 1721, 1724, 1727, 1730, 1733, 1736, 1739, 1 742, 1745, 1748, 1751, 1754, 1757, 1760, 1763, 1766, 1769, 1772, 1775, 1778, 1781, 1784, 1787, 1790, 1793, 1796, 1799, 1802, 1805, 1808, 1811, 1814, 1817, 1820, 1823, 1826, 1829, 1838, 1851, 1864, 1877, 1890, 1903, 1916, 1933, 1946, 1959, 1972, 1985, 1998, 2011, 2024, 2037, 2050, 2063, 2076, 2089, 2102, 2115, 2128, 2141, 2154, 2167, 2180, 2194, 2206, 2219, 2232, 2245, 2258, 2262, 2270, 2271, 2280, 2281, 2290, 2291, 2300, 2301, 2310, 2311, 2320, 2321, 2330, 2331, 2340, 2341, 2350, 2351, 2360, 2361, 2370, 2371, 2380, 2381, 2390, 2391, 2400, 2401, 2410, 2411, 2420, 2421, 2430, 2431, 2440, 2441,2450、2451、2460、2461、2470、2471、2480、2481、2490、2491、2500、2501、2510、2511、2520、2521、2530、2531、2540、2541、2550、2551、2560、2561、2570、2571、2580、2581、2590、2591、2600、2601、2610、2611、2620、2621、2630、2631、2640、2641、2650、2651、2660、2661、2670、2671、2680、2681、2690、2691、2700、2701、2710、2711、2720、2721、2730、2731、2740、2741、2750、2751、2760、2761、2770、2771、2780、2781、2790、2791、2800、2801、2810、2811、2820、2821、2830、2831、2840、2841、2850、2851、2860、2861、2870、2871、2880、2881、2890、2891、2900、2901、2910、2911、2920、2921、2930、2931、2940、2941、2950、2951、2960、2961、2970、2971、2980、2981、2990、2991、3000、3001、3010、3011、3020、3021、3030、3031、3040、3041、3050、3051、3060、3061、3070、3071、3080、3081、3090、3091、3100、3101、3110、3111、3120、3121、3130、3131、3140、3141、3150、3151、3160、3161、3170、3171、3180、3181、3190、3191、3200、3201、3210、3211、3220、3221、3231、3240、3241、3250、3251、3260、3261、3270、3271、3280、3281、3290、3291、3300、3301、3310、3311、3320、3321、3330、3331、3340、3341、3344、3345、3356、3367、3378、3389、3400、3411、3422、3433、3444、3455、3466、3477、3488、3499、3510、3521、3532、3543、3554、3565, 3576, 3579, 3582, 3585, 3588, 3591, 3594, 3597, 3600, 3603, 3606, 3609, 3612, 3615, 3618, 3621, 3624, 3627, 3630, 3633, 3636, 3639, 3642, 3645, 3648, 3651, 3654, 3657, 3660, 3663, 3666, 3669, 3672, 3675, 3678, 3689, 3700, 3704, 3705, 3708, 3709, 3710, Further providing antigen-binding molecules (fully bispecific antigen-binding molecules) comprising or having an amino acid sequence selected from the group consisting of 3711, 3722, 3733, 3736, 3739, 3744, 3747, 3748, 3756, 3757, 3761, and 3762, preferably 1437, or further providing antigen-binding molecules having an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the aforementioned sequence.

[0171] Covalent modification of antigen-binding molecules is also within the scope of the present invention, which usually occurs post-translation, though not always. For example, some types of covalent modification of antigen-binding molecules are introduced into the molecule by reacting specific amino acid residues of the antigen-binding molecule with an organic derivatizing agent that can react with selected side chains or N-terminal or C-terminal residues.

[0172] Cysteinyl residues most commonly react with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to produce carboxymethyl or carboxyamidemethyl derivatives. Cysteinyl residues can also be derivatized by reactions with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercrinebenzoic acid, 2-chloromercrine-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0173] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5 - 7.0 because this reagent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful and the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. Lysyl and amino-terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these reagents has the effect of inverting the charge of lysyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imido esters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0174] Arginyl residues are modified by reaction with one or more conventional reagents, particularly phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Due to the high pKa of the guanidine functional group, derivatization of arginine residues requires the reaction to be carried out under alkaline conditions. Furthermore, these reagents can react with lysine groups and the arginine epsilon-amino group.

[0175] Specific modification of tyrosyl residues may be carried out, particularly for the purpose of introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 The above-mentioned chloramine T method for iodinating tyrosyl residues with I to prepare labeled proteins for use in radioimmunoassays is preferred.

[0176] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R'-N=C=N-R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Further, aspartyl residues and glutamyl residues are converted to asparaginyl residues and glutaminyl residues by reaction with ammonium ions.

[0177] Derivatization with bifunctional substances is useful for cross-linking the antigen-binding molecules of the present invention to a water-insoluble support matrix or support surface for use in various ways. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, homobifunctional imide esters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimide-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propionimidate provide photoactivatable intermediates that can form cross-links in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are used for protein immobilization.

[0178] Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under weakly acidic conditions. Any form of these residues is included within the scope of the present invention.

[0179] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0180] Another type of covalent modification of antigen-binding molecules that falls within the scope of the present invention involves altering the glycosylation pattern of a protein. As is known in the art, the glycosylation pattern can depend on both the sequence of the protein (e.g., the presence or absence of certain glycosylated amino acid residues discussed below) or on the host cell or organism producing the protein. Individual expression systems are discussed below.

[0181] Polypeptide glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a sugar chain to an asparagine residue side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of sugar chains to asparagine side chains. Therefore, the presence of either of these tripeptide sequences in a polypeptide generates a potential glycosylation site. O-linked glycosylation refers to the addition of one sugar of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but sometimes 5-hydroxyproline or 5-hydroxylysine are used.

[0182] The addition of a glycosylation site to an antigen-binding molecule is preferably carried out by modifying the amino acid sequence to include one or more of the above-mentioned tripeptide sequences (for an N-linked glycosylation site). The modification can also be made by adding or substituting one or more serine or threonine residues into the start sequence (in the case of an O-linked glycosylation site). In short, it is preferable to modify the amino acid sequence of the antigen-binding molecule at the DNA level, particularly by mutating the DNA encoding the polypeptide with pre-selected bases so that codons that will be translated into desired amino acids are produced.

[0183] Another means of increasing the number of sugar chains on an antigen-binding molecule is by chemical or enzymatic linkage of glycosides to the protein. These procedures are advantageous in that they do not require the production of proteins in host cells that have glycosylation ability for N-linked and O-linked glycosylation. Depending on the mode of linkage used, sugars can be added to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) amide groups of glutamine. These methods are described in International Publication No. 87 / 05330 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0184] The removal of glycans present on the starting antigen-binding molecule can be performed chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or a homogeneous compound. This process cleaves almost all or all of the sugars except for the bound sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of glycans on polypeptides can be achieved by using various endo- and exo-glycosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be inhibited by the use of the compound tunicamycin, as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin inhibits the formation of protein-N-glycosidic bonds.

[0185] Other modifications of antigen-binding molecules are also considered herein. For example, another type of covalent modification of antigen-binding molecules involves linking the antigen-binding molecule to various non-proteinoid polymers, including, but not limited to, various polyols in the manner shown in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene or copolymers of polyethylene glycol and polypropylene glycol. In addition, as is known in the art, amino acid substitutions can be made at various positions within the antigen-binding molecule to facilitate the addition of polymers such as PEG.

[0186] In some embodiments, the covalent modification of the antigen-binding molecule of the present invention involves the addition of one or more labels. To reduce potential steric hindrance, label groups may be attached to the antigen-binding molecule via spacer arms of varying lengths. Various methods for labeling proteins are known in the art and can be used when carrying out the present invention. The terms “label” or “labeling group” refer to any detectable label. Generally, labels are classified into various classes depending on the assay in which the label is to be detected, and include, but are not limited to, the following: a) Radioactive isotopes or radionuclides (for example, 3 H, 14 C, 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 I) Isotopic labels that may be radioactive isotopes or heavy isotopes b) Magnetic labeling (e.g., magnetic particles) c) Redox-active moiety d) Optical dyes such as fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and fluorophores that may be either "low molecular weight" phosphors or protein phosphors (including, but not limited to, chromophores, phosphors, and fluorophores). e) Enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase) f) Biotinylation group g) A predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal-binding domain, an epitope tag, etc.).

[0187] "Fluorescent label" refers to any molecule that can be detected by its intrinsic fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Examples include, but are not limited to, Probes (Eugene, OR), FITC, Rhodamine and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, and Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes containing fluorophores are described in the Molecular Probes Handbook by Richard P. Haugland.

[0188] Suitable protein-based fluorescent labels include green fluorescent proteins containing GFP (Chalfie et al., 1994, Science 263:802-805) and EGFP (Clontech Laboratories, Inc., Genbank accession number U55762) from Renilla, Ptilosarcus, or Aequorea species, blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), and luciferase (Ichiki et al. al., 1993, J.Immunol. 150:5408-5417), β-galactosidase (Nolan et al.) Other examples include, but are not limited to, al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607, and Renilla (International Publication No. 92 / 15673, International Publication No. 95 / 07463, International Publication No. 98 / 14605, International Publication No. 98 / 26277, International Publication No. 99 / 49019, U.S. Patent Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558).

[0189] The antigen-binding molecules of the present invention may also include additional domains that are useful for isolating the molecule or related to the adaptation of its pharmacokinetic profile. Domains useful for isolating the antigen-binding molecule may be selected from a peptide motif that can be captured by an isolation method, such as an isolation column, or a secondarily introduced portion. Non-limiting embodiments of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin-binding domains (CBD tags), maltose-binding protein (MBP tags), Flag tags, Strep tags and their variants (e.g., Strep II tags), and His tags. All antigen-binding molecules disclosed herein may include a His-tagged domain, commonly known as a repeat of consecutive His residues, preferably five, more preferably six His residues (hexahistidine), in the amino acid sequence of the molecule. The His tag may be located at either the N-terminus or the C-terminus of the antigen-binding molecule, but is preferably located at the C-terminus. Most preferably, a hexahistidine tag (HHHHHH) (SEQ ID NO: 16) is attached to the C-terminus of the antigen-binding molecule according to the present invention via a peptide bond. In addition, a PLGA-PEG-PLGA conjugate system may be combined with the polyhistidine tag for sustained-release applications and improved pharmacokinetic profiles.

[0190] Amino acid sequence modifications of the antigen-binding molecules described herein are also intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecules. Amino acid sequence variants of antigen-binding molecules are generated by introducing appropriate nucleotide changes into the nucleic acid of the antigen-binding molecule or by peptide synthesis. All of the amino acid sequence modifications described below should result in antigen-binding molecules that continue to retain the desired biological activity of the unmodified parent molecule (binding to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM and CD3).

[0191] The term "amino acid" or "amino acid residue" usually refers to an amino acid selected from the group consisting of amino acids with a definition accepted in the art, such as alanine (Ala or A); arginine (Arg or R); asparagine (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 T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), but modified amino acids, synthetic amino acids, or rare amino acids may be used as needed. Generally, amino acids can be classified by the presence of nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0192] Amino acid modifications include, for example, deletions of residues from and / or insertions of residues and / or substitutions of residues within the amino acid sequence of an antigen-binding molecule. Any combination of deletions, insertions, and substitutions is made to reach the final construct if that final construct has the desired characteristics. Amino acid changes can also alter post-translational processes of the antigen-binding molecule, such as changes in the number or location of glycosylation sites.

[0193] For example, one, two, three, four, five, or six amino acids may be inserted, substituted, or deleted in each of the CDRs (naturally, depending on their length), while one, two, three, four, five, six, seven, eight, nine, ten, one, two, three, four, nine, six, seven, eight, nine, ten, one, two, three, three, four, five, six, seven, eight, nine, nine, six, six, seven, eight, nine, six, six, seven, eight, nine, or ten residues, as well as intrasequence insertions of one or more amino acid residues. Corresponding modifications may be carried out within a third domain of the antigen-binding molecule of the present invention. Insertion variants of the antigen-binding molecule of the present invention include fusion of an enzyme to the N-terminus or C-terminus of the antigen-binding molecule or fusion to a polypeptide.

[0194] The most important sites for substitutional mutagenesis are the CDRs of the heavy chain and / or light chain, particularly the hypervariable region (HDR), but modifications of the FRs in the heavy chain and / or light chain are also intended. The substitutions are preferably conservative substitutions as described herein. Preferably, depending on the length of the CDR or FR, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in the CDR, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be substituted in the framework region (FR). For example, if the CDR sequence contains 6 amino acids, 1, 2, or 3 of these amino acids are expected to be substituted. Similarly, if the CDR sequence contains 15 amino acids, 1, 2, 3, 4, 5, or 6 of these amino acids are expected to be substituted.

[0195] A useful method for identifying specific residues or regions of an antigen-binding molecule that are preferred positions for mutagenesis is what is described in Cunningham and Wells in Science, 244:1081-1085 (1989) as the "alanine scanning mutagenesis method." In this method, residues or a group of target residues within the antigen-binding molecule are identified (e.g., charged residues such as arg, asp, his, lys, and glu), and are replaced with neutral or negatively charged amino acids (most preferably, alanine or polyalanine) that affect the interaction between the amino acid and the epitope.

[0196] Next, further variants or other variants are introduced into the substitution site, i.e., in place of the substitution site, to stringently select those amino acid positions that show functional sensitivity to the substitution. Thus, the site or region into which the amino acid sequence variant is introduced is predetermined, but it is not necessary to predetermine the nature of the mutation itself. For example, alanine scanning or random mutagenesis may be performed on the target codon or target region to analyze or optimize the performance of the mutation at a given site, and variants of the expressed antigen-binding molecule are screened for the optimal combination of desired activities. Techniques for performing substitution mutations at a predetermined site within DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of variants is performed using assays for antigen-binding activity such as binding to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM or CD3.

[0197] Generally, when one or more or all of the CDRs of the heavy chain and / or light chain are substituted with amino acids, the resulting “substituted” sequence is preferably at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identical to the “original” CDR sequence. This means that the degree to which it is identical to the “substituted” sequence depends on the length of the CDR. For example, a CDR having five amino acids is preferably 80% identical to its substituted sequence because it has at least one substituted amino acid. Therefore, the CDRs of antigen-binding molecules may have different degrees of identity with respect to their substituted sequences; for example, CDRL1 may have 80% identity while CDRL3 may have 90% identity.

[0198] A preferred substitution (or replacement) is a conservative substitution. However, any substitution (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table 3 below) is conceivable, provided that the antigen-binding component retains the ability to bind to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM via a first binding domain and to CD3 epsilon via a second binding domain, and / or that the CDR is identical to the substituted sequence (at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identical to the "original" CDR sequence).

[0199] Conservative substitutions are shown in Table 3 under the heading "Preferred Substitutions." If such substitutions alter biological activity, they are referred to as "Exemplary Substitutions" in Table 3, or more substantial changes, as further described below in relation to amino acid classes, may be introduced to screen the product for desired characteristics.

[0200] [Table 3]

[0201] Substantial modifications to the biological properties of the antigen-binding molecule of the present invention are achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone of the substitution region, for example, as a sheet-like or helical three-dimensional structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the bulkiness of the side chain. Naturally occurring residues are classified into the following groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0202] Non-conservative substitutions involve exchanging one member of one class for another. Replacing any cysteine ​​residue that does not contribute to maintaining the proper three-dimensional structure of an antigen-binding molecule, generally with serine, can improve the oxidative stability of the molecule and avoid abnormal crosslinking. Conversely, adding cysteine ​​bonds to an antibody can improve its stability (especially if the antibody is an antibody fragment such as an Fv fragment).

[0203] With respect to amino acid sequences, sequence identity and / or similarity are determined by standard techniques known in the art, such as, but not limited to, the partial sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computer execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis), or by using the Best Fit sequence program, preferably with default settings, as described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, or by visual inspection. Preferably, the identity percentage is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and join penalty of 30, “Current Methods in Sequence Comparison and Analysis”, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0204] One example of a useful algorithm is PILEUP. PILEUP generates a multisequence alignment from related sequences using pairwise alignment using a progressive method. This also allows plotting a tree showing the clustering relationships used to generate the alignment. PILEUP uses a simplified version of the progressive alignment method described by Feng & Doolittle, 1987, J.Mol.Evol.35:351-360. This method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap.

[0205] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J.Mol.Biol.215:403-410; Altschul et al., 1997, Nucleic Acids Res.25:3389-3402; and Karin et al., 1993, Proc.Natl.Acad.Sci.USA90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to their default values. The adjustable parameters are set to the following values: overlap range = 1, overlap rate = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values, set by the program itself depending on the composition of individual sequences and the composition of the specific database in which the target sequence is being searched, but their values ​​can be adjusted to increase sensitivity.

[0206] Another useful algorithm is gapped BLAST, as reported in Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores, with the threshold parameter T set to 9, and a two-hit method resulting in gapless extension with a cost of 10+k for a gap length k, where Xu is set to 16 and Xg is set to 40 during the database search phase and 67 during the algorithm's output phase. Gapped alignment is provided by scores corresponding to approximately 22 bits.

[0207] Generally, the amino acid homology, similarity, or identity between individual variant CDR or VH / VL sequences is at least 60% with respect to the sequences shown herein, and more typically, the homology or identity is preferably at least 65% or 70%, more preferably at least 75% or 80%, and even more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and nearly 100%. Similarly, the “percentage of nucleic acid sequence identity (%)” for the nucleic acid sequences of binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues in the coding sequence of the antigen-binding molecule. In a specific method, the BLASTN module of WU-BLAST-2 is used, set to default parameters with overlap range and overlap percentage of 1 and 0.125, respectively.

[0208] Generally, the nucleic acid sequence homology, similarity, or identity between the nucleotide sequences encoding individual variant CDR or VH / VL sequences and the nucleotide sequences shown herein is at least 60%, and more typically, it is preferable that the homology or identity increases to at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and nearly 100%. Accordingly, the "variant CDR" or "variant VH / VL region" has specific homology, similarity or identity with respect to the parent CDR / VH / VL of the present invention and shares a biological function that includes, but is not limited to, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent CDR or VH / VL.

[0209] In one embodiment, the percentage of identity of the antigen-binding molecule according to the present invention to human germline is ≥70% or ≥75%, more preferably ≥80% or ≥85%, even more preferably ≥90%, and most preferably ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, or even further ≥96%. Identity to human antibody germline gene products is considered an important feature for reducing the risk that therapeutic proteins will induce an immune response to a drug in a patient undergoing treatment. Hwang & Foote ("Immunogenicity of engineered antibodies"; Methods 36(2005)3-10) have demonstrated that reducing the non-human portion of a drug-antigen-binding molecule leads to a reduction in the risk of inducing anti-drug antibodies in patients undergoing treatment. By comparing a vast number of clinically evaluated antibody drugs and corresponding immunogenicity data, humanization of the V region of an antibody tends to result in lower protein immunogenicity (average 5.1% in patients) than antibodies possessing the unmodified non-human V region (average 23.59% in patients). Therefore, for protein-based therapeutics in the form of antigen-binding molecules based on the V region, high sequence identity to human sequences is desirable. To determine this germline identity, the V region of VL can be aligned with the amino acid sequences of human germline V and J segments (http: / / vbase.mrc-cpe.cam.ac.uk / ) using Vector NTI software, and the amino acid sequence can be calculated as a percentage by dividing the number of identical amino acid residues by the total number of amino acid residues in VL. A similar method is possible for the VH segment (http: / / vbase.mrc-cpe.cam.ac.uk / ), however, VH CDR3 may be excluded due to its high diversity and lack of existing human germline VH CDR3 alignment partners. Subsequently, recombinant technology can be used to increase sequence identity to human antibody germline genes.

[0210] In further embodiments, the bispecific antigen-binding molecules of the present invention exhibit high monomer yields under standard research-scale conditions, for example, in a standard two-step purification process. Preferably, the monomer yield of the antigen-binding molecules according to the present invention is ≥0.25 mg / L supernatant, more preferably ≥0.5 mg / L, even more preferably ≥1 mg / L, and most preferably ≥3 mg / L supernatant.

[0211] Similarly, the yield of dimeric antigen-binding molecule isoforms and thus the monomer ratio (i.e., monomer:(monomer+dimer)) of the antigen-binding molecule can be determined. The productivity of monomer and dimeric antigen-binding molecules and the calculated monomer ratio can be obtained, for example, in the SEC purification step of the culture supernatant derived from standardized study-scale production in roller bottles. In one embodiment, the monomer ratio of the antigen-binding molecule is ≥80%, more preferably ≥85%, even more preferably ≥90%, and most preferably ≥95%.

[0212] In one embodiment, the antigen-binding molecule preferably has plasma stability (ratio of EC50 in the presence of plasma to EC50 in the absence of plasma) of ≤5 or ≤4, more preferably ≤3.5 or ≤3, even more preferably ≤2.5 or ≤2, and most preferably ≤1.5 or ≤1. The plasma stability of the antigen-binding molecule is determined by incubating the construct in human plasma at 37°C for 24 hours, followed by 51This can be tested by determining the EC50 in a chromium-releasing cytotoxicity assay. Effector cells in the cytotoxicity assay may be stimulated, enriched human CD8-positive T cells. Target cells may be, for example, human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM-transfected CHO cells. The effector cell to target cell (E:T) ratio may be selected as 10:1 or 5:1. The human plasma pool used for this purpose is derived from the blood of a healthy donor collected using an EDTA-coated syringe. Cellular components are removed by centrifugation, and the upper plasma phase is collected and then pooled. As a control, antigen-binding molecules are diluted in RPMI-1640 medium immediately before the cytotoxicity assay. Plasma stability is calculated as the ratio of EC50 (after plasma incubation) to EC50 (control).

[0213] It is even more preferable that the monomer-to-dimer conversion rate of the antigen-binding molecule of the present invention be low. The conversion rate can be measured under different conditions and analyzed by high-speed size exclusion chromatography. For example, incubation of the monomer isoform of the antigen-binding molecule can be carried out in an incubator at a concentration of, for example, 100 μg / ml or 250 μg / ml at 37°C for 7 days. Under these conditions, it is preferable that the antigen-binding molecule of the present invention exhibits a dimer ratio of ≤5%, more preferably ≤4%, even more preferably ≤3%, even more preferably ≤2.5%, even more preferably ≤2%, even more preferably ≤1.5%, and most preferably ≤1%, ≤0.5%, or even 0%.

[0214] The bispecific antigen-binding molecule of the present invention also preferably exhibits a very low dimerization rate after several freeze / thaw cycles. For example, the monomer of the antigen-binding molecule is adjusted to a concentration of 250 μg / ml in a general-purpose pharmaceutical buffer, subjected to three freeze / thaw cycles (freezing at -80°C for 30 minutes, then thawing at room temperature for 30 minutes), and then a high-speed SEC is performed to determine the proportion of the initial monomer antigen-binding molecule converted to a dimer antigen-binding molecule. Preferably, the proportion of dimers of the bispecific antigen-binding molecule is, for example, ≤5%, more preferably ≤4%, even more preferably ≤3%, even more preferably ≤2.5%, even more preferably ≤2%, even more preferably ≤1.5%, and most preferably ≤1% or even ≤0.5% after three freeze / thaw cycles.

[0215] The bispecific antigen-binding molecules of the present invention exhibit good thermal stability at aggregation temperatures of preferably ≥45°C or ≥50°C, more preferably ≥52°C or ≥54°C, even more preferably ≥56°C or ≥57°C, and most preferably ≥58°C or ≥59°C. The thermal stability parameter can be determined from the perspective of antibody aggregation temperature as follows: A 250 μg / ml antibody solution is transferred to a single-use cuvette and placed in a dynamic light scattering (DLS) instrument. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C / min while continuously acquiring the measurement radius. The antibody aggregation temperature is calculated using the increase in radius indicating protein melting and aggregation.

[0216] Alternatively, the melting temperature curve can be determined by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of the antigen-binding molecule. These experiments are performed using a MicroCal LLC (Northampton, MA, USA) VP-DSC instrument. Energy uptake of a sample containing the antigen-binding molecule is recorded from 20°C to 90°C and compared with a sample containing only the formulation buffer. The antigen-binding molecule is adjusted to a final concentration of 250 μg / ml in, for example, SEC running buffer. The overall temperature of the sample is gradually increased to record each melting curve. Energy uptake of the sample and the formulation buffer standard is recorded at each temperature T. The difference in energy uptake Cp (kcal / mole / °C) obtained by subtracting the standard from the sample is plotted against each temperature. The melting temperature is defined as the temperature at which energy uptake first reaches its maximum.

[0217] The CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM×CD3 bispecific antigen-binding molecules of the present invention are also assumed to have a turbidity of ≤0.2, preferably ≤0.15, more preferably ≤0.12, even more preferably ≤0.1, and most preferably ≤0.08 (measured by OD340 after concentrating the purified monomer antigen-binding molecule to 2.5 mg / ml and incubating overnight).

[0218] In further embodiments, the antigen-binding molecules according to the present invention are stable at physiological pH or slightly lower, i.e., at approximately pH 7.4 to 6.0. The higher the tolerance exhibited by the antigen-binding molecules at non-physiological pH, for example, approximately pH 6.0, the higher the recovery rate of antigen-binding molecules eluted from the ion-exchange column relative to the total amount of packed protein. The recovery rate of antigen-binding molecules from an ion (e.g., cation) exchange column at approximately pH 6.0 is preferably ≥30%, more preferably ≥40%, more preferably ≥50%, even more preferably ≥60%, even more preferably ≥70%, even more preferably ≥80%, even more preferably ≥90%, even more preferably ≥95%, and most preferably ≥99%.

[0219] The bispecific antigen-binding molecules of the present invention are further expected to exhibit therapeutic efficacy or antitumor activity. This can be evaluated, for example, in the tests disclosed in the generalized examples of advanced-stage human tumor xenograft models below.

[0220] On the first day of the exam, 5 x 10 6 Individual cells of a human target cell antigen (CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM)-positive cancer cell line are subcutaneously injected into the right dorsal flank of female NOD / SCID mice. The average tumor volume is approximately 100 mm². 3 When it reaches this point, in vitro-grown human CD3-positive T cells enter the peritoneal cavity of animals at a rate of approximately 2 × 10⁻¹⁶ 7 The cells are transplanted into mice by injection. Mice in solvent control group 1 do not accept effector cells and are used as a non-transplant control for comparison with solvent control group 2 (which accepts effector cells) to monitor the effect of T cells alone on tumor growth. The average tumor volume is approximately 200 mm³. 3 Antibody therapy is initiated when the tumor reaches a certain stage. The mean tumor size of each treatment group on the initiation date should not be statistically different from any other group (analysis of variance). Mice are treated by intravenous bolus injection for approximately 15–20 days with 0.5 mg / kg / day of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM × CD3 bispecific antigen-binding molecules. Tumors are measured by caliper during the study, and progression is assessed by intergroup comparison of tumor volume (TV). Tumor growth inhibition T / C[%] is determined by calculating TV as T / C% = 100 × (median TV of analysis group) / (median TV of control group 2).

[0221] Those skilled in the art know how to obtain meaningful and reproducible results while modifying or adapting specific parameters of this test, such as the number of tumor cells injected, the injection site, the number of human T cells transplanted, the amount of bispecific antigen-binding molecule administered, and the schedule. Preferably, the tumor growth inhibition T / C [%] is ≤70 or ≤60, more preferably ≤50 or ≤40, even more preferably ≤30 or ≤20, and most preferably ≤10 or ≤5 or even ≤2.5. Tumor growth inhibition is preferably close to 100%.

[0222] In a preferred embodiment of the antigen-binding molecule of the present invention, the antigen-binding molecule is a single-chain antigen-binding molecule.

[0223] Furthermore, in a preferred embodiment of the antigen-binding molecule of the present invention, the third domain is arranged in the order of amino to carboxyl, Hinge-CH2-CH3-Linker-Hinge-CH2-CH3 Includes.

[0224] In one embodiment of the present invention, each of the polypeptide monomers of the third domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 24. In a preferred embodiment or the present invention, each of the polypeptide monomers has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 24.

[0225] Furthermore, in one embodiment of the present invention, the CH2 domain of one or preferably each (both) of the third domains of the polypeptide monomer contains an intradomain cysteine ​​disulfide crosslink. As is known in the art, the term "cysteine ​​disulfide crosslink" refers to a functional group having the general structure RSSR. This linkage is also called an SS bond or disulfide crosslink and is obtained by the coupling of two thiol groups of a cysteine ​​residue. With respect to the antigen-binding molecule of the present invention, it is particularly preferable that the cysteine ​​forming the cysteine ​​disulfide crosslink in the mature antigen-binding molecule be introduced into the amino acid sequence of the CH2 domain corresponding to 309 and 321 (Kabat numbering).

[0226] In one embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain is removed. This removal of the glycosylation site is preferably achieved by an N314X substitution, where X is any amino acid other than Q. The substitution is preferably N314G. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide crosslinks at Kabat positions 309 and 321).

[0227] For example, a preferred feature of the antigen-binding molecule of the present invention compared to a bispecific hetero-Fc antigen-binding molecule known in the art (Figure 1b) is thought to be particularly related to the introduction of the above-mentioned modification in the CH2 domain. Accordingly, with respect to the construct of the present invention, it is preferable that the CH2 domain in the third domain of the antigen-binding molecule of the present invention contains intradomain cysteine ​​disulfide crosslinks at Kabat positions 309 and 321, and / or that the glycosylation site at Kabat position 314 is preferably removed by N314G substitution.

[0228] In a more preferred embodiment of the present invention, the CH2 domain in the third domain of the antigen-binding molecule of the present invention includes intradomain cysteine ​​disulfide crosslinks at Kabat positions 309 and 321, and the glycosylation site at Kabat position 314 is removed by N314G substitution. Most preferably, the polypeptide monomer of the third domain of the antigen-binding molecule of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 18.

[0229] In one embodiment, the present invention is an antigen-binding molecule, (i) The first domain contains two antibody-variable domains, and the second domain contains two antibody-variable domains; (ii) The first domain contains one antibody-variable domain, and the second domain contains two antibody-variable domains; (iii) The first domain contains two antibody-variable domains, and the second domain contains one antibody-variable domain; or (iv) The first domain contains one antibody-variable domain, and the second domain contains one antibody-variable domain, providing an antigen-binding molecule.

[0230] Accordingly, the first and second domains may be binding domains containing two antibody-variable domains, such as VH and VL domains. Examples of such binding domains containing two antibody-variable domains as described herein include, for example, the Fv fragment, scFv fragment, or Fab fragment as described herein. Alternatively, one or both of these binding domains may contain only a single variable domain. Examples of such single-domain binding domains as described herein include, for example, nanobody or single-variable-domain antibodies containing only one variable domain, which may be VHH, VH, or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0231] In preferred embodiments of the antigen-binding molecule of the present invention, the first and second domains are fused to a third domain via a peptide linker. Preferred peptide linkers are described herein and are characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 1) or a polymer thereof, i.e., (Gly4Ser)x, where x is 1 or a greater integer (e.g., 2 or 3). A particularly preferred linker in the fusion of the first and second domains to the third domain is shown in SEQ ID NO: 1.

[0232] In a preferred embodiment, the antigen-binding molecule of the present invention is composed of amino and carboxyl molecules in that order. (a) The first domain; (b) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; (c) Second domain; (d) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11, and 12; (e) The first polypeptide monomer of the third domain; (f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs. 5, 6, 7, and 8; and (g) Second polypeptide monomer of the third domain It is characterized by containing [something].

[0233] The antigen-binding molecule of the present invention comprises a first domain, which binds to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM, preferably to the extracellular domain (ECD) of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM. The term "binding to the extracellular domain of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM" in relation to the present invention is understood to imply that the binding domain binds to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM expressed on the surface of the target cell. Accordingly, the first domain of the present invention preferably binds to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM when CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM is expressed in naturally expressing cells or cell lines and / or when CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM is expressed in cells or cell lines transformed with or (stably / temporarily) transfected with CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM. In a preferred embodiment, the first binding domain also binds to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM when CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM is used as a "target" or "ligand" molecule in an in vitro binding assay such as BIAcore or Scatchard. The "target cell" may be any prokaryotic or eukaryotic cell expressing CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM on its surface, and preferably the target cell is a cell that is part of the body of a human or animal, for example, a specific cancer cell or tumor cell expressing CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM.

[0234] Preferably, the first binding domain binds to human CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM / CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM ECD. In a more preferred embodiment, the first binding domain binds to macaque CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM / CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM ECD. According to the most preferred embodiment, the first binding domain binds to CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM / CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM ECD in both human and macaque. "Extracellular domain of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM" or "CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM ECD" refers to a region or sequence of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM that does not essentially contain the transmembrane domain and cytoplasmic domain of CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM polypeptide. Those skilled in the art will understand that the transmembrane domains identified with respect to the CS1, BCMA, CD20, CD22, FLT3, CD123, CLL1, MSLN, or EpCAM polypeptide of the present invention have been identified according to criteria routinely used in the art to identify this type of hydrophobic domain. While the exact boundaries of transmembrane domains can vary, they are most likely to be approximately five or fewer amino acids at the end of any of the domains specifically mentioned herein.

[0235] Preferred binding domains for CD3 are disclosed in International Publications 2010 / 037836 and 2011 / 121110. Any binding domain for CD3 described in these applications may be used in connection with the present invention.

[0236] In accordance with the embodiments of the present invention, the multispecific antibodies are, in the case of CD123, SEQ ID NOs: 33-35, 44-46, 55-57, 66-68, 77-79, 88-90, 99-101, 110-112, 121-123, 132-134, 143-145, 154-156, 165-167, 176-178, 187-189, 198-200, 209-211, 220-222, 231-233, 242-244, 253-255, 264-266, 275-277, 286-288, 297-299, 308-310, 319-321, 330-332, 341 Selected from the group consisting of ~343, 352~354, 363~365, 374~376, 385~387, 396~398, 407~409, 418~420, 429~431, 440~442, 451~453, 462~464, 473~475, 484~486, 495~497, 506~508, 517~519, 528~530, 539~541, 550~552, 561~563, 572~574, 583~585, 594~596, 605~607, 616~618, 627~629, and in the case of FLT3, sequence numbers 638~640, 649~65 1. Selected from the group consisting of 660-662, for CS1, sequence numbers 896-898, 907-909, 918-920, 929-931, 940-942, 951-953, 962-964, 973-975, 984-986, 995-997, 1006-1008, 1017-1019, 1028-1030, 1039-1041, 1050-1052, 1061-1063, 1072-1074, 1083-1085, 1094-1096, 1105-1107, 1116-1118, 1127-1129, 1138-1140, 1149-115 Selected from the group consisting of 1, 1160~1162, 1171~1173, 1182~1184, 1193~1195, 1204~1206, 1215~1217, 1226~1228, 1237~1239, 1248~1250, 1259~1261, 1270~1272, 1281~1283, 1292~1294, 1303~1305, ~1316, 1325~1327, 1336~1338, 1347~1349, 1358~1360, 1369~1371, 1380~1382, 1391~1393, and in the case of BCMA, sequence numbers 1402~1404,It includes a first and / or second binding domain containing a VH region including CDR-H1, CDR-H2, and CDR-H3, selected from the group consisting of 1413-1415 and 1424-1426; in the case of CD22, selected from the group consisting of SEQ ID NOs: 1489-1491, 1500-1502, 1511-1513, 1522-1524, 1533-1535, and 1544-1546; and in the case of CD20, selected from the group consisting of SEQ ID NOs: 1555-1557, 1566-1568, 1577-1579, 1588-1590, 1599-1601, 1610-1612, 1621-1623, 1632-1634, 1643-1645, and 1654-1656.

[0237] In accordance with the embodiments of the present invention, the multispecific antibodies are, in the case of CD123, SEQ ID NOs: 36-38, 47-49, 58-60, 69-71, 80-82, 91-93, 102-104, 113-115, 124-126, 135-137, 146-148, 157-159, 168-170, 179-181, 190-192, 201-203, 212-214, 223-225, 234-236, 245-247, 256-258, 267-269, 278-280, 289-291, 300-302, 311-313, 322-324, 333-335, Selected from the group consisting of 344-346, 355-357, 366-368, 377-379, 388-390, 399-401, 410-412, 421-423, 432-434, 443-445, 454-456, 465-467, 476-478, 487-489, 498-500, 509-511, 520-522, 531-533, 542-544, 553-555, 564-566, 575-577, 586-588, 597-599, 608-610, 619-621, 630-632, and in the case of FLT3, sequence numbers 641-643, 6 Selected from the groups 52-654 and 663-665, for CS1, the sequence numbers are 899-901, 910-912, 921-923, 932-934, 943-945, 954-956, 965-967, 976-978, 987-989, 998-1000, 1009-1011, 1020-1022, 1031-1033, 1042-1043, 1053-1055, 1064-1066, 1075-1077, 1086-1088, 1097-1099, 1108-1110, 1119-1121, 1130-1132, 1141-1143, Selected from the group consisting of 1152-1154, 1163-1165, 1174-1176, 1185-1187, 1196-1198, 1207-1209, 1218-1220, 1229-1231, 1240-1242, 1251-1253, 1262-1264, 1273-1275, 1284-1286, 1295-1297, 1306-1308, 1317-1319, 1328-1330, 1339-1341, 1350-1352, 1361-1363, 1372-1374, 1383-1385, 1394-1396, in the case of BCMA,Selected from the group consisting of sequence numbers 1405-1407, 1416-1418, and 1427-1429, for CD22, select from the group consisting of sequence numbers 1492-1494, 1503-1505, 1514-1516, 1525-1527, 1536-1538, and 1547-1549, for CD20, select from the group consisting of sequence numbers 1558-1560, 1 It includes a first and / or second binding domain containing a VL region including CDR-L1, CDR-L2, and CDR-L3 selected from the group consisting of 569-1571, 1580-1582, 1591-1593, 1602-1604, 1613-1615, 1624-1626, 1635-1637, 1646-1648, and 1657-1659.

[0238] Similarly, in accordance with the embodiments of the present invention, the multispecific antigen-binding molecules are, in the case of CD123, SEQ ID NOs: 39, 50, 61, 72, 83, 94, 105, 116, 127, 138, 149, 160, 171, 182, 193, 204, 215, 226, 237, 248, 259, 270, 281, 292, 303, 314, 325, 336, 347, 358, 369, 380, 391, 402, 413, 424, 43 Selected from the group consisting of 5, 446, 457, 468, 479, 490, 501, 512, 523, 534, 545, 556, 567, 578, 589, 600, 611, 622, 633; ​​in the case of FLT3, selected from the group consisting of sequence numbers 644, 655, 666; in the case of CS1, selected from the group consisting of sequence numbers 902, 913, 924, 935, 946, 957, 968, 979, 990, 1001, 1012, 1023, 10 Selected from the group consisting of 34, 1045, 1056, 1067, 1078, 1089, 1100, 1111, 1122, 1133, 1144, 1155, 1166, 1177, 1188, 1199, 1210, 1221, 1232, 1243, 1254, 1265, 1276, 1287, 1298, 1309, 1320, 1331, 1342, 1353, 1364, 1375, 1386, and 1397, BCMA In the case of CD20, it includes first and second binding domains containing VH regions selected from the group consisting of SEQ ID NOs: 1408, 1419, and 1430; in the case of CD20, it includes first and second binding domains containing VH regions selected from the group consisting of SEQ ID NOs: 1495, 1506, 1517, 1528, and 1539; and in the case of CD20, it includes first and second binding domains containing VH regions selected from the group consisting of SEQ ID NOs: 1550, 1561, 1572, 1583, 1594, 1605, 1616, 1627, 1638, 1649, and 1660.

[0239] Similarly, in accordance with the embodiments of the present invention, the multispecific antigen-binding molecules are, in the case of CD123, SEQ ID NOs: 40, 51, 62, 73, 84, 95, 106, 117, 128, 139, 150, 161, 172, 183, 194, 205, 216, 227, 238, 249, 260, 271, 282, 293, 304, 315, 326, 337, 348, 359, 370, 381, 392, 403, 414, 425, 436 Selected from the group consisting of 447, 458, 469, 480, 491, 502, 513, 524, 535, 546, 557, 568, 579, 590, 601, 612, 623, 634; in the case of FLT3, selected from the group consisting of sequence numbers 645, 656, 667; in the case of CS1, selected from the group consisting of sequence numbers 903, 914, 925, 936, 947, 958, 969, 980, 991, 1002, 1013, 1024, 1035 Selected from the group consisting of 1046, 1057, 1068, 1079, 1090, 1101, 1112, 1123, 1134, 1145, 1156, 1167, 1178, 1189, 1200, 1211, 1222, 1233, 1244, 1255, 1266, 1277, 1288, 1299, 1310, 1321, 1332, 1343, 1354, 1365, 1376, 1387, 1398, in the case of BCMA. , comprising a first and / or second binding domain including a VL region selected from the group consisting of sequence numbers 1409, 1420, and 1431; in the case of CD20, selected from the group consisting of sequence numbers 1496, 1507, 1518, 1529, 1540, and 1551; in the case of CD22, selected from the group consisting of sequence numbers 1562, 1573, 1584, 1595, 1606, 1617, 1628, 1639, 1650, and 1661.

[0240] In accordance with this embodiment, the first and second domains fused to the single-chain polypeptide via a peptide linker are, in the case of CD123, SEQ ID NOs: 41, 52, 63, 74, 85, 96, 107, 118, 129, 140, 151, 162, 173, 184, 195, 206, 217, 228, 239, 250, 261, 272, 283, 294, 305, 316, 327, 338, 349, 360, 371, 382, ​​393, 404, 415, 426, 437, 448, 459, 470, 481, 492, 503, 514, 525, 536, 547, Selected from the group consisting of 558, 569, 580, 591, 602, 613, 624, 635; in the case of FTL3, selected from the group consisting of sequence numbers 646, 657, 668; in the case of FLT3×CD123, selected from the group consisting of sequence numbers 671, 674, 677, 680, 683, 686, 689, 692, 695, 698, 701, 704, 707, 710, 713, 716, 719, 722, 725, 728, 731, 734, 737, 740, 743, 746, 749, 752, 755, 758, 761, 764, 767, 770, 773, 776, 779, 782, Selected from the group consisting of 785, 788, 791, 794, 797, 800, 803, 806, 809, 812, 815, 818, 821, 824, 827, 830, 833, and in the case of CD123×FLT3, selected from the group consisting of sequence numbers 836, 839, 842, 845, 848, 851, 854, 857, 860, 863, 866, 869, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, and in the case of CS1, selected from the group consisting of sequence numbers 904, 915, 926, 937, 948, 959, 970, 981, 9 Selected from the group consisting of 92, 1003, 1014, 1025, 1036, 1047, 1058, 1069, 1080, 1091, 1102, 1113, 1124, 1135, 1146, 1157, 1168, 1179, 1190, 1201, 1212, 1223, 1234, 1245, 1256, 1267, 1278, 1289, 1300, 1311, 1322, 1333, 1344, 1355, 1366, 1377, 1388, 1399; in the case of BCMA, selected from the group consisting of sequence numbers 1410, 1421, 1432; in the case of CS1×BCMA,Selected from the group consisting of sequence number 1435, in the case of BCMA×CS1, select from the group consisting of sequence numbers 1438, 1441, 1444, 1447, 1450, 1453, 1456, 1459, 1462, and in the case of CS1×BCMA, select from the group consisting of sequence numbers 1465, 1468, 1471, 1474, 1477, 1480, 1483, 1486, C In the case of D22, the selection is made from the group consisting of sequence numbers 1497, 1508, 1519, 1530, 1541, and 1552; in the case of CD20, the selection is made from the group consisting of sequence numbers 1563, 1574, 1585, 1596, 1607, 1618, 1629, 1640, 1651, and 1662; in the case of CD22 × CD20, the selection is made from sequence numbers 1665, 1668, 1671, and 1674. Selected from the group consisting of 1677, 1680, 1683, 1686, 1689, 1692, 1695, 1698, 1701, 1704, 1707, 1710, 1713, 1716, 1719, 1722, 1725, 1728, 1731, 1734, 1737, 1740, 1743, 1746, 1749, 1752, and in the case of CD20×CD22, sequence number 175 The sequence includes a sequence selected from the group consisting of 5, 1758, 1761, 1764, 1767, 1770, 1773, 1776, 1779, 1782, 1785, 1788, 1791, 1794, 1797, 1800, 1803, 1806, 1809, 1812, 1815, 1818, 1821, and 1824, preferably selected from the group consisting of 1399 or 1435.

[0241] In one embodiment, the antigen-binding molecules of the present invention, in the case of CD123, are the group consisting of SEQ ID NOs: 42, 53, 64, 75, 86, 97, 108, 119, 130, 141, 152, 163, 174, 185, 196, 207, 218, 229, 240, 251, 262, 273, 284, 295, 306, 317, 328, 339, 350, 361, 372, 383, 394, 405, 416, 427, 438, 449, 460, 471, 482, 493, 504, 515, 526, 537, 548, 559, 570, 581, 592, 603, 614, 625, 636. Selected from, in the case of FLT3, selected from the group consisting of sequence numbers 647, 658, and 669, and in the case of FLT3×CD123, selected from sequence numbers 672, 675, 678, 681, 684, 687, 690, 693, 696, 699, 702, 705, 708, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 759, 762, 765, 768, 771, 774, 777, 780, 783, 786, 789, 792, 795, 798, 801, 804, 807, 810, 81 Selected from the group consisting of 3, 816, 819, 822, 825, 828, 831, 834, and in the case of CD123×FLT3, selected from the group consisting of sequence numbers 837, 840, 843, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, and in the case of CS1, selected from the group consisting of sequence numbers 905, 916, 927, 938, 949, 960, 971, 982, 993, 1004, 1015, 1026, 1037, 1048, 1059, 1070, 10 Selected from the group consisting of 81, 1092, 1103, 1114, 1125, 1136, 1147, 1158, 1169, 1180, 1191, 1202, 1213, 1224, 1235, 1246, 1257, 1268, 1279, 1290, 1301, 1312, 1323, 1334, 1345, 1356, 1367, 1378, 1389, 1400; in the case of BCMA, selected from the group consisting of sequence numbers 1411, 1422, 1433; in the case of CS1×BCMA, selected from the group consisting of sequence number 1436; in the case of BCMA×CS1, sequence number 1439,Selected from the group consisting of 1442, 1445, 1448, 1451, 1454, 1457, 1460, 1463; for CS1×BCMA, selected from the group consisting of sequence numbers 1466, 1469, 1472, 1475, 1478, 1481, 1484, 1487; for CD22, selected from the group consisting of sequence numbers 1498, 1509, 1520, 1531, 1542 Selected from the group consisting of 1553, in the case of CD20, selected from the group consisting of sequence numbers 1564, 1575, 1586, 1597, 1608, 1619, 1630, 1641, 1652, 1663, in the case of CD22×CD20, sequence numbers 1666, 1669, 1672, 1675, 1678, 1681, 1684, 1687, 1690, 16 Selected from the group consisting of 93, 1696, 1699, 1702, 1705, 1708, 1711, 1714, 1717, 1720, 1723, 1726, 1729, 1732, 1735, 1738, 1741, 1744, 1747, 1750, 1753, and in the case of CD20×CD22, sequence numbers 1756, 1759, 1762, 1765, 176 It is characterized by having an amino acid sequence selected from the group consisting of 8, 1771, 1774, 1777, 1780, 1783, 1786, 1789, 1792, 1795, 1798, 1801, 1804, 1807, 1810, 1813, 1816, 1819, 1822, and 1825, preferably selected from the group consisting of 1400 or 1436.

[0242] The present invention further provides polynucleotide / nucleic acid molecules encoding the antigen-binding molecule of the present invention. A polynucleotide is a biomacromolecule composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotides having different biological functions. A nucleotide is an organic molecule that functions as a monomer or subunit of a nucleic acid molecule such as DNA or RNA. Nucleic acid molecules or polynucleotides can be double-stranded and single-stranded, linear and cyclic. Preferably, they are contained within a vector contained in a host cell. The host cell can express the antigen-binding molecule after transformation or transfection using, for example, the vector or polynucleotide of the present invention. For this purpose, the polynucleotide or nucleic acid molecule is operably linked to a control sequence.

[0243] The genetic code is a set of rules that translate information encoded within genetic material (nucleic acids) into proteins. Biological decoding in living cells is carried out by ribosomes, which use tRNA molecules—which carry amino acids and read three nucleotides from mRNA at once—to link the amino acids in the order specified by the mRNA. This code defines how a sequence of three nucleotides, called a codon, specifies the next amino acid to be added during protein synthesis. With some exceptions, a three-nucleotide codon in a nucleic acid sequence specifies one amino acid. Because most genes are encoded with the exact same code, this particular code is often referred to as the reference genetic code or standard genetic code. While the genetic code determines the protein sequence of a given coding region, other genomic regions can influence when and where these proteins are produced.

[0244] Furthermore, the present invention provides vectors comprising the polynucleotide / nucleic acid molecule of the present invention. A vector is a nucleic acid molecule used as a medium for transferring (foreign) genetic material into cells. The term “vector” includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Generally, genetically engineered vectors include an origin of replication, a multicloning site, and a selection marker. The vector itself is generally a nucleotide sequence, generally a DNA sequence, containing an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Recent vectors may include additional features such as promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags, in addition to the transgene insert and backbone. Vectors called expression vectors (expression constructs) are specifically for the expression of transgenes in target cells and generally have a regulatory sequence.

[0245] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of an operablely linked coding sequence in a particular host organism. Suitable regulatory 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.

[0246] Nucleic acids are "operably linked" if they have a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide if it is expressed as a protein precursor involved in polypeptide secretion; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous and, in the case of a secretion leader, contiguous and within the read frame. Enhancers, however, do not need to be contiguous. Linking is done by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotide adapters or linkers are used, according to conventional practice.

[0247] "Transfection" is a method of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. This term is most often used in non-viral methods in eukaryotic cells. Transduction is often used to describe the viral transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells usually involves creating transient pores, or "holes," in the cell membrane to allow uptake of the substance. Transfection can be performed using calcium phosphate, by electroporation, by compressing the cell, or by mixing cationic lipids with substances that produce liposomes, fusing them with the cell membrane, and accumulating cargo inside.

[0248] The term "transformation" is used to describe the nonviral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and non-animal eukaryotic cells, including plant cells. Therefore, transformation is a genetic modification of a bacterial or non-animal eukaryotic cell resulting from direct uptake from its periphery across the cell membrane and subsequent integration of exogenous genetic material (nucleic acid molecules). Transformation can be induced by artificial means. For transformation to occur, the cell or bacterium must be in a competent state where transformation can occur as a timed response to environmental conditions such as starvation and cell density.

[0249] Furthermore, the present invention provides host cells transformed or transfected with the polynucleotide / nucleic acid molecules or vectors of the present invention. As used herein, the terms “host cell” or “recipient cell” are intended to include any individual cell or cell culture that may or may have been a recipient of the vectors, exogenous nucleic acid molecules and polynucleotides encoding the antigen-binding molecules of the present invention; and / or the antigen-binding molecules themselves. The introduction of each substance into a cell is carried out by transformation, transfection, etc. The term “host cell” is also intended to include single-cell offspring or potential offspring. In subsequent generations, certain modifications may occur due to spontaneous, accidental or intentional mutations, or due to environmental influences, and such offspring may not actually be completely identical to the parent cell (morphologically or with respect to the genome or total DNA set), but are still included within the scope of the terms as used herein. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, as well as bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque, or human cells.

[0250] The antigen-binding molecule of the present invention can be produced within bacteria. After expression, the antigen-binding molecule of the present invention can be isolated from the E. coli cell paste in the soluble fraction and purified, for example, by affinity chromatography and / or size exclusion. Final purification can be carried out, for example, in the same manner as the purification method for antibodies expressed in CHO cells.

[0251] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for the antigen-binding molecules of the present invention. Saccharomyces cerevisiae or common 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, for example, Schizosaccharomyces pombe, K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 16045) Hosts of the genus Kluyveromyces, such as K. thermotolerans and K. marxianus (36906); yarrowia (European Patent No. 402226); Pichia pastoris (European Patent No. 183070); Candida; Trichoderma reesia (European Patent No. 244234); Neurospora crassa; Schwanniomyces occidentalis Hosts include the genus Schwanniomyces (such as Schwanniomyces occidentalis), as well as filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as A. nidulans and A. niger.

[0252] Suitable host cells for the expression of the glycosylated antigen-binding molecules of the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants, as well as corresponding acceptable insect host cells derived from hosts such as the armyworm (Spodoptera frugiperda), Aedes aegypti, Aedes albopictus, Drosophila melanogaster, and silkworm (Bombyx mori), have been identified. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as the viruses of this specification according to the present invention, particularly for transfection of armyworm (Spodoptera frugiperda) cells.

[0253] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, 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 skilled in the art. See, for example, Hiatt 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 et al. (1996) Plant Mol Biol 32:979-986.

[0254] However, there is the greatest interest in vertebrate cells, and the proliferation of vertebrate cells under culture (tissue culture) conditions has become a standard procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, 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)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, 1413 8065); Mouse mammary tumor cells (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad.Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and Human hepatoma strain (Hep G2).

[0255] In further embodiments, the present invention provides a process for generating the antigen-binding molecule of the present invention, comprising culturing host cells of the present invention under conditions that enable the expression of the antigen-binding molecule of the present invention, and recovering the produced antigen-binding molecule from the culture.

[0256] As used herein, the term “culture” refers to the maintenance, differentiation, growth, proliferation, and / or propagation of cells in vitro under preferred conditions in a culture medium. The term “expression” includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion, any step involved in the generation of the antigen-binding molecules of the present invention.

[0257] When using recombinant technology, antigen-binding molecules can be produced intracellularly in the perimembranous space of the cell membrane or secreted directly into the culture medium. If antigen-binding molecules are produced intracellularly, the first step is to remove host cells or particulate fragments of lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the perimembranous space of Escherichia coli (E. coli). Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell fragments can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant from such an expression system is generally concentrated first using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit protein degradation, protease inhibitors such as PMSF may be included in one of the aforementioned steps, and antibiotics may be included to prevent the growth of exogenous contaminating bacteria.

[0258] The antigen-binding molecules of the present invention, prepared from host cells, can be recovered or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Depending on the recovered antibody, other protein purification techniques are also available, such as fractionation on an ion-exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE®, chromatography on anion or cation exchange resin (e.g., polyaspartate column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation. If the antigen-binding molecules of the present invention contain a CH3 domain, Bakerbond ABX resin (JTBaker, Phillipsburg, NJ) is useful for purification.

[0259] Affinity chromatography is a preferred purification technique. While agarose is the matrix to which affinity ligands bind in most cases, other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than those achievable with agarose.

[0260] Furthermore, the present invention provides a pharmaceutical composition comprising an antigen-binding molecule of the present invention or an antigen-binding molecule produced according to the process of the present invention. In the pharmaceutical composition of the present invention, the homogeneity of the antigen-binding molecule is preferably ≥80%, more preferably ≥81%, ≥82%, ≥83%, ≥84%, or ≥85%, even more preferably ≥86%, ≥87%, ≥88%, ≥89%, or ≥90%, still more preferably ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%, and most preferably ≥96%, ≥97%, ≥98%, or ≥99%.

[0261] As used herein, the term “pharmaceutical composition” refers to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions of the present invention contain one or more antigen-binding molecules of the present invention, preferably in a therapeutically effective amount. Preferably, the pharmaceutical composition further comprises one or more suitable formulations of pharmaceutically effective carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. The components of the acceptable composition are preferably nontoxic to the recipient at the doses and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0262] The compositions of the present invention may contain pharmaceutically acceptable carriers. Generally, as used herein, “pharmaceutically acceptable carriers” means any aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, e.g., phosphate-buffered saline (PBS) solutions, water, suspensions, emulsions such as oil / water emulsions, various types of wetting agents, liposomes, dispersion media, and coatings suitable for pharmaceutical administration, particularly parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well known conventional methods.

[0263] Certain embodiments provide pharmaceutical compositions comprising the antigen-binding molecule of the present invention and one or more excipients, such as those described exemplary in this section and elsewhere in this specification. Excipients can be used in the present invention to accommodate a wide range of purposes, such as processes of the present invention for adjusting the physical, chemical, or biological properties of a formulation, such as viscosity, and / or improving its efficacy, and / or stabilizing such a formulation, as well as processes for degradation and damage caused by stress during, for example, manufacturing, transport, storage, preparation before use, administration, and thereafter.

[0264] In certain embodiments, the pharmaceutical composition may contain formulation materials intended to modify, sustain, or protect, for example, the pH, molar osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or osmosis of the composition (see REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (ARGenrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulation materials may include, but are not limited to, the following: • Charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and / or histidine, such as glycine, alanine, glutamine, asparagine, threonine, proline, and 2-phenylalanine. • Antimicrobial agents such as antibacterial agents and antifungal agents • Antioxidants such as ascorbic acid, methionine, sodium sulfite, or sodium bisulfite; Buffers, buffer systems, and buffering agents used to maintain a composition at a physiological pH or slightly lower, preferably a pH lower than 4.0 to 6.5; examples of buffers include borates, bicarbonates, tris-HCl, citrates, phosphates or other organic acids, succinates, phosphates, and histidines; for example, Tris buffer at approximately pH 7.0 to 8.5; Non-aqueous solvents, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate; • Aqueous carriers containing water, alcohol / aqueous solution, emulsion, or suspension, including physiological saline and a buffer medium; • Biodegradable polymers such as polyester; • Fillers such as mannitol or glycine; • Chelating agents such as ethylenediaminetetraacetic acid (EDTA); • Isotonic agents and absorption retarders; • Complexing agents, e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin) • Injectable agent; Monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); the carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol, or xylitol; • (Low molecular weight) proteins, polypeptides, or protein carriers, such as human or bovine serum albumin, gelatin, or preferably immunoglobulins of human origin; • Colorants and fragrances; • Sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate. • Diluent; ·emulsifier; (Hydrophilic polymers such as polyvinylpyrrolidone) • Salt-forming counterions such as sodium; • Antimicrobial agents, antioxidants, chelating agents, and preservatives such as inert gases; examples include benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide. • Metal complexes such as Zn-protein complexes; • Solvents and co-solvents (such as glycerin, propylene glycol, or polyethylene glycol); Sugars and sugar alcohols, such as trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol, stachyose, mannose, sorbose, xylose, ribose, myoinisitose, galactose, lactitol, ribitol, myoinisitol, galactitol, glycerol, cyclitol (e.g., inositol), polyethylene glycol; and polyhydric sugar alcohols; • Suspending agent; • Surfactants or wetting agents, such as Pluronic, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, and tyloxapal; the surfactant may preferably be a detergent having a molecular weight of >1.2 kD and / or a polyether having a molecular weight of >3 kD; non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000; • Stabilizers such as sucrose or sorbitol; • Isotonic enhancers, such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, or sorbitol; Parenteral delivery vehicle containing sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or fixative oil; • Intravenous delivery vehicles containing body fluids, nutritional supplements, and electrolyte supplements (such as those based on ringer's dextrose).

[0265] In connection with the present invention, a pharmaceutical composition that may preferably be a liquid composition, a solid composition obtained by freeze-drying, or a reconstituted liquid composition is (a) an antigen-binding molecule comprising at least three domains, The first domain binds to the target cell surface antigen and has an isoelectric point (pI) in the range of 4 to 9.5; • The second domain binds to the second antigen and has a pI in the range of 8-10, preferably 8.5-9.0; and Optionally, the third domain comprises two polypeptide monomers, each containing a hinge, a CH2 domain, and a CH3 domain, and the two polypeptide monomers are fused to each other via a peptide linker, forming an antigen-binding molecule; (b) at least one type of buffering agent; (c) at least one type of sugar; and (d) at least one surfactant The pharmaceutical composition contains [the specified ingredient], and its pH is in the range of 3.5 to 6.

[0266] In connection with the present invention, it is further assumed that at least one buffering agent is present in a concentration range of 5 to 200 mM, more preferably in a concentration range of 10 to 50 mM. In connection with the present invention, it is assumed that at least one sugar is selected from the group consisting of monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched dextran, or linear unbranched dextran. In connection with the present invention, it is also assumed that the disaccharide is selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, and combinations thereof. In connection with the present invention, it is further assumed that the sugar alcohol is sorbitol. In connection with the present invention, it is assumed that at least one sugar is present in a concentration range of 1 to 15% (m / V), preferably in a concentration range of 9 to 12% (m / V).

[0267] In connection with the present invention, it is also assumed that at least one surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, Pluronic F68, Triton X-100, polyoxyethylene, PEG3350, PEG4000, and combinations thereof. In connection with the present invention, it is further assumed that at least one surfactant is present at a concentration in the range of 0.004 to 0.5% (m / V), preferably in the range of 0.001 to 0.01% (m / V). In connection with the present invention, it is assumed that the pH of the composition is in the range of 4.0 to 5.0, preferably 4.2. In connection with the present invention, it is also assumed that the pharmaceutical composition has a molar osmotic pressure concentration in the range of 150 to 500 mOsm. In connection with the present invention, it is further assumed that the pharmaceutical composition further comprises an excipient selected from the group consisting of one or more polyols and one or more amino acids. In relation to the present invention, it is assumed that the one or more excipients mentioned above are present in a concentration range of 0.1 to 15% (w / V).

[0268] Pharmaceutical compositions, (a) Antigen-binding molecules as described above, (b) 10 mM glutamate or acetate, (c) 9% (m / V) sucrose or 6% (m / V) sucrose and 6% (m / V) hydroxypropyl-β-cyclodextrin, (d) 0.01% (m / V) of polysorbate 80 It is also assumed in connection with the present invention that the liquid pharmaceutical composition contains [the specified substance] and has a pH of 4.2.

[0269] In connection with the present invention, it is further assumed that the antigen-binding molecule is present in a concentration range of 0.1 to 8 mg / ml, preferably 0.2 to 2.5 mg / ml, and more preferably 0.25 to 1.0 mg / ml.

[0270] It will be apparent to those skilled in the art that different components of a pharmaceutical composition (e.g., those listed above) may have different effects, for example, amino acids may act as buffers, stabilizers and / or antioxidants; mannitol may act as fillers and / or isotonic enhancers; and sodium chloride may act as delivery vehicles and / or isotonic enhancers.

[0271] The compositions of the present invention may, in addition to the polypeptides of the present invention as defined herein, contain further biologically active agents depending on the intended use of the composition. Such agents may include drugs known in the art that act on the gastrointestinal system, drugs that act as cell proliferation inhibitors, drugs that prevent hyperurikemia, drugs that inhibit immune responses (e.g., corticosteroids), drugs that modulate inflammatory responses, drugs that act on the circulatory system, and / or cytokines. Furthermore, the antigen-binding molecules of the present invention may also be used in combination therapy, i.e., in combination with other anticancer drugs.

[0272] In certain embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art, for example, depending on the intended route of administration, the form of delivery, and the desired dose. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES (cited above). In certain embodiments, such a composition may affect the physical state, stability, in vivo release rate, and in vitro clearance rate of the antigen-binding molecule of the present invention. In certain embodiments, the main medium or carrier in the pharmaceutical composition may be essentially aqueous or non-aqueous. For example, suitable vehicles or carriers may be water for injection, saline solution, or artificial cerebrospinal fluid, supplemented optionally with other materials common in parenteral administration compositions. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the antigen-binding molecule of the composition of the present invention may be prepared for storage by optionally mixing a selected composition having a desired degree of purity with a compounding agent (REMINGTON'S PHARMACEUTICAL SCIENCES cited above) in the form of a lyophilized cake or aqueous solution. Furthermore, in certain embodiments, the antigen-binding molecule of the present invention can be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0273] When parenteral administration is intended, the therapeutic composition for use in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired antigen-binding molecule of the present invention in a pharmaceutically acceptable vehicle. A vehicle particularly suitable for parenteral injection is sterile distilled water, in which the antigen-binding molecule of the present invention is formulated as a sterile isotonic solution appropriately preserved therein. In certain embodiments, the formulation may include a formulation of the desired molecule with a drug that can provide controlled or sustained release of the product that can be delivered via depot injection, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid having an effect of increasing the duration of action in circulation may also be used. In certain embodiments, the desired antigen-binding molecule may be introduced using an implantable drug delivery device.

[0274] Further pharmaceutical compositions are apparent to those skilled in the art, including formulations containing the antigen-binding molecule of the present invention in sustained-release or controlled-release formulations. Techniques for formulating various other sustained-release or controlled-release means, such as liposome carriers, biodegradable microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, International Patent Application PCT / US Patent Application Publication 93 / 00829, which describes the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions. Sustained-release formulations may include a semipermeable polymer matrix in the form of molded articles, such as films or microcapsules. The sustained-release matrix may include polyester, hydrogel, polylactide (disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. 058481), copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, cited above), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. 133,988). The sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the art. For example, see Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Publication No. 036,676; and Publication Nos. 088,046 and 143,949.

[0275] Antigen-binding molecules can also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. Ed. (1980).

[0276] Pharmaceutical compositions used for in vivo administration are typically provided as sterile formulations. Sterilization can be achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Parenteral compositions can be stored in lyophilized form or in solution. Parenteral compositions are generally filled into containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be penetrated by subcutaneous needles.

[0277] Another aspect of the present invention, as described in International Patent Application Publication No. 06138181A2 (PCT / US Patent Application Publication No. 2006 / 022599), includes a self-buffering antigen-binding molecule of the formulation of the present invention that can be used as a pharmaceutical composition. Various explanations are available regarding protein stabilization and useful pharmaceutical materials and methods therein, for example, Arakawa et al., “Solvent interactions in pharmaceutical formulations”, Pharm Res. 8(3):285-91 (1991); Kendrick et al., “Physical stabilization of proteins in aqueous solution”, in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002); and Randolph et al., “Surfactant-protein interactions”, Pharm Biotechnol. 13:159-75 (2002), particularly with respect to protein pharmaceuticals and processes for veterinary and / or human medical applications, and especially the parts relating to the same excipients and processes as the self-buffering protein formulations according to the present invention.

[0278] Salts may be used, according to certain embodiments of the present invention, for example, to adjust the ionic strength and / or isotonicity of a formulation, and / or to improve the solubility and / or physical stability of a protein or other component of a composition according to the present invention. As is well known, ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein and shielding charged and polar groups in the protein, thereby reducing the intensity of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize denatured proteins, particularly by binding to denatured peptide bonds (--CONH) of the protein. Furthermore, ionic interactions with charged and polar groups in the protein can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0279] The effects of different ionic species on proteins vary considerably. Several classifications of ions and their effects on proteins have been devised and can be used in the formulation of pharmaceutical compositions according to the present invention. One example is the Hofmeister series, which ranks ionic solutes and polar nonionic solutes according to their effect on the stereostructural stability of proteins in solution. Stabilizing solutes are called "cosmotropic." Destabilizing solutes are called "chaotropic." Cosmotropes are generally used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins from solution ("salting out"). Chaotropes are generally used to denature and / or solubilize proteins ("salting out"). The relative effects of ions on "salting out" and "salting out" define the position of ions in the Hofmeister series.

[0280] Free amino acids can be used as fillers, stabilizers, and antioxidants, and for other standard uses, in antigen-binding molecules of the formulations of the present invention according to various embodiments of the present invention. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulations. Glycine is useful for ensuring proper cake structure and properties in lyophilization. Arginine may be useful for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0281] Polyols include sugars, such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols, such as glycerol and propylene glycol, and, for the purposes of this specification, polyethylene glycol (PEG) and related substances. Polyols are cosmotropic. They are useful stabilizers for protecting proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols are also useful for adjusting the isotonicity of formulations. Among the polyols, mannitol is useful in selected embodiments of the present invention, and is commonly used to ensure the structural stability of cakes in lyophilized formulations. Mannitol ensures the structural stability of cakes. Generally, it is used in conjunction with lyophilization protectants, such as sucrose. Sorbitol and sucrose are among the preferred agents for adjusting isotonicity and as stabilizers for protecting against freeze-thaw stress during transport or bulk preparation in the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can saccharify surface lysine and arginine residues. Therefore, they are generally not included in the preferred polyols for use according to the present invention. In addition, sugars that form such reactive species, such as sucrose, are also not included in the preferred polyols of the present invention because they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation. PEG is useful for stabilizing proteins and as a cryoprotective substance, and in this respect it can be used in the present invention.

[0282] Embodiments of the antigen-binding molecule in the formulation of the present invention further include surfactants. Protein molecules are prone to adsorption to surfaces and denaturation and resulting aggregation at gas-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to the protein concentration. These harmful interactions are generally inversely proportional to the protein concentration and are usually aggravated by physical agitation, such as that occurring during product transport and handling. Surfactants are conventionally used to prevent, minimize, or reduce surface adsorption. Useful surfactants in this regard in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188. Surfactants are also commonly used to control the ste...

Claims

1. A multi-targeting single-chain antigen-binding molecule comprising at least three binding domains, (i.) The first binding domain is scFv and contains a paratope that binds immunospecifically to the first target cell surface antigen (TAA1), (ii.) The second binding domain is scFv and contains a paratope that binds immunospecifically to the second target cell surface antigen (TAA2), and (iii.) The third binding domain is scFv and contains a paratope that immunospecifically binds to the extracellular epitope of human and / or macaque CD3ε chain. TAA1 and TAA2 are respectively selected from the groups consisting of CS1 and BCMA, BCMA and CS1, FLT3 and CD123, CD123 and FLT3, CD20 and CD22, CD22 and CD20, EpCAM and MSLN, MSLN and EpCAM, MSLN and CDH3, and CDH3 and MSLN. The first binding domain and the second binding domain are linked by a peptide linker having a length of 5 to 18 amino acids, and the peptide linker between the first binding domain and the second binding domain is S(G ４ S) ｎ , (G ４ S) ｎ G ４ｎ and G ５ｎ (where n is equal to 1, 2 or 3) selected from the group, the first binding domain can bind to TAA1 and the second binding domain can bind to TAA2 simultaneously, TAA1 and TAA2 are located on the same target cell, and The antigen-binding molecule comprises a fourth domain containing two polypeptide monomers, each containing a hinge, a CH2, and a CH3 domain, and the two polypeptide monomers are fused to each other via a peptide linker. The fourth domain described above consists of amino and carboxyl molecules, Hinge - CH2 - CH3 - Linker - Hinge - CH2 - CH3 Includes, Each of the polypeptide monomers in the fourth domain has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 24, or each of the polypeptide monomers has an amino acid sequence selected from SEQ ID NOs: 17 to 24. The CH2 domain includes intradomain cysteine ​​disulfide bridges, and The first, second, third, and fourth binding domains are arranged in the order of amino to carboxyl, forming a multi-targeting single-chain antigen-binding molecule.

2. The peptide linker between the first binding domain and the second binding domain is S(G ４ S) ｎ , (G ４ S) ｎ , G ４ｎ and G ５ｎ (where n is equal to 1 or 2), or the peptide linker is SG ４ S, the multi-targeted antigen-binding molecule according to claim 1.

3. (i) The first and second binding domains each contain two antibody variable domains, and the third binding domain each contains two antibody variable domains; (ii) The first and second binding domains each contain one antibody variable domain, and the third binding domain contains two antibody variable domains; (iii) The first and second binding domains each contain two antibody variable domains, and the third binding domain each contains one antibody variable domain; or (iv) The first binding domain comprises one antibody variable domain, and the second binding domain comprises one antibody variable domain, and / or The multi-targeting antigen-binding molecule according to claim 1, wherein the first, second, and third binding domains are fused to the fourth domain via a peptide linker.

4. From amino to carboxyl, (a) The first binding domain; (b) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3, 9, and 11-12, or the amino acid sequence of SEQ ID NO: 11; (c) The second binding domain; (d) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3; and (e) The third binding domain A multi-targeting antigen-binding molecule according to any one of claims 1 to 3, comprising:

5. From amino to carboxyl, (f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 9, 10, 11 and 12. (g) the first polypeptide monomer of the fourth domain; (h) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8; and (i) The second polypeptide monomer of the fourth domain A multi-targeting antigen-binding molecule according to any one of claims 1 to 4, further comprising:

6. The first and second combined domains are, (a) FLT3 and CD123, or CD123 and FLT3, wherein the first and second binding domains are sequence numbers 33-35 and 36-38, 44-46 and 47-49, 55-57 and 58-60, 66-68 and 69-71, 77-79 and 80-82, 88-90 and 91-93, 99-101 and 102-104, 110-112 and 113-115, 121-123 and 124-126, 132-134 and 135-137, 143-145 and 146-148, 154-156 and 157-159, 165-167 and 168-17 0, 176-178 and 179-181, 187-189 and 190-192, 198-200 and 201-203, 209-211 and 212-214, 220-222 and 223-225, 231-233 and 234-236, 242-244 and 245-247, 253-255 and 256-258, 264-266 and 267-269, 275-277 and 278-280, 286-288 and 289-291, 297-299 and 300-302, 308-310 and 311-313, 319-321 and 322-324, 330-332 and 3 33-335, 341-343 and 344-346, 352-354 and 355-357, 363-365 and 366-368, 374-376 and 377-379, 385-387 and 388-390, 396-398 and 399-401, 407-409 and 410-412, 418-420 and 421-423, 429-431 and 432-434, 440-442 and 443-445, 451-453 and 454-456, 462-464 and 465-467, 473-475 and 476-478, 484-486 and 487-489, 495-4 97 and 498-500, 506-508 and 509-511, 517-519 and 520-522, 528-530 and 531-533, 539-541 and 542-544, 550-552 and 553-555, 561-563 and 564-566, 572-574 and 575-577, 583-585 and 586-588, 594-596 and 597-599, 605-607 and 608-610, 616-618 and 619-621, 627-629 and 630-632, 638-640 and 641-643, 649-651 and 652-654,FLT3 and CD123, or CD123 and FLT3, including a VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of 660-662 and 663-665, and a VL region containing CDR-L1, CDR-L2 and CDR-L3. (b) CS1 and BCMA, or BCMA and CS1, wherein the first and second binding domains are sequence numbers 896-898 and 899-901, 907-909 and 910-912, 918-920 and 921-923, 929-931 and 932-934, 940-942 and 943-945, 951-953 and 954-956, 962-964 and 965-967, 973-975 and 976-978, 984-986 and 987-989, 995-997 and 998-1000, 1006-1008 and 1009-1011, 1017-1 019 and 1020-1022, 1028-1030 and 1031-1033, 1039-1041 and 1042-1043, 1050-1052 and 1053-1055, 1061-1063 and 1064-1066, 1072-1074 and 1075-1077, 1083-1085 and 1086-1088, 1094-1096 and 1097-1099, 1105-1107 and 1108-1110, 1116-1118 and 1119-1121, 1127-1129 and 1130-1132, 1138-1140 and 1141-1143, 1149 ~1151 and 1152~1154, 1160~1162 and 1163~1165, 1171~1173 and 1174~1176, 1182~1184 and 1185~1187, 1193~1195 and 1196~1198, 1204~1206 and 1207~1209, 1215~1217 and 1218~1220, 1226~1228 and 1229~1231, 1237~1239 and 1240~1242, 1248~1250 and 1251~1253, 1259~1261 and 1262~1264, 1270~1272 and 1273~1275, 12 81-1283 and 1284-1286, 1292-1294 and 1295-1297, 1303-1305 and 1306-1308, 1314-1316 and 1317-1319, 1325-1327 and 1328-1330, 1336-1338 and 1339-1341, 1347-1349 and 1350-1352, 1358-1360 and 1361-1363, 1369-1371 and 1372-1374, 1380-1382 and 1383-1385, 1391-1393 and 1394-1396, 1402-1404 and 1405-1407,A VH region including CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of 1413-1415, 1416-1418, 1424-1426, and 1427-1429, and a VL region including CDR-L1, CDR-L2, and CDR-L3, comprising CS1 and BCMA, or BCMA and CS1, (c) CD20 and CD22, or CD22 and CD20, wherein the first and second binding domains are sequence numbers 1489-1491 and 1492-1494, 1500-1502 and 1503-1505, 1511-1513 and 1514-1516, 1522-1524 and 1525-1527, 1533-1535 and 1536-1538, 1544-1546 and 1547-1549, 1555-1557 and 1558-1560, 1566-1568 and 1569-1571, 1577-1579 and 1580-1582, 1588-1 590 and 1591-1593, 1599-1601 and 1602-1604, 1610-1612 and 1613-1615, 1621-1623 and 1624-1626, 1632-1634 and 1635-1637, 1643-1645 and 1646-1648, 1654-1656 and 1657-1659, 1827-1829 and 1830-1832, 1840-1842 and 1843-1845, 1853-1855 and 1856-1858, 1866-1868 and 1869-1871, 1879-1881 and 1882-1884, 1892 ~1894 and 1895-1897, 1905-1907 and 1908-1910, 1922-1924 and 1925-1927, 1935-1937 and 1938-1940, 1948-1950 and 1951-1953, 1961-1963 and 1964-1966, 1974-1976 and 1977-1979, 1987-1989 and 1990-1992, 2000-2002 and 2003-2005, 2013-2015 and 2016-2018, 2026-2028 and 2029-2031, 2039-2041 and 2042-2044, 20 52-2054 and 2055-2057, 2065-2067 and 2068-2070, 2078-2080 and 2081-2083, 2091-2093 and 2094-2096, 2104-2106 and 2107-2109, 2117-2119 and 2120-2122, 2130-2132 and 2133-2135, 2143-2145 and 2146-2148, 2156-2158 and 2159-2161, 2169-2171 and 2172-2174, 2182-2184 and 2185-2187, 2195-2197 and 2198-2200,CD20 and CD22, or CD22 and CD20, include a VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of 2208-2210 and 2211-2213, 2221-2223 and 2224-2226, 2234-2236 and 2237-2239, 2247-2249 and 2250-2252, and a VL region containing CDR-L1, CDR-L2 and CDR-L3. (d) CDH3 and MSLN, or MSLN and CDH3, wherein the first and second binding domains are sequence numbers 3467-3469 and 3470-3472, 3478-3480 and 3481-3483, 3489-3491 and 3492-3494, 3500-3502 and 3503-3505, 3511-3513 and 3514-3516, 3522-3524 and 3525-3527, 3533-3535 and 35 CDH3 and MSLN, or MSLN and CDH3, including a VH region including CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of 36-3538, 3544-3546 and 3547-3549, 3555-3557 and 3558-3560, 3566-3568 and 3569-3571, 3690-3692 and 3693-3695, and a VL region including CDR-L1, CDR-L2 and CDR-L3, and (e) MSLN and EpCAM, or EpCAM and MSLN, wherein the first and second binding domains are sequence numbers 3478-3480 and 3481-3483, 3489-3491 and 3492-3494, 3500-3502 and 3503-3505, 3511-3513 and 3514-3516, 3522-3524 and 3525-3527, 3533-3535 and 3536-3538, 3544-3546 and MSLN and EpCAM, or EpCAM and MSLN, including a VH region containing CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of 3547-3549, 3555-3557 and 3558-3560, 3566-3568 and 3569-3571, 3679-3681 and 3682-3684, 3690-3692 and 3693-3695, and a VL region containing CDR-L1, CDR-L2 and CDR-L3. A multi-targeting antigen-binding molecule according to any one of claims 1 to 5, comprising TAA1 and TAA2.

7. The first and second binding domains are sequence numbers 39 and 40, 50 and 51, 61 and 62, 72 and 73, 83 and 84, 94 and 95, 105 and 106, 116 and 117, 127 and 128, 138 and 139, 149 and 150, 160 and 161, 171 and 172, 182 and 183, 193 and 194, 204 and 205, 215 and 216, 226 and 227, 237 and 238, 248 and 249, 259 and 260, 270 and 271, 281 and 282, 292 and 293, 303 and 304, 314 and 315, 325 and 3 26, 336 and 337, 347 and 348, 358 and 359, 369 and 370, 380 and 381, 391 and 392, 402 and 403, 413 and 414, 424 and 425, 435 and 436, 446 and 447, 457 and 458, 468 and 469, 479 and 480, 490 and 491, 501 and 502, 512 and 513, 523 and 524, 534 and 535, 545 and 546, 556 and 557, 567 and 568, 578 and 579, 589 and 590, 600 and 601, 611 and 612, 622 and 623, 633 and 634, 644 and 645, 655 and 656, 666 and 667, 902 and 903, 913 and 914, 924 and 925, 935 and 936, 946 and 947, 957 and 958, 968 and 969, 979 and 980, 990 and 991, 1001 and 1002, 1012 and 1013, 1023 and 1024, 1034 and 1035, 1045 and 1046, 1056 and 1057, 1067 and 1068, 1078 and 1079, 1089 and 1090, 1100 and 1101, 1111 and 1112, 1122 and 1123, 113 3 and 1134, 1144 and 1145, 1155 and 1156, 1166 and 1167, 1177 and 1178, 1188 and 1189, 1199 and 1200, 1210 and 1211, 1221 and 1222, 1232 and 1233, 1243 and 1244, 1254 and 1255, 1265 and 1266, 1276 and 1277, 1287 and 1288, 1298 and 1299, 1309 and 1310, 1320 and 1321, 1331 and 1332, 1342 and 1343, 1353 and 1354, 1364 and 1365, 1375 and 1376,1386 and 1387, 1397 and 1398, 1408 and 1409, 1419 and 1420, 1430 and 1431, 1495 and 1496, 1506 and 1507, 1517 and 1518, 1528 and 1529, 1539 and 1540, 1550 and 1551, 1561 and 1562, 1572 and 1573, 1583 and 1584, 1594 and 1595, 1605 and 1606, 1616 and 1617, 1627 and 1628, 1638 and 1639, 1649 and 1650, 1660 and 1661, 1833 and 1834, 1846 and 1847, 1859 and 1860, 1872 and 1873, 1885 and 1886, 1898 and 1899, 1911 and 1912, 1928 and 1929, 1941 and 1942, 1954 and 1955, 1967 and 1968, 1980 and 1981, 1993 and 1994, 2006 and 2007, 2019 and 2020, 2032 and 2033, 2045 and 2046, 2058 and 2059, 2071 and 2072, 2084 and 2085 , 2097 and 2098, 2110 and 2111, 2123 and 2124, 2136 and 2137, 2149 and 2150, 2162 and 2163, 2175 and 2176, 2188 and 2189, 2201 and 2202, 2214 and 2215, 2227 and 2228, 2240 and 2241, 2253 and 2254, 3352 and 3353, 3363 and 3364, 3374 and 3375, 3385 and 3386, 3396 and 3397, 3407 and 3408, 3418 and 3419, 3429 A multi-targeted antigen-binding molecule according to any one of claims 1 to 6, comprising a VH region and a VL region selected from the group consisting of 3430, 3440 and 3441, 3451 and 3452, 3462 and 3463, 3473 and 3474, 3484 and 3485, 3495 and 3496, 3506 and 3507, 3517 and 3518, 3528 and 3529, 3539 and 3540, 3550 and 3551, 3561 and 3562, 3572 and 3573, 3686 and 3685, 3696 and 3697.

8. The first and / or second binding domains are sequence numbers 41, 52, 63, 74, 85, 96, 107, 118, 129, 140, 151, 162, 173, 184, 195, 206, 217, 228, 239, 250, 261, 272, 283, 294, 305, 316, 327, 338, 349, 360, 371, 382, ​​393, 404, 415, 426, 437, 448, 459, 470, 481, 492, 503, 514, 525, 536, 547, 558, 569, 580, 591, 602, 613, 624, 635, 646, 657, 668 ,671,674,677,680,683,686,689,692,695,698,701,704,707,710,713,716,719,722,725,728,731,734,737,740,743,746,749,752,755,758,761 ,764,767,770,773,776,779,782,785,788,791,794,797,800,803,806,809,812,815,818,821,824,827,830,833,836,839,842,845,848,851,854, 857, 860, 863, 866, 869, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 904, 915, 926, 937, 948, 959, 970, 981, 992, 1003, 1014, 1025, 1036, 1047, 1058, 1069, 1080, 1091, 1102, 1113, 1124, 1135, 1146, 1157, 1168, 1179, 1190, 1201, 1212, 1223, 1234, 1245, 1256, 1267, 1278, 1289, 1300, 1311, 1322, 1333, 1344, 1355, 1366, 1377, 1388, 1399, 1410, 1421, 1432, 1435, 1438, 1441, 1444, 1447, 1450, 1453, 1456, 1459, 1462, 1465, 1468, 1471, 1474, 1477, 1480, 1483, 1486, 1497, 1508, 1519, 1530, 1541, 1552, 1563, 1574, 1585, 1596, 1607, 1618, 1629, 1640, 1651, 1662, 1665, 1668, 1671, 1674, 1677,１６８０、１６８３、１６８６、１６８９、１６９２、１６９５、１６９８、１７０１、１７０４、１７０７、１７１０、１７１３、１７１６、１７１９、１７２２、１７２５、１７２８、１７３１、１７３４、１７３７、１７４０、１７４３、１７４６、１７４９、１７５２、１７５５、１７５８、１７６１、１７６４、１７６７、１７７０、１７７３、１７７６、１７７９、１７８２、１７８５、１７８８、１７９１、１７９４、１７９７、１８００、１８０３、１８０６、１８０９、１８１２、１８１５、１８１８、１８２１、１８２４、１８３５、１８４８、１８６１、１８７４、１８８７、１９００、１９１３、１９３０、１９４３、１９５６、１９６９、１９８２、１９９５、２００８、２０２１、２０３４、２０４７、２０６０、２０７３、２０８６、２０９９、２１１２、２１２５、２１３８、２１５１、２１６４、２１７７、２１９０、２２０３、２２１６、２２２９、２２４２、２２５５、２２６４、２２６５、２２７４、２２７５、２２８４、２２８５、２２９４、２２９５、２３０４、２３０５、２３１４、２３１５、２３２４、２３２５、２３３４、２３３５、２３４４、２３４５、２３５４、２３５５、２３６４、２３６５、２３７４、２３７５、２３８４、２３８５、２３９４、２３９５、２４０４、２４０５、２４１４、２４１５、２４２４、２４２５、２４３４、２４３５、２４４４、２４４５、２４５４、２４５５、２４６４、２４６５、２４７４、２４７５、２４８４、２４８５、２４９４、２４９５、２５０４、２５０５、２５１４、２５１５、２５２４、２５２５、２５３４、２５３５、２５４４、２５４５、２５５４、２５５５、２５６４、２５６５、２５７４、２５７５、２５８４、２５８５、２５９４、２５９５、２６０４、２６０５、２６１４、２６１５、２６２４、２６２５、２６３４、２６３５、２６４４、２６４５、２６５４、２６５５、２６６４、２６６５、２６７４、２６７５、２６８４、２６８５、２６９４、２６９５、２７０４、２７０５、２７１４、２７１５、２７２４、２７２５、２７３４、２７３５、２７４４、２７４５、２７５４、２７５５、２７６４、２７６５、２７７４、２７７５、２７８４、２７８５、２７９４、２７９５、２８０４、２８０５、２８１４、２８１５、２８２４、２８２５、２８３４、２８３５、２８４４、２８４５、2854, 2855, 2864, 2865, 2874, 2875, 2884, 2885, 2894, 2895, 2904, 2905, 2914, 2915, 2924, 2925, 2934, 2935, 2944, 2945, 2954, 2955, 2964, 2965, 2974, 2975, 2984, 2985, 2994, 2995, 3004, 3005, 3014, 3015, 3024, 3025, 3034, 3035, 3044, 3045, 3054, 3055 ,3064,3065,3074,3075,3084,3085,3094,3095,3104,3105,3114,3115,3124,3125,3134,3135,3144,3145,3154,3155,3164,3165,3174,3175,3184,3185,3194,3195,3204,3205,3214,3215,3224,3225,3234,3235,3244,3245,3254,3255,3264,326 5, 3274, 3275, 3284, 3285, 3294, 3295, 3304, 3305, 3314, 3315, 3324, 3325, 3334, 3335, 3354, 3365, 3376, 3387, 3398, 3409, 3420, 3431, 3442, 3453, 3464, 3475, 3486, 3497, 3508, 3519, 3530, 3541, 3552, 3563, 3574, 3577, 3580, 3583, 3586, 3589, 3592, 35 A multi-targeted antigen-binding molecule according to any one of claims 1 to 7, comprising an scFv sequence selected from the group consisting of 95, 3598, 3601, 3604, 3607, 3610, 3613, 3616, 3619, 3622, 3625, 3628, 3631, 3634, 3637, 3640, 3643, 3646, 3649, 3652, 3655, 3658, 3661, 3664, 3667, 3670, 3673, 3676, 3687, 3698, 3701, and 3706.

9. The molecule comprises a first and / or second target-binding domain and a third effector-binding domain, wherein the two or three binding domains are sequence numbers 42, 53, 64, 75, 86, 97, 108, 119, 130, 141, 152, 163, 174, 185, 196, 207, 218, 229, 240, 251, 262, 273, 284, 295, 306, 317, 328, 339, 350, 361, 372, 383, 394, 405, 416, 427, 438, 449, 460, 471, 482, 493, 504, 515, 526, 537, 548, 559, 570, 581, 592, 603, 614, 625, 636, 647, 658, 669, 672, 675, 678, 681, 684, 687, 690, 693, 696, 699, 702, 705, 708, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 759, 762, 765, 768, 771, 774, 777, 780, 783, 786, 789, 792, 795, 798, 801, 804, 807, 810, 813, 816, 819, 822, 8 25, 828, 831, 834, 837, 840, 843, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 905, 916, 927, 9 38, 949, 960, 971, 982, 993, 1004, 1015, 1026, 1037, 1048, 1059, 1070, 1081, 1092, 1103, 1114, 1125, 1136, 1147, 1158, 1169, 1180, 1191, 1202, 1213, 1 224, 1235, 1246, 1257, 1268, 1279, 1290, 1301, 1312, 1323, 1334, 1345, 1356, 1367, 1378, 1389, 1400, 1411, 1422, 1433, 1436, 1439, 1442, 1445, 1448, 1451, 1454, 1457, 1460, 1463, 1466, 1469, 1472, 1475, 1478, 1481, 1484, 1487, 1498, 1509, 1520, 1531, 1542, 1553, 1564, 1575, 1586, 1597, 1608, 1619,１６３０、１６４１、１６５２、１６６３、１６６６、１６６９、１６７２、１６７５、１６７８、１６８１、１６８４、１６８７、１６９０、１６９３、１６９６、１６９９、１７０２、１７０５、１７０８、１７１１、１７１４、１７１７、１７２０、１７２３、１７２６、１７２９、１７３２、１７３５、１７３８、１７４１、１７４４、１７４７、１７５０、１７５３、１７５６、１７５９、１７６２、１７６５、１７６８、１７７１、１７７４、１７７７、１７８０、１７８３、１７８６、１７８９、１７９２、１７９５、１７９８、１８０１、１８０４、１８０７、１８１０、１８１３、１８１６、１８１９、１８２２、１８２５、１８３６、１８４９、１８６２、１８７５、１８８８、１９０１、１９１４、１９３１、１９４４、１９５７、１９７０、１９８３、１９９６、２００９、２０２２、２０３５、２０４８、２０６１、２０７４、２０８７、２３００、２１１３、２１２６、２１３９、２１５２、２１６５、２１７８、２１９１、２２０４、２２１７、２２３０、２２４３、２２５６、２２６０、２２６６、２２６７、２２７６、２２７７、２２８６、２２８７、２２９６、２２９７、２３０６、２３０７、２３１６、２３１７、２３２６、２３２７、２３３６、２３３７、２３４６、２３４７、２３５６、２３５７、２３６６、２３６７、２３７６、２３７７、２３８６、２３８７、２３９６、２３９７、２４０６、２４０７、２４１６、２４１７、２４２６、２４２７、２４３６、２４３７、２４４６、２４４７、２４５６、２４５７、２４６６、２４６７、２４７６、２４７７、２４８６、２４８７、２４９６、２４９７、２５０６、２５０７、２５１６、２５１７、２５２６、２５２７、２５３６、２５３７、２５４６、２５４７、２５５６、２５５７、２５６６、２５６７、２５７６、２５７７、２５８６、２５８７、２５９６、２５９７、２６０６、２６０７、２６１６、２６１７、２６２６、２６２７、２６３６、２６３７、２６４６、２６４７、２６５６、２６５７、２６６６、２６６７、２６７６、２６７７、２６８６、２６８７、２６９６、２６９７、２７０６、２７０７、２７１６、２７１７、２７２６、２７２７、２７３６、２７３７、２７４６、２７４７、２７５６、２７５７、２７６６、２７６７、２７７６、２７７７、２７８６、２７８７、２７９６、２７９７、2806, 2807, 2816, 2817, 2826, 2827, 2836, 2837, 2846, 2847, 2856, 2857, 2866, 2867, 2876, 2877, 2886, 2887, 2896, 2897, 2906, 2907, 2916, 2917, 2926, 2927, 2936, 2937, 2946, 2947, 2956, 2957, 2966, 2967, 2976, 2977, 2986, 2987, 2996, 2997, 3006, 3007, 3016, 3017, 30 26, 3027, 3036, 3037, 3046, 3047, 3056, 3057, 3066, 3067, 3076, 3077, 3086, 3087, 3096, 3097, 3106, 3107, 3116, 3117, 3126, 3127, 3136, 3137, 3146, 3147, 3156, 3157, 3166, 3167, 3176, 3177, 3186, 3187, 3196, 3197, 3206, 3207, 3216, 3217, 3226, 3227, 3236, 3237, 3246 ,3247,3256,3257,3266,3267,3276,3277,3286,3287,3296,3297,3306,3307,3316,3317,3326,3327,3336,3337,3355,3366,3377,3388,3399,3410,3421,3432,3443,3454,3465,3476,3487,3498,3509,3520,3531,3542,3553,3564,3575,3578,3581,3584,3587,3 A multi-targeted antigen-binding molecule according to any one of claims 1 to 8, having a sequence selected from the group consisting of 590, 3593, 3596, 3599, 3602, 3605, 3608, 3611, 3614, 3617, 3620, 3623, 3626, 3629, 3632, 3635, 3638, 3641, 3644, 3647, 3650, 3653, 3656, 3659, 3662, 3665, 3668, 3671, 3674, 3677, 3688, 3699, 3702, 3703, and 3707.

10. The molecule comprises a first and / or second target-binding domain, a third effector-binding domain and a fourth domain that provides an extended half-life, wherein the two or three binding domains and the fourth domain are sequence numbers 43, 54, 65, 76, 87, 98, 109, 120, 131, 142, 153, 164, 175, 186, 197, 208, 219, 230, 241, 252, 263, 274, 285, 296, 307, 318, 329, 340, 351, 362, 373, 384, 395, 406, 417, 428, 439, 450, 46 1, 472, 483, 494, 505, 516, 527, 538, 549, 560, 571, 582, 593, 604, 615, 626, 637, 648, 659, 670, 673, 676, 679, 682, 685, 688, 691, 694, 697, 700, 703, 7 06, 709, 712, 715, 718, 721, 724, 727, 730, 733, 736, 739, 742, 745, 748, 751, 754, 757, 760, 763, 766, 769, 772, 775, 778, 781, 784, 787, 790, 793, 796, 7 99, 802, 805, 808, 811, 814, 817, 820, 823, 826, 829, 832, 835, 838, 841, 844, 847, 850, 853, 856, 859, 862, 865, 868, 871, 906, 917, 928, 939, 950, 961, 972, 983, 994, 1005, 1016, 1027, 1038, 1049, 1060, 1071, 1082, 1093, 1104, 1115, 1126, 1137, 1148, 1159, 1170, 1181, 1192, 1203, 1214, 1225, 1236, 12 47, 1258, 1269, 1280, 1291, 1302, 1313, 1324, 1335, 1346, 1357, 1368, 1379, 1390, 1401, 1412, 1423, 1434, 1437, 1440, 1443, 1446, 1449, 1452, 1455, 1 458, 1461, 1464, 1467, 1470, 1473, 1476, 1479, 1482, 1485, 1488, 1499, 1510, 1521, 1532, 1543, 1554, 1565, 1576, 1587, 1598, 1609, 1620, 1631, 1642,１６５３、１６６４、１６６７、１６７０、１６７３、１６７６、１６７９、１６８２、１６８５、１６８８、１６９１、１６９４、１６９７、１７００、１７０３、１７０６、１７０９、１７１２、１７１５、１７１８、１７２１、１７２４、１７２７、１７３０、１７３３、１７３６、１７３９、１７４２、１７４５、１７４８、１７５１、１７５４、１７５７、１７６０、１７６３、１７６６、１７６９、１７７２、１７７５、１７７８、１７８１、１７８４、１７８７、１７９０、１７９３、１７９６、１７９９、１８０２、１８０５、１８０８、１８１１、１８１４、１８１７、１８２０、１８２３、１８２６、１８３８、１８５１、１８６４、１８７７、１８９０、１９０３、１９１６、１９３３、１９４６、１９５９、１９７２、１９８５、１９９８、２０１１、２０２４、２０３７、２０５０、２０６３、２０７６、２０８９、２１０２、２１１５、２１２８、２１４１、２１５４、２１６７、２１８０、２１９４、２２０６、２２１９、２２３２、２２４５、２２５８、２２６２、２２７０、２２７１、２２８０、２２８１、２２９０、２２９１、２３００、２３０１、２３１０、２３１１、２３２０、２３２１、２３３０、２３３１、２３４０、２３４１、２３５０、２３５１、２３６０、２３６１、２３７０、２３７１、２３８０、２３８１、２３９０、２３９１、２４００、２４０１、２４１０、２４１１、２４２０、２４２１、２４３０、２４３１、２４４０、２４４１、２４５０、２４５１、２４６０、２４６１、２４７０、２４７１、２４８０、２４８１、２４９０、２４９１、２５００、２５０１、２５１０、２５１１、２５２０、２５２１、２５３０、２５３１、２５４０、２５４１、２５５０、２５５１、２５６０、２５６１、２５７０、２５７１、２５８０、２５８１、２５９０、２５９１、２６００、２６０１、２６１０、２６１１、２６２０、２６２１、２６３０、２６３１、２６４０、２６４１、２６５０、２６５１、２６６０、２６６１、２６７０、２６７１、２６８０、２６８１、２６９０、２６９１、２７００、２７０１、２７１０、２７１１、２７２０、２７２１、２７３０、２７３１、２７４０、２７４１、２７５０、２７５１、２７６０、２７６１、２７７０、２７７１、２７８０、２７８１、２７９０、２７９１、２８００、２８０１、２８１０、２８１１、2820, 2821, 2830, 2831, 2840, 2841, 2850, 2851, 2860, 2861, 2870, 2871, 2880, 2881, 2890, 2891, 2900, 2901, 2910, 2911, 2920, 2921, 2930, 2931, 2940, 2941, 2950, ​​2951, 2960, 2961, 2970, 2971, 2980, 2981, 2990, 2991, 3000, 3001, 3010, 3011, 3020, 3021, 3030, 3031, 3040 ,3041,3050,3051,3060,3061,3070,3071,3080,3081,3090,3091,3100,3101,3110,3111,3120,3121,3130,3131,3140,3141,3150,3151,3160,3161,3170,3171,3180,3181,3190,3191,3200,3201,3210,3211,3220,3221,3231,3240,3241,3250,3251,3260,3261,3270 ,3271,3280,3281,3290,3291,3300,3301,3310,3311,3320,3321,3330,3331,3340,3341,3344,3345,3356,3367,3378,3389,3400,3411,3422,3433,3444,3455,3466,3477,3488,3499,3510,3521,3532,3543,3554,3565,3576,3579,3582,3585,3588,3591,3594,359 A multi-targeted antigen-binding molecule according to any one of claims 1 to 9, having a sequence selected from the group consisting of 7, 3600, 3603, 3606, 3609, 3612, 3615, 3618, 3621, 3624, 3627, 3630, 3633, 3636, 3639, 3642, 3645, 3648, 3651, 3654, 3657, 3660, 3663, 3666, 3669, 3672, 3675, 3678, 3689, 3700, 3704, 3705, 3708, 3709, 3710, and 3711.

11. A pharmaceutical composition comprising a multi-targeted antigen-binding molecule according to any one of claims 1 to 10.

12. A multi-targeted antigen-binding molecule according to any one of claims 1 to 10, for use in the prevention, treatment, or remission of a disease selected from proliferative disorders, neoplastic disorders, cancer, or immunodeficiency.

13. The multi-targeted antigen-binding molecule according to claim 12, wherein the disease is selected from multiple myeloma (MM), acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

Images