Humanized or chimeric CD3 antibodies

Abstract

The present invention relates to humanized or chimeric antibodies that bind to CD3, as well as bispecific antibodies, compositions, pharmaceutical compositions, uses of the antibodies in treating diseases, and methods of treatment.

Description

Field of the Invention

[0001] The present invention relates to humanized or chimeric antibodies that bind to human CD3, compositions comprising the humanized or chimeric antibodies, and uses of the humanized or chimeric antibodies in treating diseases. Background of the Invention

[0002] Cluster of differentiation 3 (CD3) has been known for many years and is therefore of interest in many areas. In particular, antibodies are known to be raised against CD3 or the T-cell receptor complex of which CD3 is a part. In vitro characterization of recombinant chimeric CD3 isotype variants as well as a number of humanized OKT3 effector function variant antibodies has been described [1].

[0003] CD3 antibodies, such as muromonab-CD3, have been widely used to treat acute allogeneic transplant rejection. Furthermore, in the absence of ongoing immunosuppressive drug therapy, treatment with the anti-CD3 monoclonal antibody hOKT3γ1(Ala-Ala) resulted in improved C-peptide responses and clinical parameters for at least 2 years after the onset of type 1 diabetes [2].

[0004] A promising approach to improve targeted antibody therapy is by delivering cytotoxic cells specifically directed against cancer cells expressing antigens. This concept of using T cells to effectively kill tumor cells has been described [3]. However, initial clinical studies were quite disappointing, primarily due to low efficacy, severe side effects (cytokine burst), and immunogenicity of bispecific antibodies [4]. Advances in the design and application of bispecific antibodies have partially overcome the initial barrier of cytokine burst and improved clinical effectiveness without dose-limiting toxicity [5].

[0005] For example, certain bispecific antibodies that target an antigen on tumor cells with one arm and CD3 on T cells with the other arm and contain an active Fc fragment that provides Fc receptor binding have been shown to induce tumor cell killing. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the Fc region of the antibody potentially forms, leading to tumor cell killing [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heavy chain heterodimer and has been found to be successful in treating cancer-related ascites after intraperitoneal administration [6]. However, the mouse / rat hybrid is immunogenic [7] and cannot be used for long-term treatment in humans. Frequent treatment-related adverse events have been attributed to symptoms related to catumaxomab-induced cytokine release (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) [8]-[9], which are related to the potent polyclonal T cell activation caused by catumaxomab due to its active Fc fragment. Another antibody is ertumaxomab (HER2xCD3), which induces cytotoxicity in cell lines with HER2 expression. Ertumaxomab is already in Phase II clinical development for metastatic breast cancer

[10] -

[11] .

[0006] The efficacy of CD3 bispecific antibodies and other CD3 bispecific antibody-based formats depends on several properties of the bispecific antibody, such as the affinity of the CD3 arm and / or the target affinity of the second arm and the target copy number on the target cell. When CD3 affinity is low, some CD3 bispecific antibodies show high efficacy (EpCamxCD3-Bortoletto 2002PMID12385030, MT103 / Blinatumomab vs TandAb-Molhoj 2007PMID 17083975), while other CD3 bispecifics use high CD3 affinity to show high efficacy (Reusch 2015, Mabs, PMID 25875246). High CD3 affinity is required in some cases, such as when providing bispecific antibodies comprising an anti-CD3 targeting arm and a second arm for a selected tumor-associated antigen to activated T cells expanded in vitro from a patient. In the latter case, when the product is infused back into the patient to mediate cytolysis of tumor cells, the CD3 affinity should be high to maintain interaction with the expanded T cells (Reusch 2006 Clin Cancer Res PMID 16397041). However, compared to low-affinity ligands, high-affinity antibodies against CD3 are much less effective in TCR triggering at low copy numbers because they show a stoichiometry of approximately 1:1 and a linear dose-response curve, indicating a single cycle rather than a continuous triggering mode of T cell response (Viola 1996 Science, PMID 8658175). In other words, the low affinity of the CD3 arm allows T cells to flexibly move from one target and / or target cell to another (Hoffman 2005, PMID: 15688411).

[0007] Low CD3 affinity can potentially prevent the biased localization of bispecific antibodies to T cells (due to first encounter in the circulation) and thus improve biodistribution and minimize interference with normal T cell immune responses. Depending on the target and target copy number of the second arm, the indication and / or route of administration, the desired CD3 affinity can be customized to increase the maximum efficacy of the product. A panel of CD3 variants covering a range of CD3 affinities may be essential for meeting these specific customization requirements by the antibody product.

[0008] CD3 antibodies that cross-react with cynomolgus and / or rhesus CD3 have been described

[12] -

[13] , however further improvements in these cross-reactive antibodies are needed. Summary of the Invention

[0009] It is an object of the present invention to provide humanized or chimeric CD3 antibodies with optimized affinity for CD3. Accordingly, it is an object of the present invention to provide humanized or chimeric CD3 antibodies that are optimized compared to a reference antibody, such as the antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. Thus, such antibodies may have reduced or increased affinity for CD3 compared to the reference antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. Another object of the present invention is to provide antibodies that have a lower binding affinity for CD3 compared to the antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. The inventors have discovered that antibodies that have reduced binding affinity for the CD3 peptide set forth in SEQ ID NO: 402 compared to the reference antibody having the VH region sequence set forth in SEQ ID NO: 4 maintain the same or similar cytotoxic activity in vitro and in vivo. Another object of the present invention is to provide a CD3 antibody having reduced binding affinity for CD3 compared to a reference antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8, but retaining the same cytolytic activity as the reference antibody. Another object of the present invention is to provide an antibody having higher binding affinity for CD3 than the antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8.

[0010] In one aspect, the invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises a mutation in one of the three CDR sequences of a reference antibody having the VH CDR sequences shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3, wherein the mutation is located at a position selected from the group consisting of: T31M, T31P, N57, H101, G105, S110, and Y114, wherein the positions are numbered according to the reference sequence of SEQ ID NO: 4. Amino acids in SEQ ID NO: 4 are numbered according to the direct numbering scheme from the first amino acid to number 125 in the N-terminal to C-terminal direction. Figure 2 The numerical numbers corresponding to the positions in SEQ ID NO: 4 are shown in FIG. In addition, the VH CDR regions have been annotated according to the IMGT definition.

[0011] In one embodiment of the invention, the antibody has reduced or increased binding affinity for human CD3 compared to a reference antibody having VH CDR sequences shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3.

[0012] In some embodiments of the invention, an antibody having reduced binding affinity for a human CD3 molecule, eg, a CD3 peptide, eg, SEQ ID NO: 402, compared to a reference antibody can have the same cytolytic activity against target cells as the reference antibody.

[0013] In one embodiment of the invention, the antibody comprises a mutation at a position corresponding to N57 of SEQ ID NO: 4. In one embodiment, the mutation is N57E.

[0014] In one embodiment of the invention, the antibody comprises a mutation at the position corresponding to H101G of SEQ ID NO: 4. In one embodiment, the mutation is H101G or H101N.

[0015] In one embodiment of the invention, the antibody comprises a mutation at the position corresponding to G105 of SEQ ID NO: 4. In one embodiment, the mutation is G105P.

[0016] In one embodiment of the invention, the antibody comprises a mutation at a position corresponding to Y114 of SEQ ID NO: 4. In one embodiment, the mutation is Y114M, Y114R or Y114V.

[0017] In one embodiment, the invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions having CDR sequences selected from the group consisting of:

[0018] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 12, 2, 3;

[0019] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 14, 2, 3;

[0020] c) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 16, 2, 3;

[0021] d) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 18, 2, 3;

[0022] e) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 20, 2, 3;

[0023] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 22, 2, 3;

[0024] g) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 24, 2, 3;

[0025] h) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 26, 2, 3;

[0026] i) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 28, 2, 3;

[0027] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 30, 2, 3;

[0028] k) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 32, 2, 3;

[0029] l) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 34, 2, 3;

[0030] m) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 36, 2, 3;

[0031] n) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 38, 2, 3;

[0032] o) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 40, 2, 3;

[0033] p) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 42, 2, 3;

[0034] q) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 44, 2, and 3;

[0035] r) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 46, 2, 3;

[0036] s) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 48, 2, 3;

[0037] t) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 50, 2, 3;

[0038] u) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 52, 2, 3;

[0039] v) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3;

[0040] w) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 56, 2, 3;

[0041] x) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 58, 2, and 3;

[0042] y) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 60, 2, 3;

[0043] z) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 62, 2, 3;

[0044] aa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 64, 2, or 3;

[0045] bb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 66, 2, and 3;

[0046] cc) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 68, 2, 3;

[0047] dd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 70, 2, 3;

[0048] ee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 72, 2, 3;

[0049] ff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 74, 2, 3;

[0050] gg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 76, 2, and 3;

[0051] hh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 78, 2, and 3;

[0052] ii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 80, 2, 3;

[0053] jj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 82, 2, 3;

[0054] kk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 84, 2, 3;

[0055] 11) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 86, 2, 3;

[0056] mm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 88, 2, 3;

[0057] nn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 90, 2, 3;

[0058] oo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:92, 2, 3;

[0059] pp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 2, 3;

[0060] qq) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 96, 2, and 3;

[0061] rr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 98, 2, 3;

[0062] ss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 100, 3;

[0063] tt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 102, and 3;

[0064] uu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 104, 3;

[0065] vv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 106, 3;

[0066] ww) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 108, 3;

[0067] xx) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 110, 3;

[0068] yy) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 112, and 3;

[0069] zz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 114, 3;

[0070] aaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 116, 3;

[0071] bbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 118, 3;

[0072] ccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 120, and 3;

[0073] ddd) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 122, and 3;

[0074] eee) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 124, and 3;

[0075] fff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 126, 3;

[0076] ggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 128, 3;

[0077] hhh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 130, 3;

[0078] iii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 132, 3;

[0079] jjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 134, 3;

[0080] kkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 136, 3;

[0081] 111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 138, and 3;

[0082] mmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 140, 3;

[0083] nnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 142, 3;

[0084] ooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 144, 3;

[0085] ppp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 146, 3;

[0086] qqq) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 148, 3;

[0087] rrr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 150, 3;

[0088] sss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 152, 3;

[0089] ttt) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 154, 3;

[0090] uuu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 156, 3;

[0091] vvv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 158, 3;

[0092] www) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,160,3;

[0093] xxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 162, 3;

[0094] yyy) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,164,3;

[0095] zzz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,166,3;

[0096] aaaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 168, 3;

[0097] bbbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 170;

[0098] cccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 172;

[0099] dddd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 174;

[0100] eeee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176;

[0101] ffff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 178;

[0102] gggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 180;

[0103] hhhh) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 182;

[0104] iiii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 184;

[0105] jjjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 186;

[0106] kkkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 188;

[0107] 111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 190;

[0108] mmmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 192;

[0109] nnnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 194;

[0110] oooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 196;

[0111] pppp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 198;

[0112] qqqq) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 200;

[0113] rrrr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 202;

[0114] ssss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 204;

[0115] tttt) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 206;

[0116] uuuu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 208;

[0117] vvvv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 210;

[0118] wwww) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 212;

[0119] xxxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 214;

[0120] yyyy) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 216;

[0121] zzzz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 218;

[0122] aaaaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 220;

[0123] bbbbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 222;

[0124] ccccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 224;

[0125] ddddd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 226;

[0126] eeeee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 228;

[0127] fffff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 230;

[0128] ggggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 232;

[0129] hhhhh) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 234;

[0130] iiiii) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 236;

[0131] jjjjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 238;

[0132] kkkkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 240;

[0133] 1111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 242;

[0134] mmmmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244;

[0135] nnnnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 246;

[0136] ooooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 248;

[0137] ppppp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 250;

[0138] qqqqq) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 252;

[0139] rrrrr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 254;

[0140] sssss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 256;

[0141] ttttt) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 258;

[0142] uuuuu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 260;

[0143] vvvvv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 262;

[0144] wwwww) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 264;

[0145] xxxxx) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 266;

[0146] yyyyy) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 268;

[0147] zzzzz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 270;

[0148] aaaaaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 272;

[0149] bbbbbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 274;

[0150] cccccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 276;

[0151] dddddd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 278;

[0152] eeeeee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 280;

[0153] ffffff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 282;

[0154] gggggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284;

[0155] hhhhhh) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 286;

[0156] iiiiii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 288;

[0157] jjjjjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 290;

[0158] kkkkkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292;

[0159] 1111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 294;

[0160] mmmmmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 296;

[0161] nnnnnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298; and

[0162] oooooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 300.

[0163] That is, in the first aspect of the present invention, the inventors have found that humanized or chimeric antibodies of the sequences have an optimized binding affinity for the CD3 peptide SEQ ID NO: 402 compared to a reference antibody (e.g., an antibody designated by the VH sequence SEQ ID NO: 4 and the VL sequence SEQ ID NO: 8). As shown in Example 7, the reference antibody designated by the VH sequence SEQ ID NO: 4 and the VL sequence SEQ ID NO: 8 has an affinity of 1.5x10 -8 In some embodiments of the present invention, the binding affinity of the antibody to the CD3 peptide of SEQ ID NO: 402 is less than 1.5×10 -8 M, for example, 1.6×10 -8 M to 9.9×10 -8 The binding affinity of M or for example 1.0×10 -7 to 9.9×10 -7The binding affinity of the antibody to the CD3 peptide of SEQ ID NO: 402 is greater than 1.5×10 -8 M, for example, 1.4×10 -8 to 1.0×10 -8 M, for example, 9.9×10 -9 to 1×10 -9 M or for example 9.9×10 -9 to 1×10 -9 M. Binding affinity corresponds to K D value.

[0164] In one aspect of the invention, the invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2 and CDR3 regions having CDR sequences selected from one of the following:

[0165] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M];

[0166] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0167] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0168] d) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 176 [H101G];

[0169] e) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N];

[0170] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0171] g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A];

[0172] h) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G];

[0173] i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M];

[0174] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R];

[0175] k) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and

[0176] l) CDR1, CDR2 and CDR3 sequences that have a total of at least 90% or at least 95% amino acid sequence identity with any one of the three CDR sequences shown in a) to k), with the proviso that the CDR1, CDR2 and CDR3 sequences do not have the sequences shown in SEQ ID NOs: 1, 2 or 3.

[0177] On the other hand, the present invention relates to a humanized or chimeric antibody, wherein the binding region comprises a light chain variable (VL) region, wherein the VL region comprises CDR1, CDR2 and CDR3 regions having the CDRs shown in SEQ ID NO:6, GTN,7.

[0178] In another aspect, the present invention relates to a method for reducing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable region (VH) region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 1, 2, and 3, the method comprising introducing a mutation into one of the three CDR sequences of the reference antibody, wherein the mutation is selected from a mutation in one of the following positions, wherein the positions are numbered according to the reference sequence of SEQ ID NO: 4.

[0179] In one embodiment of the invention, the method comprises introducing a mutation in a VH CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the invention, the method comprises introducing a mutation in a VH CDR2 region corresponding to N57E. In another embodiment of the invention, the method comprises introducing a mutation in a VH CDR3 region selected from H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V.

[0180] In one embodiment, the CD3 is human CD3 epsilon.

[0181] In another aspect, the invention relates to a bispecific antibody comprising a first binding region of an antibody of the invention, and a second binding region that binds to a different target than the first antigen binding region.

[0182] In another aspect, the present invention relates to nucleic acid constructs encoding one or more amino acid sequences of the present invention.

[0183] In another aspect, the present invention relates to an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized or chimeric antibody of the present invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody of the present invention, or (iii) both (i) and (ii).

[0184] In another aspect, the present invention relates to a host cell comprising the expression vector of the present invention.

[0185] In another aspect, the invention relates to a composition comprising an antibody or bispecific antibody of the invention.

[0186] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody or bispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0187] In another aspect, the present invention relates to an antibody or bispecific antibody, composition or pharmaceutical composition of the invention for use as a medicament.

[0188] In another aspect, the present invention relates to an antibody or bispecific antibody, composition or pharmaceutical composition of the present invention for use in treating a disease.

[0189] In another aspect, the invention relates to a method of treating a disease comprising administering to a subject in need thereof an antibody or bispecific antibody, composition or pharmaceutical composition of the invention.

[0190] In another aspect, the invention relates to a method of administering an antibody or bispecific antibody, wherein the antibody or bispecific antibody is administered subcutaneously or topically.

[0191] In one aspect, the invention relates to a method for diagnosing a disease characterized by involvement or accumulation of cells expressing CD3, comprising administering to a subject a humanized or chimeric antibody, composition, or pharmaceutical composition of the invention, optionally wherein the humanized or chimeric antibody is labeled with a detectable agent.

[0192] In another aspect, the present invention relates to a method for producing the antibody or bispecific antibody of the present invention, comprising the steps of: a) culturing the host cell of the present invention, and b) purifying the antibody from the culture medium.

[0193] In another aspect, the invention relates to a diagnostic composition comprising the antibody or bispecific antibody according to any of the embodiments disclosed herein.

[0194] In one embodiment, the diagnostic composition is a companion diagnostic for screening and selecting patients who will benefit from bispecific antibody therapy.

[0195] In another aspect, the present invention relates to a method for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising the steps of: a) contacting the sample with an antibody or bispecific antibody of the present invention under conditions allowing formation of a complex between the antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

[0196] In another aspect, the present invention relates to a kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising i) an antibody or bispecific antibody of the present invention, and ii) instructions for use of the kit.

[0197] In another aspect, the invention relates to an anti-idiotypic antibody or anti-idiotypic antibody pair that binds to an antibody of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0198] Figure 1 Figure 3: Heatmap showing binding ratios of mutants versus wt UniTE-huCD3-H1L1-T41K molecules. Ratios above 1 indicate stronger binding than wt, while ratios below 1 indicate weaker binding than wt. Binding was determined on Freestyle 293-F cells transfected with CD3 / TCR-LC13.

[0199] Figure 2 : Alignment of selected CD3 affinity variants from the generated library with mutations in VH. CDRs are underlined in the humanized wild-type sequence (SEQ ID NO: 4) HuCD3-H1. Highlighted amino acids are substitutions.

[0200] Figure 3 : T cell binding curves of selected VH affinity variants of humanized CD3 (UniTE-huCD3-H1L1-T41K) antibody determined by flow cytometry. The described affinity variants cover a wide range of T cell binding capacity between wild-type response and undetectable response.

[0201] Figure 4 : T cell binding curves of selected VH affinity variants of humanized CD3 (UniTE-huCD3-H1L1-T41K) antibody determined by flow cytometry, showing very low undetectable T cell binding.

[0202] Figure 5: T cell binding curves of selected VH affinity variants of humanized CD3 (BisG1-huCD3-H1L1-X-FEAL / 1014-Herceptin-FEAR) antibodies determined by flow cytometry. The described affinity variants cover a wide range of T cell binding capacities between wild-type responses and undetectable responses.

[0203] Figure 6: Cytotoxicity of CD3 affinity variants against solid tumor cell lines measured by Alamar Blue assay. (A) NCI-N87 cells, effector cell (T cell): tumor cell (NCI-N87 cell) ratio = 3:1, incubated for 48 hours, n = 2 donors, (B) SKOV3 cells, T cell: SKOV3 cell ratio = 4:1, incubated for 48 hours, n = 2 donors, (C) MDA-MB-231 cells, T cell: MDA-MB-231 cell ratio = 8:1, incubated for 48 hours, n = 2 donors. The depicted affinity variants tested cover a wide range of cytotoxicity between the wild-type response and no cytotoxicity observed against all tumor cell lines tested.

[0204] Figure 7 Figure 2: Cytotoxicity of CD3 affinity variants against hematological (Daudi) cell lines measured by chromium release assay. T cell:Daudi cell ratio = 10:1, 24 h incubation, 1 donor. The depicted tested affinity variants spanned a wide range of cytotoxicity between wild-type responses and no cytotoxicity was observed against the tumor cell lines tested.

[0205] Figure 8: Cytotoxicity of CD3xHER2 bispecific antibodies in the NCI-N87 human PBMC co-implantation model in NOD-SCID mice. At two different dose levels of CD3 affinity antibodies (0.5 and 0.05 mg / kg), HLA-A matched human unstimulated PBMCs were co-inoculated with NCI-N87 tumor cells as a source of human T cells in NOD-SCID mice. Humanized WT CD3 (huCD3) and 4 different CD3 affinity variants (N57E, H101K, S110A, Y114M) were tested. (A) Average tumor volume over time after treatment with 0.05 mg / kg antibody (n=4 per group). (B) Average tumor volume over time after treatment with 0.5 mg / kg antibody (n=4 per group). (C) Average tumor volume on day 44 after treatment with 0.05 mg / kg antibody on day 0 (n=4 per group). (D) Average tumor volume on day 44 after treatment with 0.5 mg / kg antibody on day 0 (n=4 per group). Data from C and D have been combined. Detailed Description of the Invention

[0206] In one aspect, the present invention relates to humanized or chimeric antibodies that bind to human CD3 with optimized affinity for CD3. Accordingly, an object of the present invention is to provide optimized humanized or chimeric CD3 antibodies compared to a reference antibody, such as an antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. Another object of the present invention is to provide antibodies that have optimized in vivo efficacy compared to a reference antibody, such as an antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. Another object of the present invention is to provide antibodies that have lower binding affinity for CD3 than the antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. Another object of the present invention is to provide antibodies that have higher binding affinity for CD3 than the antibody specified by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8.

[0207] In one aspect, the invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises a mutation in one of the three CDR sequences of a reference antibody having the CDR sequences shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3, wherein the mutation is located at a position selected from the group consisting of: T31M, T31P, N57, H101, G105, S110, and Y114, wherein the positions are numbered according to the reference sequence of SEQ ID NO: 4. Amino acids in SEQ ID NO: 4 are numbered according to the direct numbering scheme from the first amino acid to number 125 in the N-terminal to C-terminal direction. Figure 2 The numerical numbers corresponding to the positions in SEQ ID NO: 4 are shown in FIG. In addition, the CDR regions have been annotated according to the IMGT definition.

[0208] In one embodiment of the invention, the antibody has reduced or increased binding affinity for human CD3 compared to a reference antibody having VH CDR sequences shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3.

[0209] In some embodiments of the invention, an antibody with decreased binding affinity for a CD3 molecule, such as a CD3 peptide, such as SEQ ID NO: 402, compared to a reference antibody can have the same cytolytic activity against target cells as the reference antibody.

[0210] In one embodiment of the invention, the antibody comprises a T31M or T31P mutation. Position T31 is according to SEQ ID NO:4.

[0211] In one embodiment of the invention, the antibody comprises a mutation at position N57. Position N57 is according to SEQ ID NO: 4. In one embodiment, the mutation is N57E.

[0212] In one embodiment of the invention, the antibody comprises a mutation at position H101. Position H101 is according to SEQ ID NO: 4. In one embodiment, the mutation is H101G or H101N.

[0213] In one embodiment of the invention, the antibody comprises a mutation at position G105. Position G105 is according to SEQ ID NO: 4. In one embodiment, the mutation is G105P.

[0214] In one embodiment of the invention, the antibody comprises a mutation at position Y114. Position Y114 is according to SEQ ID NO: 4. In one embodiment, the mutation is Y114M, Y114R or Y114V.

[0215] As shown in Example 7, the reference antibody designated by SEQ ID NO: 4 and VL sequence SEQ ID NO: 8 has a KD value corresponding to 1.5×10 -8 The binding affinity of M to the CD3 peptide of SEQ ID NO: 402.

[0216] In some embodiments of the present invention, the binding affinity of the antibody to the CD3 peptide of SEQ ID NO: 402 is less than 1.5×10 -8 M, for example, 1.6×10 -8 M to 9.9×10 -8 The binding affinity of M or for example 1.0×10 -7 to 9.9×10 -7 The binding affinity of the antibody to the CD3 peptide of SEQ ID NO: 402 is greater than 1.5×10 -8 M, for example, 1.4×10 -8 to 1.0×10 -8 M, for example, 9.9×10 -9 to 1×10 -9 M.

[0217] In one embodiment, the invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions having CDR sequences selected from the group consisting of:

[0218] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 12, 2, 3;

[0219] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 14, 2, 3;

[0220] c) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 16, 2, 3;

[0221] d) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 18, 2, 3;

[0222] e) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 20, 2, 3;

[0223] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 22, 2, 3;

[0224] g) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 24, 2, 3;

[0225] h) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 26, 2, 3;

[0226] i) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 28, 2, 3;

[0227] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 30, 2, 3;

[0228] k) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 32, 2, 3;

[0229] l) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 34, 2, 3;

[0230] m) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 36, 2, 3;

[0231] n) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 38, 2, 3;

[0232] o) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 40, 2, 3;

[0233] p) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 42, 2, 3;

[0234] q) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 44, 2, and 3;

[0235] r) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 46, 2, 3;

[0236] s) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 48, 2, 3;

[0237] t) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 50, 2, 3;

[0238] u) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 52, 2, 3;

[0239] v) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3;

[0240] w) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 56, 2, 3;

[0241] x) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 58, 2, and 3;

[0242] y) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 60, 2, 3;

[0243] z) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 62, 2, 3;

[0244] aa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 64, 2, or 3;

[0245] bb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 66, 2, and 3;

[0246] cc) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 68, 2, 3;

[0247] dd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 70, 2, 3;

[0248] ee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 72, 2, 3;

[0249] ff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 74, 2, 3;

[0250] gg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 76, 2, and 3;

[0251] hh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 78, 2, and 3;

[0252] ii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 80, 2, 3;

[0253] jj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 82, 2, 3;

[0254] kk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 84, 2, 3;

[0255] 11) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 86, 2, 3;

[0256] mm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 88, 2, 3;

[0257] nn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 90, 2, 3;

[0258] oo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:92, 2, 3;

[0259] pp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 94, 2, 3;

[0260] qq) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 96, 2, and 3;

[0261] rr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 98, 2, 3;

[0262] ss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 100, 3;

[0263] tt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 102, and 3;

[0264] uu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 104, 3;

[0265] vv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 106, 3;

[0266] ww) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 108, 3;

[0267] xx) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 110, 3;

[0268] yy) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 112, and 3;

[0269] zz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 114, 3;

[0270] aaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 116, 3;

[0271] bbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 118, 3;

[0272] ccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 120, and 3;

[0273] ddd) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 122, and 3;

[0274] eee) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 124, and 3;

[0275] fff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 126, 3;

[0276] ggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 128, 3;

[0277] hhh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 130, 3;

[0278] iii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 132, 3;

[0279] jjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 134, 3;

[0280] kkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 136, 3;

[0281] 111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 138, and 3;

[0282] mmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 140, 3;

[0283] nnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 142, 3;

[0284] ooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 144, 3;

[0285] ppp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 146, 3;

[0286] qqq) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 148, 3;

[0287] rrr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 150, 3;

[0288] sss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 152, 3;

[0289] ttt) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 154, 3;

[0290] uuu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 156, 3;

[0291] vvv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 158, 3;

[0292] www) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,160,3;

[0293] xxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 162, 3;

[0294] yyy) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,164,3;

[0295] zzz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,166,3;

[0296] aaaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 168, 3;

[0297] bbbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 170;

[0298] cccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 172;

[0299] dddd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 174;

[0300] eeee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176;

[0301] ffff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 178;

[0302] gggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 180;

[0303] hhhh) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 182;

[0304] iiii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 184;

[0305] jjjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 186;

[0306] kkkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 188;

[0307] 111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 190;

[0308] mmmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 192;

[0309] nnnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 194;

[0310] oooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 196;

[0311] pppp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 198;

[0312] qqqq) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 200;

[0313] rrrr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 202;

[0314] ssss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 204;

[0315] tttt) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 206;

[0316] uuuu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 208;

[0317] vvvv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 210;

[0318] wwww) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 212;

[0319] xxxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 214;

[0320] yyyy) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 216;

[0321] zzzz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 218;

[0322] aaaaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 220;

[0323] bbbbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 222;

[0324] ccccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 224;

[0325] ddddd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 226;

[0326] eeeee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 228;

[0327] fffff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 230;

[0328] ggggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 232;

[0329] hhhhh) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 234;

[0330] iiiii) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 236;

[0331] jjjjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 238;

[0332] kkkkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 240;

[0333] 1111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 242;

[0334] mmmmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244;

[0335] nnnnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 246;

[0336] ooooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 248;

[0337] ppppp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 250;

[0338] qqqqq) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 252;

[0339] rrrrr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 254;

[0340] sssss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 256;

[0341] ttttt) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 258;

[0342] uuuuu) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 260;

[0343] vvvvv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 262;

[0344] wwwww) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 264;

[0345] xxxxx) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 266;

[0346] yyyyy) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 268;

[0347] zzzzz) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 270;

[0348] aaaaaa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 272;

[0349] bbbbbb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 274;

[0350] cccccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 276;

[0351] dddddd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 278;

[0352] eeeeee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 280;

[0353] ffffff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 282;

[0354] gggggg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284;

[0355] hhhhhh) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 286;

[0356] iiiiii) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 288;

[0357] jjjjjj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 290;

[0358] kkkkkk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292;

[0359] 1111) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 294;

[0360] mmmmmm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 296;

[0361] nnnnnn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298; and

[0362] oooooo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 300.

[0363] In one embodiment, the invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions having CDR sequences selected from one of the following:

[0364] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M];

[0365] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0366] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0367] d) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 176 [H101G];

[0368] e) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N];

[0369] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0370] g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A];

[0371] h) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G];

[0372] i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M];

[0373] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R];

[0374] k) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and

[0375] l) CDR1, CDR2 and CDR3 sequences that have a total of at least 90% or at least 95% amino acid sequence identity with any one of the three CDR sequences shown in a) to k), with the proviso that the CDR1, CDR2 and CDR3 sequences do not have the sequences shown in SEQ ID NOs: 1, 2 or 3.

[0376] In one embodiment, the invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions having CDR sequences selected from one of the following:

[0377] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M];

[0378] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0379] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0380] d) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 176 [H101G];

[0381] e) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N];

[0382] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0383] g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A];

[0384] h) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G];

[0385] i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M];

[0386] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R];

[0387] k) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V], and

[0388] 1) The CDR1, CDR2 and CDR3 sequences described in a) to k) have a total of up to 5 additional mutations or substitutions, up to 4 additional mutations or substitutions, up to 3 additional mutations or substitutions, up to 2 additional mutations or substitutions, or up to 1 additional mutation or substitution in the three CDR sequences, and the mutations or substitutions preferably do not alter the binding affinity for human CD3.

[0389] In one embodiment, the invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises a VH sequence selected from the group consisting of:

[0390] a) the VH sequence shown in SEQ ID NO: 13;

[0391] b) the VH sequence shown in SEQ ID NO: 15;

[0392] c) the VH sequence shown in SEQ ID NO: 17;

[0393] d) the VH sequence shown in SEQ ID NO: 19;

[0394] e) the VH sequence shown in SEQ ID NO: 21;

[0395] f) the VH sequence shown in SEQ ID NO: 23;

[0396] g) the VH sequence shown in SEQ ID NO: 25;

[0397] h) the VH sequence shown in SEQ ID NO: 27;

[0398] i) the VH sequence shown in SEQ ID NO: 29;

[0399] j) the VH sequence shown in SEQ ID NO: 31;

[0400] k) the VH sequence shown in SEQ ID NO: 33;

[0401] 1) the VH sequence shown in SEQ ID NO: 35;

[0402] m) the VH sequence shown in SEQ ID NO: 37;

[0403] n) the VH sequence shown in SEQ ID NO: 39;

[0404] o) the VH sequence shown in SEQ ID NO: 41;

[0405] p) the VH sequence shown in SEQ ID NO: 43;

[0406] q) the VH sequence shown in SEQ ID NO: 45;

[0407] r) the VH sequence shown in SEQ ID NO: 47;

[0408] s) the VH sequence shown in SEQ ID NO: 49;

[0409] t) the VH sequence shown in SEQ ID NO: 51;

[0410] u) the VH sequence shown in SEQ ID NO: 53;

[0411] v) the VH sequence shown in SEQ ID NO: 55;

[0412] w) the VH sequence shown in SEQ ID NO: 57;

[0413] x) the VH sequence shown in SEQ ID NO: 59;

[0414] y) the VH sequence shown in SEQ ID NO: 61;

[0415] z) the VH sequence shown in SEQ ID NO: 63;

[0416] aa) the VH sequence shown in SEQ ID NO: 65;

[0417] bb) the VH sequence shown in SEQ ID NO: 67;

[0418] cc) the VH sequence shown in SEQ ID NO: 69;

[0419] dd) the VH sequence shown in SEQ ID NO: 71;

[0420] ee) the VH sequence shown in SEQ ID NO: 73;

[0421] ff) the VH sequence shown in SEQ ID NO: 75;

[0422] gg) the VH sequence shown in SEQ ID NO: 77;

[0423] hh) the VH sequence shown in SEQ ID NO: 79;

[0424] ii) the VH sequence shown in SEQ ID NO: 81;

[0425] jj) the VH sequence shown in SEQ ID NO: 83;

[0426] kk) the VH sequence shown in SEQ ID NO: 85;

[0427] 11) the VH sequence shown in SEQ ID NO: 87;

[0428] mm) the VH sequence shown in SEQ ID NO: 89;

[0429] nn) the VH sequence shown in SEQ ID NO: 91;

[0430] oo) the VH sequence shown in SEQ ID NO: 93;

[0431] pp) the VH sequence shown in SEQ ID NO: 95;

[0432] qq) VH sequence shown in SEQ ID NO: 97;

[0433] rr) the VH sequence shown in SEQ ID NO: 99;

[0434] ss) VH sequence shown in SEQ ID NO: 101;

[0435] tt) VH sequence shown in SEQ ID NO: 103;

[0436] uu) the VH sequence shown in SEQ ID NO: 105;

[0437] vv) the VH sequence shown in SEQ ID NO: 107;

[0438] ww) VH sequence shown in SEQ ID NO: 109;

[0439] xx) the VH sequence shown in SEQ ID NO: 111;

[0440] yy) the VH sequence shown in SEQ ID NO: 113;

[0441] zz) the VH sequence shown in SEQ ID NO: 115;

[0442] aaa) the VH sequence shown in SEQ ID NO: 117;

[0443] bbb) the VH sequence shown in SEQ ID NO: 119;

[0444] ccc) the VH sequence shown in SEQ ID NO: 121;

[0445] ddd) the VH sequence shown in SEQ ID NO: 123;

[0446] eee) the VH sequence shown in SEQ ID NO: 125;

[0447] fff) the VH sequence shown in SEQ ID NO: 127;

[0448] ggg) the VH sequence shown in SEQ ID NO: 129;

[0449] hhh) the VH sequence shown in SEQ ID NO: 131;

[0450] iii) the VH sequence shown in SEQ ID NO: 133;

[0451] jjj) VH sequence shown in SEQ ID NO: 135;

[0452] kkk) VH sequence shown in SEQ ID NO: 137;

[0453] 111) the VH sequence shown in SEQ ID NO: 139;

[0454] mmm) VH sequence shown in SEQ ID NO: 141;

[0455] nnn) the VH sequence shown in SEQ ID NO: 143;

[0456] ooo) the VH sequence shown in SEQ ID NO: 145;

[0457] ppp) the VH sequence shown in SEQ ID NO: 147;

[0458] qqq) VH sequence shown in SEQ ID NO: 149;

[0459] rrr) the VH sequence shown in SEQ ID NO: 151;

[0460] sss) VH sequence shown in SEQ ID NO: 153;

[0461] ttt) VH sequence shown in SEQ ID NO: 155;

[0462] uuu) the VH sequence shown in SEQ ID NO: 157;

[0463] vvv) the VH sequence shown in SEQ ID NO: 159;

[0464] www) VH sequence shown in SEQ ID NO: 161;

[0465] xxx) the VH sequence shown in SEQ ID NO: 163;

[0466] yyy) VH sequence shown in SEQ ID NO: 165;

[0467] zzz) VH sequence shown in SEQ ID NO: 167;

[0468] aaaa) the VH sequence shown in SEQ ID NO: 169;

[0469] bbbb) VH sequence shown in SEQ ID NO: 171;

[0470] cccc) the VH sequence shown in SEQ ID NO: 173;

[0471] dddd) VH sequence shown in SEQ ID NO: 175;

[0472] eeee) VH sequence shown in SEQ ID NO: 177;

[0473] ffff) VH sequence shown in SEQ ID NO: 179;

[0474] gggg) VH sequence shown in SEQ ID NO: 181;

[0475] hhhh) VH sequence shown in SEQ ID NO: 183;

[0476] iiii) the VH sequence shown in SEQ ID NO: 185;

[0477] jjjj) VH sequence shown in SEQ ID NO: 187;

[0478] kkkk) VH sequence shown in SEQ ID NO: 189;

[0479] 1111) the VH sequence shown in SEQ ID NO: 191;

[0480] mmmm) VH sequence shown in SEQ ID NO: 193;

[0481] nnnn) VH sequence shown in SEQ ID NO: 195;

[0482] oooo) the VH sequence shown in SEQ ID NO: 197;

[0483] pppp) VH sequence shown in SEQ ID NO: 199;

[0484] qqqq) VH sequence shown in SEQ ID NO: 201;

[0485] rrrr) VH sequence shown in SEQ ID NO: 203;

[0486] ssss) VH sequence shown in SEQ ID NO: 205;

[0487] tttt) VH sequence shown in SEQ ID NO: 207;

[0488] uuuu) VH sequence shown in SEQ ID NO: 209;

[0489] vvvv) the VH sequence shown in SEQ ID NO: 211;

[0490] wwww) VH sequence shown in SEQ ID NO: 213;

[0491] xxxx) the VH sequence shown in SEQ ID NO: 215;

[0492] yyyy) VH sequence shown in SEQ ID NO: 217;

[0493] zzzz) VH sequence shown in SEQ ID NO: 219;

[0494] aaaaa) the VH sequence shown in SEQ ID NO: 221;

[0495] bbbbb) the VH sequence shown in SEQ ID NO: 223;

[0496] ccccc) the VH sequence shown in SEQ ID NO: 225;

[0497] ddddd) VH sequence shown in SEQ ID NO: 227;

[0498] eeeee) VH sequence shown in SEQ ID NO: 229;

[0499] fffff) VH sequence shown in SEQ ID NO: 221;

[0500] ggggg) the VH sequence shown in SEQ ID NO: 223;

[0501] hhhhh) VH sequence shown in SEQ ID NO: 225;

[0502] iiiii) the VH sequence shown in SEQ ID NO: 227;

[0503] jjjjj) VH sequence shown in SEQ ID NO: 229;

[0504] kkkkk) VH sequence shown in SEQ ID NO: 231;

[0505] 1111) the VH sequence shown in SEQ ID NO: 233;

[0506] mmmmm) VH sequence shown in SEQ ID NO: 235;

[0507] nnnnn) VH sequence shown in SEQ ID NO: 237;

[0508] ooooo) the VH sequence shown in SEQ ID NO: 239;

[0509] ppppp) VH sequence shown in SEQ ID NO: 241;

[0510] qqqqq) VH sequence shown in SEQ ID NO: 243;

[0511] rrrrr) VH sequence shown in SEQ ID NO: 245;

[0512] sssss) VH sequence shown in SEQ ID NO: 247;

[0513] ttttt) VH sequence shown in SEQ ID NO: 249;

[0514] uuuuu) VH sequence shown in SEQ ID NO: 251;

[0515] vvvvv) VH sequence shown in SEQ ID NO: 253;

[0516] wwwww) VH sequence shown in SEQ ID NO: 255;

[0517] xxxxx) the VH sequence shown in SEQ ID NO: 257;

[0518] yyyyy) VH sequence shown in SEQ ID NO: 259;

[0519] zzzzz) VH sequence shown in SEQ ID NO: 261;

[0520] aaaaaa) the VH sequence shown in SEQ ID NO: 263;

[0521] bbbbbb) VH sequence shown in SEQ ID NO: 265;

[0522] cccccc) VH sequence shown in SEQ ID NO: 267;

[0523] dddddd) VH sequence shown in SEQ ID NO: 269;

[0524] eeeeee) VH sequence shown in SEQ ID NO: 271;

[0525] ffffff) VH sequence shown in SEQ ID NO: 273;

[0526] gggggg) VH sequence shown in SEQ ID NO: 275;

[0527] hhhhhh) VH sequence shown in SEQ ID NO: 277;

[0528] iiiiii) the VH sequence shown in SEQ ID NO: 279;

[0529] jjjjjj) VH sequence shown in SEQ ID NO: 281;

[0530] kkkkkk) VH sequence shown in SEQ ID NO: 283;

[0531] 1111) VH sequence shown in SEQ ID NO: 285;

[0532] mmmmmm) VH sequence shown in SEQ ID NO: 287;

[0533] nnnnnn) VH sequence shown in SEQ ID NO: 289;

[0534] oooooo) the VH sequence shown in SEQ ID NO: 291;

[0535] pppppp) VH sequence shown in SEQ ID NO: 293;

[0536] qqqqqq) VH sequence shown in SEQ ID NO: 295;

[0537] rrrrrr) VH sequence shown in SEQ ID NO: 297;

[0538] ssssss) VH sequence shown in SEQ ID NO: 299; and

[0539] tttttt) VH sequence shown in SEQ ID NO:301.

[0540] In one embodiment, the invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises a VH sequence selected from the group consisting of:

[0541] a) the VH sequence shown in SEQ ID NO: 55[T31M],

[0542] b) the VH sequence shown in SEQ ID NO: 59[T31P],

[0543] c) the VH sequence shown in SEQ ID NO: 107[N57E],

[0544] d) the VH sequence shown in SEQ ID NO: 177 [H101G],

[0545] e) the VH sequence shown in SEQ ID NO: 185 [H101N],

[0546] f) the VH sequence shown in SEQ ID NO: 221[G105P],

[0547] g) the VH sequence shown in SEQ ID NO: 237 [S110A],

[0548] h) the VH sequence shown in SEQ ID NO: 245 [S110G],

[0549] i) the VH sequence shown in SEQ ID NO: 285[Y114M],

[0550] j) the VH sequence shown in SEQ ID NO: 293[Y114R], and

[0551] k) The VH sequence shown in SEQ ID NO: 299 [Y114V].

[0552] In one embodiment of the invention, a humanized or chimeric antibody comprises a binding region, wherein the binding region comprises a light chain variable (VL) region, wherein the VL region comprises CDR1, CDR2, and CDR3 having a CDR sequence selected from the group consisting of:

[0553] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 7;

[0554] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 302, GTN, 7;

[0555] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 304, GTN, 7;

[0556] d) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 306, GTN, 7;

[0557] e) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 308, GTN, 7;

[0558] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 310, GTN, 7;

[0559] g) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 312, GTN, 7;

[0560] h) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 314, GTN, 7;

[0561] i) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 316, GTN, 7;

[0562] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 318, GTN, 7;

[0563] k) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 320, GTN, 7;

[0564] l) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 322, GTN, 7;

[0565] m) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 324, GTN, 7;

[0566] n) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 326, GTN, 7;

[0567] o) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 328, GTN, 7;

[0568] p) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 330, GTN, 7;

[0569] q) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 332;

[0570] r) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 334;

[0571] s) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 336;

[0572] t) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 338;

[0573] u) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 340;

[0574] v) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 342;

[0575] w) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 344;

[0576] x) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 346;

[0577] y) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 348;

[0578] z) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 350;

[0579] aa) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 352;

[0580] bb) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 354;

[0581] cc) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 356;

[0582] dd) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 358;

[0583] ee) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 360;

[0584] ff) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 362;

[0585] gg) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 364;

[0586] hh) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 366;

[0587] ii) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 368;

[0588] jj) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 370;

[0589] kk) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 372;

[0590] 11) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 374;

[0591] mm) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 376;

[0592] nn) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 378;

[0593] oo) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 380;

[0594] pp) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 382;

[0595] qq) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 384;

[0596] rr) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 386;

[0597] ss) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 388;

[0598] tt) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 390;

[0599] uu) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 392; and

[0600] vv) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, GTN, 394.

[0601] In another embodiment of the invention, the humanized or chimeric antibody comprises a binding region comprising a light chain variable (VL) region, wherein the VL region comprises one of the following VL sequences:

[0602] a) the VL sequence shown in SEQ ID NO: 8; and

[0603] b) The VL sequence shown in SEQ ID NO: 10.

[0604] The term "antibody" as used herein means an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions and has a half-life of a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more; about 3, 4, 5, 6, 7 or more days, or any other relevant functionally defined time (e.g., a time sufficient to induce, promote, enhance and / or regulate a physiological response associated with an antibody that binds to an antigen and / or a time sufficient for the antibody to recruit effector activity). The binding region (or binding domain, which can also be used herein, the two terms have the same meaning) that interacts with the antigen comprises the variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant region of the antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells and T cells) and components of the complement system (e.g., C1q, the first complement of the classical pathway of complement activation). As noted above, the term antibody, as used herein, unless otherwise indicated or clearly contradicted by the context, includes fragments of antibodies that retain the ability to specifically interact with (e.g., bind to) an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) Fab' or Fab fragments, a V-binding fragment comprising a V-binding fragment of a full-length antibody. L 、V H 、C L and C H 1 domain, or a monovalent antibody described in WO2007059782 (Genmab A / S); (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a fragment consisting essentially of V H and CH 1 domain; and (iv) a V domain consisting essentially of a single arm of an antibody L and V H In addition, although the two domains V L and V H are encoded by separate genes, but they can be joined using recombinant methods by a synthetic linker that enables them to be made into a single protein chain, where V L The VH region is paired with the VH region to form a monovalent molecule (called a single-chain antibody or single-chain Fv (scFv), see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are included in the term antibody, unless otherwise noted or the context clearly indicates. Although such fragments are generally included in the meaning of antibodies, they are collectively and independently unique features of the present invention, exhibiting different biological properties and functions. These and other useful antibody fragments in the context of the present invention are further discussed herein. It should also be understood that the term antibody, unless otherwise indicated, also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen provided by any known technology, such as enzymatic cleavage, peptide synthesis and recombinant technology. The antibodies produced may have any isotype.

[0605] As used herein, the terms "immunoglobulin heavy chain", "heavy chain of an immunoglobulin" or "heavy chain" refer to one of the chains of an immunoglobulin. A heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the isotype of the immunoglobulin. The heavy chain constant region is typically composed of three domains, CH1, CH2 and CH3. The heavy chain constant region may further comprise a hinge region. As used herein, the term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of light (L) chains and a pair of heavy (H) chains, all four chains possibly interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, for example,

[14] ). Within the structure of an immunoglobulin (e.g., IgG), the two heavy chains are interconnected by disulfide bonds in the so-called "hinge region". Like the heavy chain, each light chain is typically composed of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically comprises one domain, CL. Furthermore, the VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions, whose sequences can be highly variable and / or form structurally defined loops), also known as complementarity determining regions (CDRs), which alternate with more conserved regions known as framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

[15] ). CDR sequences can be determined using methods provided by IMGT

[16] -

[17] .

[0606] As used herein, the term "isotype" refers to an immunoglobulin (sub) class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f) [SEQ ID NO: 407], encoded by a heavy chain constant region gene. Thus, in one embodiment, the antibody comprises an immunoglobulin heavy chain of the IgG1 class or any allotype thereof. Furthermore, each heavy chain isotype may be combined with a kappa (κ) or lambda (λ) light chain.

[0607] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term for different types of antibody modifications, and it is a method well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques described in

[18] . Thus, chimeric antibodies can be recombinant antibodies that have been genetically engineered. Some chimeric antibodies can be engineered both genetically and enzymatically. The production of chimeric antibodies is within the knowledge of those skilled in the art, and thus the production of chimeric antibodies of the present invention can be achieved by methods other than those described herein. Chimeric monoclonal antibodies are developed for therapeutic applications to reduce antibody immunogenicity. They can generally comprise a non-human (e.g., mouse) variable region that is specific for the antigen of interest, and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" used in the context of chimeric antibodies refers to the region that contains the CDRs and framework regions of both the heavy and light chains of an immunoglobulin.

[0608] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology with a human variable domain. This can be achieved by transplanting the six non-human antibody complementary determining regions (CDRs) that together form the antigen binding site onto the homologous human receptor framework region (FR) (see

[19] -

[20] ). In order to fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the framework residues of the parent antibody (i.e., the non-human antibody) into the human framework region (back mutation). Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise a non-human CDR sequence, a substantially human framework region optionally comprising one or more amino acid back mutations to a non-human amino acid sequence, and a complete human constant region. Optionally, additional amino acid modifications that are not necessarily back mutations may be applied to obtain a humanized antibody with preferred characteristics (e.g., affinity and biochemical properties).

[0609] A humanized or chimeric antibody according to any aspect or embodiment of the invention may be referred to as a "humanized or chimeric CD3 antibody," a "humanized or chimeric antibody of the invention," a "CD3 antibody," or a "CD3 antibody of the invention," all of which have the same meaning and intent unless otherwise contradicted by the context.

[0610] The amino acid sequence of non-human antibodies differs from that of human antibodies, and therefore non-human antibodies have the potential to be immunogenic when administered to human patients. However, despite the non-human origin of the antibody, its CDR segments are responsible for the antibody's ability to bind to its target antigen, and humanization aims to maintain the antibody's specificity and binding affinity. Therefore, humanization of non-human therapeutic antibodies is performed to minimize their immunogenicity in humans, while at the same time maintaining the specificity and binding affinity of the non-human antibody.

[0611] As used herein, the term "binding region" refers to a region of an antibody that is capable of binding to any molecule, such as a polypeptide, for example, present on a cell, bacterium, or virion.

[0612] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target, typically with a specific affinity corresponding to about 10 -6 M or smaller, such as 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or smaller, about 10 -10 M or smaller or about 10 -11 M or even smaller K D and with an affinity corresponding to a K that is at least 10-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, than its affinity for binding to a non-specific antigen (e.g., BSA, casein) that is not the predetermined antigen or a closely related antigen. D The affinity of the antibody is lower depending on the K D , so that when the antibody K D When the affinity for an antigen is very low (i.e., the antibody is highly specific), the affinity for the antigen may be at least 10,000 times lower than the affinity for a nonspecific antigen. D "(M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

[0613] As used herein, the term "human CD3" refers to the human cluster of differentiation 3 protein, which is part of the T cell co-receptor protein complex and is composed of four different chains. CD3 is also present in other species, and therefore the term "CD3" may be used herein and is not limited to human CD3, unless the context contradicts. In mammals, the complex contains one CD3γ (gamma) chain (human CD3γ chain Swissprot P09693 or cynomolgus monkey CD3γ Swissprot Q95LI7), one CD3δ (delta) chain (human CD3δ Swissprot P04234 or cynomolgus monkey CD3δ Swissprot Q95LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766; or cynomolgus monkey CD3ε Swissprot Q95LI5), rhesus monkey CD3ε (Swissprot G7NCB9), and one CD3ζ-chain (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TK0). These chains associate with a molecule called the T-cell receptor (TCR) and generate activation signals in T lymphocytes. TCR and CD3 molecules together constitute the TCR complex.

[0614] It is within the knowledge of the skilled person that the amino acid sequences referred to by the Swissprot number include a signal peptide, which is removed after the protein is translated. Thus, proteins present on the cell surface, such as CD3, do not include a signal peptide. In particular, the amino acid sequences listed in Table 1 do not contain such a signal peptide. Such proteins listed in Table 1 may be referred to as "mature proteins". Thus, SEQ ID NO: 398 shows the amino acid sequence of mature human CD3 δ (delta), SEQ ID NO: 399 shows the amino acid sequence of mature human CD3 ε (epsilon), SEQ ID NO: 403 shows the amino acid sequence of mature cynomolgus monkey CD3 ε, and SEQ ID NO: 404 shows the amino acid sequence of mature rhesus monkey CD3 ε. Thus, the term "mature" as used herein refers to a protein that does not contain any signal or leader sequence.

[0615] It is well known that signal peptide sequence homology, length and cleavage site position vary significantly between different proteins. Signal peptides can be determined by different methods, for example SEQ ID NO: 399 of the present invention has been determined based on the SignalP application (available from http: / / www.cbs.dtu.dk / services / SignalP / ).

[0616] In a specific embodiment, the humanized or chimeric antibodies of the invention bind to the epsilon chain of CD3, e.g., the epsilon chain of human CD3 (SEQ ID NO: 399). In yet another specific embodiment, the humanized or chimeric antibodies bind to an epitope within amino acids 1-27 of the N-terminal portion of human CD3 epsilon (SEQ ID NO: 402). In such specific embodiments, the antibodies may further cross-react with other non-human primate species, such as cynomolgus monkeys (cynomolgus CD3 epsilon SEQ ID NO: 403) and / or rhesus monkeys (rhesus CD3 epsilon SEQ ID NO: 404).

[0617] Compared to the original antibody, the antibodies of the present invention comprising the CDR sequences defined herein and further comprising framework regions may differ in sequence outside of the CDR sequences while still retaining full binding ability. Thus, the present invention also relates to antibodies comprising variable region amino acid sequences that have a certain sequence identity to any of the sequences described herein.

[0618] The term "sequence identity" as used in the context of the present invention refers to the percentage identity between two sequences as a function of the number of identical positions shared by the sequences (i.e. % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller

[21] . In addition, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm

[22] . Multiple alignments are preferably performed using the Clustal W algorithm

[23] (e.g. in Vector NTI Software version 11.5; used by Invitrogen Inc.).

[0619] Thus, in one embodiment of the invention, an antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions having three CDR sequences selected from one of the following groups:

[0620] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M];

[0621] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0622] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0623] d) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 176 [H101G];

[0624] e) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N];

[0625] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0626] g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A];

[0627] h) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G];

[0628] i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M];

[0629] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R];

[0630] k) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and

[0631] l) CDR1, CDR2 and CDR3 sequences that have a total of at least 90% or at least 95% amino acid sequence identity with any one of the three CDR sequences shown in a) to k), with the proviso that the CDR1, CDR2 and CDR3 sequences do not have the sequences shown in SEQ ID NOs: 1, 2 or 3.

[0632] As shown in Sequence Table 1 of this document, the VH region consists of a 125 amino acid sequence. Thus, a second VH sequence consisting of 125 amino acids identical to one of the first VH sequences listed above at 124 amino acid positions has 99.2% sequence identity with the first VH sequence. A second sequence consisting of 125 amino acids identical to one of the first VH sequences listed above at 120 amino acid positions has 96% sequence identity with the first VH sequence. A second sequence consisting of 125 amino acids identical to one of the first VH sequences listed above at 115 amino acid positions has 92% sequence identity with the first VH sequence.

[0633] In a specific embodiment thereof, the VH region has at least 96% amino acid sequence identity with at least one VH sequence specified in said group.

[0634] In one embodiment of the present invention, the mutations are located in the framework regions of the VH region. Thus, in some embodiments, the three CDR sequences of the VH region are 100% identical to those of the antibodies of the present invention, but amino acid changes may occur in the framework regions of the VH region. When the CDRs are contained in the reference frame of SEQ ID NO: 407, such amino acid changes in the framework regions may preferably not alter the binding affinity of the antibody for CD3.

[0635] Mutations in the VH sequence that result in changes in sequence identity may preferably be conservative, physical or functional amino acids. Replacing an amino acid with a similar amino acid may increase the likelihood of retaining the functionality of the parent antibody.

[0636] In one embodiment of the invention, the antibody is a humanized antibody.

[0637] In one embodiment of the invention, the antibody is a full-length antibody.

[0638] The humanized antibodies of the present invention can be produced by comparing the heavy chain and light chain variable region amino acid sequences with a database of human germline variable region sequences to identify heavy chain and light chain human sequences with an appropriate degree of homology for use as human variable framework regions. A series of humanized heavy chain and light chain variable regions can be designed by grafting, for example, mouse CDRs onto the framework regions (identified as described above) and, if necessary, by backmutation (mutation of one or more human amino acid residues at designated positions in the framework regions back to non-human amino acids) to specific mouse residue sequences identified as being critical for restoring antibody binding efficacy. The humanized heavy chain and light chain variable regions are then designed using computer technology: iTope TM and TCED TM The variant sequence with the lowest occurrence of possible T-cell epitopes as determined by (

[24] ,

[25] and

[26] ) was selected.

[0639] In addition, the humanized antibodies of the present invention may also be "deimmunized". Deimmunization may be necessary because the presence of human T cell epitopes within protein sequences, such as the humanized antibodies of the present invention, may increase the immunogenicity risk profile when they have the potential to activate helper T cells. Such activation of helper T cells can be avoided by deimmunization. Deimmunization can be performed by introducing mutations into the amino acid sequence of the humanized antibody to remove T cell epitopes without significantly reducing the binding affinity of the antibody.

[0640] Thus, in one embodiment of the present invention, humanized antibodies can be produced by a method comprising the following steps: (i) comparing a non-human fully variable heavy chain sequence and / or fully variable light chain sequence to a database of human germline sequences, (ii) selecting the human germline sequence with the highest homology to the non-human sequence to obtain the humanized sequence, (iii) optimizing the humanized sequence by back mutation, if necessary, and (iv) expressing the sequence in a suitable expression system.

[0641] Therefore, the full-length antibodies of the present invention can be produced by a method comprising the following steps: (i) comparing non-human variable heavy chain sequences and variable light chain sequences to a database of human germline sequences, (ii) selecting the human germline sequence with the highest homology to the non-human sequence, (iii) transplanting the non-human CDRs to the selected human germline to obtain a humanized sequence, (iv) optimizing the humanized sequence by back mutation if necessary, (v) identifying the constant heavy and light chain sequences, and (vi) expressing the complete heavy chain sequence and the complete light chain sequence in a suitable expression system. The full-length antibodies of the present invention can therefore be produced as described in Example 1. It is within the knowledge of the skilled person to produce full-length antibodies starting from CDR sequences or complete variable region sequences. Therefore, the skilled person will know how to produce full-length antibodies of the present invention.

[0642] As used herein, the term "complete heavy chain sequence" refers to a sequence consisting of a variable heavy chain and a constant heavy chain sequence.

[0643] As used herein, the term "complete light chain sequence" refers to a sequence consisting of a variable light chain and a constant light chain sequence.

[0644] Back mutations can be introduced by standard DNA mutagenesis. Such standard techniques for DNA mutagenesis are described in

[18] . Alternatively, commercially available kits such as Quickchange TM Site-Directed Mutagenesis Kit (Stratagene), or desired back mutations can be introduced by de novo DNA synthesis.

[0645] Thus, in one embodiment, the antibody is a humanized antibody.

[0646] Chimeric antibodies can be produced by replacing all constant region sequences of non-human (e.g., mouse) antibodies with constant region sequences derived from humans. Therefore, the complete non-human variable region sequence is retained in the chimeric antibody. Therefore, the chimeric antibody of the present invention can be produced by a method comprising the following steps: expressing non-human variable heavy chain (SEQ ID NO: 405), non-human variable light chain sequence (SEQ ID NO: 406), human constant heavy chain and human constant light chain sequence in a suitable expression system, and thereby producing a full-length chimeric antibody. Alternative methods can be used. Such methods for producing chimeric antibodies are within the knowledge of the technician, and therefore the technician will know how to produce the chimeric antibody of the present invention. Therefore, in order to prepare the chimeric antibody of the present invention, a mutation of the present invention will be introduced into the non-human (e.g., mouse) VH or VL sequence.

[0647] Thus, in one embodiment, the antibody is a chimeric antibody.

[0648] In one embodiment, the antibody is a full-length antibody.The term "full-length antibody" as used herein refers to an antibody (e.g., a parent or variant antibody) that contains all heavy and light chain constant and variable domains corresponding to those naturally occurring in a wild-type antibody of that isotype.

[0649] In one embodiment, the antibody comprises an Fc region comprising first and second immunoglobulin heavy chains.

[0650] The term "Fc region" as used herein refers to a region comprising at least a hinge region, a CH2 region, and a CH3 region in the direction from the N-terminus to the C-terminus. The Fc region may further comprise a CH1 region at the N-terminus of the hinge region.

[0651] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to Eu numbering as described in Kabat.

[0652] Unless otherwise indicated or contradicted by context, the amino acids of the constant region sequences are numbered herein according to the Eu-numbering index (described in

[27] ) and may be referred to as "Eu numbering according to Kabat", "Eu numbering according to Kabat" or "according to the Eu numbering system".

[0653] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering system. However, the CH1 region may also be of any other subtype described herein.

[0654] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering system. However, the CH2 region may also be of any other subtype described herein.

[0655] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering system. However, the CH3 region may also be of any other subtype described herein.

[0656] In one embodiment, the isotype of the immunoglobulin heavy chain is selected from IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain can be any allotype within each immunoglobulin class, such as IgG1m(f) (SEQ ID NO: 407). Therefore, in a specific embodiment, the isotype of the immunoglobulin heavy chain is IgG1 or any allotype thereof, such as IgG1m(f) (SEQ ID NO: 407).

[0657] When targeting the antigen CD3, which is part of the T-cell receptor (TCR), a T cell-specific mechanism of cell killing is desirable. Other effector functions, such as complement activation, may be undesirable, and therefore reducing effector function is desirable. C1q binding is the first step in the complement cascade and is therefore used as an indicator of the complement-dependent cytotoxicity (CDC) ability of an antibody. If C1q binding to the antibody can be avoided, activation of the complement cascade can also be avoided.

[0658] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.9%, or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA. In a preferred embodiment, the antibody comprises an Fc region that has been modified such that binding of C1q to the antibody is reduced by at least 99% to 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0659] As used herein, the term "modified" refers to an amino acid sequence in an Fc region that is different from the amino acid sequence of a wild-type Fc region. That is, amino acid residues at specified positions in the wild-type Fc region have been substituted, deleted, or inserted to alter, for example, the binding site for C1q, the binding site for other effector molecules, or binding to an Fc receptor (FcR). Such modifications of the amino acid sequence can be made by replacing one or more amino acids with conservative amino acids, or by replacing one or more amino acids with alternative amino acids that are physically and / or functionally similar to the amino acids present in the wild-type region. Substitutions can also be made by replacing one or more amino acids with non-conservative amino acids.

[0660] In the context of the present invention, amino acids can be described as conservative or non-conservative amino acids and can be classified accordingly. Amino acid residues can also be divided into types defined by optional physical and functional properties. Therefore, the type of amino acid can be reflected in one or both of the following tables:

[0661] Conserved amino acid residues

[0662] acidic residues D and E basic residues K, R, and H Hydrophilic uncharged residues S, T, N, and Q Aliphatic uncharged residues G, A, V, L, and I Nonpolar uncharged residues C, M and P Aromatic residues F, Y and W

[0663] Alternative physical and functional classifications of amino acid residues

[0664]

[0665] In the context of the present invention, substitutions in an antibody, e.g., a humanized or chimeric antibody, are represented as follows:

[0666] Original amino acid – position – substituted amino acid;

[0667] Any amino acid residue is indicated using either the three-letter code or the one-letter code, including the codes Xaa and X, with reference to generally accepted amino acid nomenclature. Thus, the notation "L234F" or "Leu234Phe" means that the antibody comprises a substitution of phenylalanine for leucine at amino acid position 234.

[0668] The substitution of an amino acid at a given position for any other amino acid is indicated as follows:

[0669] Original amino acid – position; or for example, "L234".

[0670] For modifications in which the original amino acid and / or the substituted amino acid may include more than one but not all amino acids, the more than one amino acid may be separated by "," or " / ". For example, replacing leucine at position 234 with phenylalanine, arginine, lysine or tryptophan:

[0671] “Leu234Phe, Arg, Lys, Trp” or “Leu234Phe / Arg / Lys / Trp” or “L234F,R,K,W” or “L234F / R / K / W” or “L234 to F, R, K or W”.

[0672] In the context of the present invention such nomenclature are used interchangeably but have the same meaning and purpose.

[0673] In addition, the term "replacement" includes replacement into any of the other 19 natural amino acids, or other amino acids, such as non-natural amino acids. For example, the amino acid L at replacement position 234 includes the following various replacements: 234A, 234C, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P, and 234Y. That is, in this way, it is equivalent to naming 234X, wherein X indicates any amino acid other than the original amino acid. These replacements may also be referred to as L234A, L234C, etc., or L234A, C, etc., or L234A / C / , etc. The nomenclature is similarly applicable to each and all positions mentioned herein, and any of such replacements is particularly included herein.

[0674] Antibodies of the present invention may also comprise deletions of amino acid residues. Such deletions may be indicated as "del" and include, for example, L234del. Thus, in such embodiments, the leucine at position 234 is deleted from the amino acid sequence.

[0675] The terms "amino acid" and "amino acid residue" are used interchangeably herein.

[0676] In one embodiment of the invention, the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions having three CDR sequences selected from one of the following groups:

[0677] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M];

[0678] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0679] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0680] d) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 176 [H101G];

[0681] e) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N];

[0682] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0683] g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A];

[0684] h) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G];

[0685] i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M];

[0686] j) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R];

[0687] k) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and

[0688] 1) The CDR1, CDR2 and CDR3 sequences described in a) to k) have a total of up to 5 additional mutations or substitutions, up to 4 additional mutations or substitutions, up to 3 additional mutations or substitutions, up to 2 additional mutations or substitutions, or up to 1 additional mutation or substitution in the three CDR sequences, and the mutations or substitutions preferably do not alter the binding affinity for human CD3.

[0689] In one embodiment of the invention, the additional mutations or substitutions are of conservative, physical or functional amino acids.

[0690] In some embodiments, binding to CD3 can be binding to full-length CD3, such as CD3 present on T cells. In other embodiments, binding to CD3 can be binding to a CD3 peptide, such as that set forth in SEQ ID NO: 402. Binding to the CD3 peptide and whether any additional mutations may modify binding to CD3 can be determined by biolayer interferometry as disclosed in Example 7.

[0691] In one embodiment, the antibody comprises an Fc region comprising first and second immunoglobulin heavy chains.

[0692] As used herein, the term "CIq binding" refers to the binding of CIq to an antibody when the antibody is bound to its antigen. As used herein, the term "binding to its antigen" refers to the binding of an antibody to its antigen both in vivo and in vitro.

[0693] As used herein, the term "reduced" when referring to CIq binding refers to the ability of an antibody of the invention to reduce, minimize or even completely inhibit CIq binding to the antibody when compared to CIq binding to the wild-type antibody.

[0694] As used herein, the terms "reduced" or "reduced" or any variant thereof, when used in relation to the binding affinity of an antibody that binds to human CD3, refer to a lower binding affinity compared to a reference binding affinity. In this case, the reference binding affinity may be the binding affinity of a reference antibody designated by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8 when bound to the CD3 peptide of SEQ ID NO: 402, and determined by biolayer interferometry as described in Example 7.

[0695] As used herein, the term "binding affinity" relates to the binding of an antibody to a predetermined antigen or target, which typically corresponds to the K D The term "K D "(M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

[0696] As used herein, the term "wild-type antibody" refers to an antibody that is identical to the antibody to be tested except that it is not inactive when used in a comparative assay of the antibodies of the present invention. In this context, the term "inactive" refers to a modified Fc region that has reduced or no C1q binding, i.e., reduced or no Fc-mediated T-cell proliferation as determined in a PBMC-based functional assay (i.e., T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay) when C1q binding is determined by ELISA; and / or reduced or no Fc-mediated CD69 expression as determined in a PBMC-based functional assay. Thus, a wild-type antibody comprises amino acids naturally occurring in an immunoglobulin heavy chain, i.e., an antibody that does not comprise any amino acid modifications that may alter or reduce the ability of the antibody to interact with, for example, C1q, an Fc receptor, etc. Thus, such a wild-type antibody will remain an activating antibody capable of binding, for example, C1q. Wild-type antibodies and antibodies of the present invention may comprise amino acid modifications that are not those that affect the ability of the antibody to induce effector function, to prepare the antibody into a bispecific antibody, etc.

[0697] The term "ELISA" as used herein refers to enzyme-linked immunosorbent assay, which is a test that uses antibodies and a color change to identify a substance. A first specific antibody is attached to the surface of a plate. Thereby a protein from the sample is added, wherein binding to the first specific antibody is tested. A second antibody is added that binds to the antibody from the sample. The second antibody is linked to an enzyme, and in a final step a substance comprising a substrate for the enzyme is added. The subsequent reaction produces a detectable signal, most commonly a color change of the substrate. The concept of the ELISA method is well known in the art, and various ways of performing ELISA are considered part of the method for evaluating the antibodies of the present invention. In particular, the ability of the antibodies of the present invention to bind to C1q can be determined by ELISA, comprising the steps of: (i) coating the antibody onto a 96-well plate, (ii) adding 3% serum, (iii) adding anti-human C1q antibody, (iv) developing the plate, and (v) measuring the OD 405 nm. Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA comprising the steps of: (i) coating the antibody onto a 96-well plate, (ii) adding 3% serum, (iii) adding anti-human C1q, (iv) developing the plate, and (v) measuring OD 405 nm.

[0698] As used herein, the term "Fc receptor" or "FcR" refers to a protein found on the surface of certain cells. An FcR binds to the Fc region of an antibody. There are several different types of FcRs, which are categorized by the type of antibody they recognize. For example, Fcγ (gamma) receptors bind to IgG-type antibodies.

[0699] As used herein, the term "Fcγ receptor," "Fc gamma receptor," or "FcγR" refers to a group of Fc receptors that belong to the immunoglobulin superfamily and are the most important Fc receptors for inducing phagocytosis of opsonized (coated) microorganisms. This family includes several members, FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), which differ in antibody affinity due to their different molecular structures.

[0700] Fc-mediated effector functions form part of the biological activity of human immunoglobulin G (IgG) molecules. Examples of such effector functions include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are triggered by binding of various effector molecules to the Fc region. In the context of the present invention, "Fc binding," "Fc receptor binding," "FcR binding," and "antibody Fc region binding to FcR" refer to the binding of the Fc region to Fc receptors (FcRs) or effector molecules. The terms "FcγR binding" and "FcγRI binding" refer to binding to or binding to Fc gamma receptors and Fc gamma receptor I with the Fc region, respectively. When the CD3 antibody binds to T cells, the wild-type Fc region of the CD3 antibody binds to FcRs present on other cells (e.g., monocytes), which results in nonspecific Fc-mediated T cell activation. Such nonspecific Fc-mediated T cell activation may be unwanted. T cells may also be activated by targeted or target-specific T cell activation. For the treatment of many indications such as cancer, such targeted T cell activation can be highly desirable. The term "targeted T cell activation" as used herein refers to directing T cells to specific cells such as tumor cells by using a bispecific antibody, wherein the bispecific antibody comprises a first binding region that binds to a specific target such as a tumor target on a tumor cell, and a second binding region that binds to a T cell-specific target such as CD3. Therefore, T cell targeting to specific cells such as tumor cells can be promoted by using a bispecific antibody, wherein one of the binding regions binds to CD3 present on the T cell and the other binding region binds to a target-specific antigen, such as a target-specific antigen on a tumor cell. Nevertheless, nonspecific Fc-mediated T cell activation may still exist, and therefore such unwanted nonspecific Fc-mediated T cell activation by Fc-mediated cross-linking should be avoided, and can be inactivated by preparing an Fc region that is inactive for such activity. Therefore, the interaction between the inactive Fc region and the Fc receptor present is prevented.

[0701] The antibodies of the present invention may include modifications to the Fc region. When an antibody includes such modifications, it may become an inactive or non-activated antibody. As used herein, the terms "inactive," "inactive," or "non-activating" refer to an Fc region that is at least unable to bind to any Fcγ receptor, induce Fc-mediated crosslinking via FcRs, or induce FcR-mediated target antigen crosslinking via the Fc region, or is unable to bind to C1q. The inactivity of the Fc region of a humanized or chimeric CD3 antibody is suitably tested using a monospecific form of the antibody, but the inactive Fc region identified in this manner can be used in bispecific or other humanized or chimeric multispecific CD3 antibodies.

[0702] Several variants can be constructed to prepare Fc regions of antibodies that are inactive for interaction with Fc gamma receptors and C1q for use in therapeutic antibody development. Examples of such variants are described herein.

[0703] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that the antibody mediates reduced Fc-mediated T-cell proliferation by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

[0704] The term "reduce" when referring to T-cell proliferation refers to the ability of the antibodies of the present invention to reduce, minimize, or even completely inhibit T-cell proliferation when compared to T-cell proliferation bound by wild-type antibodies. The ability of an antibody to reduce T-cell proliferation can be evaluated by a PBMC-based functional assay. In one embodiment, the assay is performed using human PBMCs. In another embodiment, the assay is performed using cynomolgus monkey PBMCs. In yet another embodiment, the assay is performed using rhesus monkey PBMCs. Because the antibodies of the present invention are cross-reactive, the PBMC-based assays described herein can be performed using PBMCs of any species to demonstrate a reduction in T-cell proliferation, as long as the PBMCs of the species used are within the cross-reactivity spectrum of the antibody, such as humans, cynomolgus monkeys, or rhesus monkeys.

[0705] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay for evaluating the functional characteristics of an antibody of the invention, such as the ability of the antibody to affect T-cell proliferation or CD69 expression, wherein the only cells present are peripheral blood mononuclear cells. Thus, in one embodiment, T-cell proliferation is measured by a method comprising the following steps: incubating PBMCs with 1-1000 ng / mL of antibody at 37°C for three days in a 5% (vol / vol) CO2 humidified incubator; adding a compound, such as BrdU, which is incorporated into the DNA of the proliferating cells; incubating for 5 hours, pelleting the cells, drying the cells, optionally storing the cells at 4°C, coating the cells onto an ELISA plate, incubating with anti-BrdU-peroxidase for 90 minutes at room temperature, developing with 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) for about 30 minutes, stopping the reaction by adding 100 μL 2% oxalic acid, and measuring the absorbance at 405 nm in a suitable microplate reader.

[0706] As used herein, the term "proliferation" refers to cell growth in the context of cell division.

[0707] As used herein, the term "BrdU" refers to 5-bromo-2'-deoxyuridine, a homologue of thymidine. When BrdU is added to a cell culture for a limited period of time (e.g., 4 hours), it is incorporated into the DNA of proliferating cells. After fixing the cells, the incorporated BrdU can be detected in an ELISA using anti-BrdU-peroxidase. BrdU incorporation is therefore a measure of proliferation.

[0708] In one embodiment, the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0709] Specifically, the term "reduce" when referring to the expression level of the T cell activation marker CD69 refers to a reduction in the expression level of CD69 compared to the expression level of CD69 when T cells are bound to a wild-type antibody that binds to CD3 and interacts with an Fc receptor. The ability of an antibody to reduce CD69 expression can be evaluated by a functional assay based on PBMC. Therefore, in one embodiment, the expression of CD69 is measured by a method comprising the following steps: incubating PBMCs with an antibody in the range of 1-1000 ng / mL at 37°C in a 5% (vol / vol) CO2 humidified incubator for 16-24 hours, washing the cells, staining the cells with mouse anti-human CD28-PE and mouse-anti-human CD69-APC antibodies at 4°C, and measuring CD69 expression on CD28-positive cells by flow cytometry.

[0710] The term "CD69" as used herein refers to cluster of differentiation 69, a human transmembrane C-type lectin protein encoded by the CD69 gene. Activation of T lymphocytes and natural killer (NK) cells in vivo and in vitro induces expression of CD69. CD69 functions as a signaling receptor involved in cell activation events including proliferation, serves as a signaling receptor in lymphocytes including natural killer cells and platelets, and induces specific genes.

[0711] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay for evaluating the functional characteristics of an antibody of the invention, such as the ability of the antibody to affect T-cell proliferation or CD69 expression, wherein the only cells present are peripheral blood mononuclear cells. The PBMC-based functional assay comprises the following steps: (i) incubating PBMCs with the antibody at 37°C in a 5% (vol / vol) CO2 humidified incubator for approximately 16-24 hours, (ii) washing the cells, (iii) staining the cells with mouse anti-human CD28-PE and mouse-anti-human CD69-APC antibodies at 4°C, and (iv) when evaluating CD69 expression, measuring CD69 expression on CD28-positive cells by flow cytometry.

[0712] Amino acids in the Fc region that play a major role in interactions with C1q and Fc gamma receptors can be modified. Examples of amino acid positions that can be modified include positions L234, L235, and P331. Combinations such as L234F / L235E / P331S can significantly reduce binding to human CD64, CD32A, CD16, and C1q.

[0713] Thus, in one embodiment, the amino acid at at least one position corresponding to L234, L235, and P331 may be A, A, and S, respectively ([1],

[28] ). In addition, amino acid substitutions of L234F and L235E may result in an Fc region with abolished interactions with Fc gamma receptors and C1q (

[29] -

[30] ). Thus, in one embodiment, the amino acids at the positions corresponding to L234 and L235 may be F and E, respectively. Amino acid substitution of D265A may reduce binding to all Fc gamma receptors and prevent ADCC (

[31] ). Thus, in one embodiment, the amino acid at the position corresponding to D265 may be A. Binding to C1q may be abolished by mutating positions D270, K322, P329, and P331. Mutating any of these positions to D270A, K322A, P329A, or P331A may render the antibody deficient in CDC activity (

[32] ). Thus, in one embodiment, the amino acids at at least one of the positions corresponding to D270, K322, P329, and P331 may be A, A, A, and A, respectively.

[0714] An alternative approach to minimizing the interaction of the Fc region with the Fc gamma receptor and C1q is by removing the glycosylation site of the antibody. Mutating position N297 to, for example, Q, A, and E removes the glycosylation site that is critical for the IgG-Fc gamma receptor interaction. Thus, in one embodiment, the amino acid at the position corresponding to N297 may be G, Q, A, or E (

[33] ). Another alternative approach to minimizing the interaction of the Fc region with the Fc gamma receptor may be achieved by the following mutations: P238A, A327Q, P329A, or E233P / L234V / L235A / G236del (

[31] ).

[0715] Alternatively, although human IgG2 and IgG4 subclasses are considered naturally deficient in their interactions with C1q and Fc gamma receptors, interactions with Fc gamma receptors (Fc gamma receptors) have been reported (

[34] -

[35] ). Mutations that abolish these residual interactions can be made in both isotypes, resulting in a reduction in unwanted side effects associated with FcR binding. For IgG2, these include L234A and G237A, and for IgG4, L235E. Thus, in one embodiment, the amino acids at the positions corresponding to L234 and G237 in the human IgG2 heavy chain can be A and A, respectively. In one embodiment, the amino acid at the position corresponding to L235 in the human IgG4 heavy chain can be E.

[0716] Other approaches to further minimize interactions with Fc gamma receptors and CIq in IgG2 antibodies include those described in

[36] and

[37] .

[0717] The hinge region of antibodies may also be important for interactions with Fc gamma receptors and complement (

[38] -

[39] ). Therefore, mutations or deletions in the hinge region may affect the effector functions of antibodies.

[0718] As used herein, the term "cross-linking" refers to the indirect bridging of an antibody Fab arm (monovalent or bivalent) that binds to a target antigen through the Fc region of a cell-bound antibody bearing an FcR. Thus, an antibody that binds to its target antigen on a cell bearing the target antigen can cross-link to another cell expressing an FcR.

[0719] As used herein, the term "non-specific killing" refers to killing of cells by the cytotoxic function of T cells or other effector cells through tumor target antigen-independent activation of the cells. Thus, non-specific killing means that cells bearing tumor targets can be killed, for example, by cytotoxic T cells, rather than by antibodies that bind to the tumor target, for example, by inducing CDC.

[0720] An inactivated Fc region can be obtained by modifying one or more of at least five specific amino acid positions in the Fc region.

[0721] In one embodiment, the antibody comprises an Fc region comprising first and second immunoglobulin heavy chains.

[0722] Thus, in one embodiment, the antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of the first and second immunoglobulin heavy chains one or more amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are not L, L, D, N and P, respectively.

[0723] In one embodiment, in both the first and the second heavy chain one or more of the amino acids at the positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are other than L, L, D, N and P, respectively.

[0724] In another embodiment, in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0725] As used herein, the term "amino acid corresponding to a position" refers to the numbering of amino acid positions in a human IgG1 heavy chain. Unless otherwise indicated or contradicted by context, the amino acids of the constant region sequences are numbered herein according to the Eu numbering index (described in

[27] ). Thus, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence is an amino acid or segment that is aligned with the other amino acids or segments using a standard sequence alignment program such as ALIGN, ClustalW or similar programs, typically with default settings, and has at least 50%, at least 80%, at least 90% or at least 95% identity with a human IgG1 heavy chain. It is well known in the art how to align sequences or segments within a sequence and thereby determine the position in a sequence that corresponds to an amino acid position of the present invention.

[0726] In the context of the present invention, amino acids may be defined as described above.

[0727] The term "amino acid is not" or similar terms when referring to an amino acid in a heavy chain should be understood to mean that the amino acid is any other amino acid than the specific amino acid mentioned. For example, the amino acid at the position corresponding to L234 of the human IgG1 heavy chain is not L, meaning that the amino acid may be any other naturally occurring or non-naturally occurring amino acid besides L.

[0728] In one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0729] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0730] In one embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0731] The term "hydrophobic" as used herein with reference to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0732] The term "polar" as used herein with reference to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T.

[0733] In another embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is an aliphatic uncharged, aromatic or acidic amino acid.

[0734] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V.

[0735] The term "aromatic" as used herein with respect to amino acid residues refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of F, T and W.

[0736] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of D and E.

[0737] In a specific embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V and W.

[0738] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0739] In one embodiment, in both the first and the second heavy chains the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0740] In one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is a hydrophobic or polar amino acid.

[0741] The term "hydrophobic" as used herein with reference to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in both the first and the second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0742] As used herein, the term "polar" with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T. In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0743] In one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T.

[0744] In another embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is an aliphatic uncharged, aromatic or acidic amino acid.

[0745] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V.

[0746] The term "aromatic" as used herein with respect to amino acid residues refers to any amino acid residue selected from F, T and W. Thus, in one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from F, T and W.

[0747] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from D and E. Thus, in one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from D and E.

[0748] In a specific embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V and W.

[0749] In a further embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain is not N.

[0750] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N, and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0751] In one embodiment the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain is not N in both said first and second heavy chains.

[0752] In one embodiment, in both the first and the second heavy chain the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N, and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0753] In a further embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0754] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of a human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0755] In one embodiment, in at least one of said first and second heavy chains the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V.

[0756] In one embodiment, in at least one of said first and second heavy chains the amino acids at positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0757] The term "hydrophobic" as used herein with reference to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y.

[0758] As used herein, the term "polar" with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of C, D, E, H, K, N, Q, R, S and T.

[0759] In a specific embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W and Y.

[0760] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0761] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of a human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0762] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to L234 and L235 of a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0763] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y.

[0764] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each chosen from the group consisting of C, D, E, H, K, N, Q, R, S and T.

[0765] In a specific embodiment, in both the first and the second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0766] In another embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0767] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, G, I and V.

[0768] The term "aromatic" as used herein with respect to amino acid residues refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of F, T and W.

[0769] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of D and E.

[0770] In a specific embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to L234 and L235 are each selected from the group consisting of A, D, E, F, G, I, T, V and W.

[0771] In one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E; or A and A, respectively.

[0772] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of a human IgG1 heavy chain are F and E, respectively; or A and A, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0773] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E; or A and A, respectively.

[0774] In one embodiment, in both the first and the second heavy chains, the amino acids at positions corresponding to L234 and L235 of a human IgG1 heavy chain are F and E, respectively; or A and A, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0775] In a specific embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0776] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to positions L234 and L235 in a human IgGl heavy chain are F and E, respectively.

[0777] In one embodiment, in at least one of said first and second heavy chains, at least the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0778] In one embodiment, in both said first and second heavy chains at least the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0779] In one embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are other than L, L and D, respectively.

[0780] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 of a human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0781] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V and W, and the amino acid corresponding to position D265 is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V and W.

[0782] In one embodiment, in at least one of said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgGl heavy chain are hydrophobic or polar amino acids.

[0783] The term "hydrophobic" as used herein with respect to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y.

[0784] As used herein, the term "polar" with respect to amino acid residues refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of C, D, E, H, K, N, Q, R, S and T, and the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T.

[0785] In a specific embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0786] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to L234, L235 and D265 of a human IgGl heavy chain are hydrophobic or polar amino acids.

[0787] In one embodiment, in both the first and the second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y.

[0788] In one embodiment, in both the first and the second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of C, D, E, H, K, N, Q, R, S and T, and the amino acid at the position corresponding to position D265 in the human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T.

[0789] In a specific embodiment, in both the first and the second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgGl heavy chain are each selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0790] In another embodiment, in at least one of said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0791] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, G, I and V.

[0792] The term "aromatic" as used herein with respect to amino acid residues refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are each selected from the group consisting of F, T and W.

[0793] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are each selected from the group consisting of D and E.

[0794] In a specific embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V and W, and the amino acids at the positions corresponding to L234 and L235 are each selected from the group consisting of A, D, E, F, G, I, T, V and W.

[0795] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgGl heavy chain are other than L, L and D, respectively.

[0796] In one embodiment, in both the first and the second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 of a human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0797] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to L234, L235 and D265 of a human IgGl heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0798] In one embodiment, in both the first and the second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, G, I and V.

[0799] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgGl heavy chain are each selected from the group consisting of D and E.

[0800] In a specific embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V and W, and the amino acids at the positions corresponding to L234 and L235 are each selected from the group consisting of A, D, E, F, G, I, T, V and W.

[0801] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A; or A, A and A, respectively.

[0802] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 of a human IgG1 heavy chain are F, E and A, respectively; or A, A and A, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0803] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A; or A, A and A, respectively.

[0804] In one embodiment, in both the first and the second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 of a human IgG1 heavy chain are F, E and A, or A, A and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0805] In a specific embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively.

[0806] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgGl heavy chain are F, E and A respectively.

[0807] In one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A, respectively.

[0808] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A respectively.

[0809] In another embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are F, E, A, Q and S, respectively.

[0810] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgGl heavy chain are F, E, A, Q and S respectively.

[0811] In one embodiment, an antibody according to the invention comprises a VH sequence as set forth in any one of SEQ ID NOs: 107; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293; a VL sequence as set forth in SEQ ID NO: 8, and in at least one or both heavy chains, the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively. Thus provided are embodiments of anti-CD3 antibodies that have reduced affinity for human CD3 epsilon compared to a reference antibody comprising the VH and VL sequences set forth in SEQ ID NOs: 4 and 8, and wherein the antibody further comprises a non-activating Fc region.

[0812] In a specific embodiment, an antibody according to the invention comprises a VH sequence as set forth in any one of SEQ ID NOs: 107; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293, a VL sequence as set forth in SEQ ID NO: 10, and in at least one or both heavy chains, the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively. Thus, an embodiment of an anti-CD3 antibody having reduced affinity for human CD3 epsilon compared to a reference antibody comprising the VH and VL sequences set forth in SEQ ID NOs: 4 and 8 is provided, wherein the antibody further comprises a non-activating Fc region and a VL region that allows for enhanced production.

[0813] In another embodiment, the antibody according to the present invention comprises a VH sequence as shown in SEQ ID NO: 221, a VL sequence as shown in SEQ ID NO: 8 or 10, and in at least one or both heavy chains the amino acids corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively.

[0814] In one embodiment of the present invention, the human IgG1 heavy chain has the IgG1m(f) sequence as shown in SEQ ID NO: 407. In another embodiment, the amino acids in the positions corresponding to positions L234, L235, and D265 in human IgG1m(f) as shown in SEQ ID NO: 407 are F, E, and A, respectively.

[0815] In one embodiment of the present invention, the human IgG1 heavy chain has the IgG1m(f) sequence shown in SEQ ID NO:409.

[0816] In one aspect, an antibody according to the invention comprises the human IgLC2 / IgLC3 constant domain lambda light chain of SEQ ID NO: 408.

[0817] In one aspect, the antibodies of the present invention can be modified in the light chain (LC) and / or heavy chain (HC) to increase expression levels and / or production yields. In one embodiment, the antibodies of the present invention can be modified in the light chain (LC). Such modifications are known in the art and can be performed according to the methods described in, for example, Zheng, L., Goddard, J.-P., Baumann, U., & Reymond, J.-L. (2004) Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3), 807–14. doi: 10.1016 / j.jmb.2004.06.014.

[0818] In one aspect, the antibodies according to the invention can be modified in the VH region and / or the VL region to modify the affinity of the antibody, for example to reduce or increase the affinity of the antibody. In some cases this may be advantageous and result in increased efficacy. Specifically, a low affinity CD3 arm may have an effect on the motility of circulating T cells neutralizing tumor sites, thereby resulting in better engagement of tumor cells with T cells, see et al., Molecular Immunology 44 (2007). In particular, this can be used in a bispecific format in which a CD3 antibody is used as one of the binding arms. Modifications that result in reduced antibody affinity are known in the art, see, for example, Webster et al., Int J Cancer Suppl. 1988; 3: 13-6.

[0819] Thus, in one embodiment, an antibody of the invention comprises a light chain variable (VL) region comprising CDR1, CDR2 and CDR3 having a sequence as shown in SEQ ID NO: 6, GTN, 7, and a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2 and CDR3 having a CDR sequence selected from one of the following groups;

[0820] a) the CDR sequence shown in SEQ ID NO: 54, 2, 3 [T31M];

[0821] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0822] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0823] d) CDR1, CDR2 and

[0824] CDR3 sequence;

[0825] e) CDR1, CDR2 and

[0826] CDR3 sequence;

[0827] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0828] g) CDR1, CDR2 and

[0829] CDR3 sequence;

[0830] h) CDR1, CDR2 and

[0831] CDR3 sequence;

[0832] i) CDR1, CDR2 and

[0833] CDR3 sequence;

[0834] j) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R]; and

[0835] k) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V].

[0836] In another aspect, the present invention provides an antibody that binds to human CD3, comprising a binding region comprising a light chain variable (VL) region and a heavy chain variable (VH) region having a sequence shown in SEQ ID NO 10, wherein CDR1, CDR2, and CDR3 of the VH region have a sequence selected from one of the following groups:

[0837] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M];

[0838] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0839] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0840] d) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 176 [H101G];

[0841] e) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N];

[0842] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0843] g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A];

[0844] h) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G];

[0845] i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M];

[0846] j) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R]; and

[0847] k) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V].

[0848] Thus provided is an embodiment comprising a T41K mutation in the VL region as shown in SEQ ID NO: 10, thereby allowing increased production of said antibody.

[0849] In one aspect, the present invention relates to a multispecific antibody comprising at least a first binding region of an antibody according to any aspect or embodiment described herein, and one or more binding regions that bind to one or more different targets than the first binding region. Such a multispecific antibody may be a bispecific antibody.

[0850] Thus, in one aspect, the invention relates to a bispecific antibody comprising a first binding region of the antibody of any aspect or embodiment described herein, and a second binding region that binds a different target than the first binding region.

[0851] The term "multispecific antibody" refers to an antibody that is specific for at least two different, for example at least three, generally non-overlapping, epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different targets, such targets can be on the same cell or on different cells or cell types.

[0852] The term "bispecific antibody" refers to an antibody that has specificity for at least two different, usually non-overlapping, epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different targets, such targets can be on the same cell or on different cells or cell types.

[0853] In one embodiment, the bispecific antibody comprises a first and a second heavy chain.

[0854] Embodiments involving modifications of the Fc region and embodiments involving specific amino acid replacements are contemplated as part of any bispecific antibody of the invention. Thus, in one embodiment, at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any embodiment described herein (e.g., those described with respect to providing an inactive Fc region). In one embodiment, both the first and second heavy chains comprise one or more amino acids modified as defined in any embodiment described herein (e.g., those described with respect to providing an inactive Fc region). Thus, the bispecific antibody comprises an Fc region modified according to any aspect or embodiment described herein; or at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any aspect or embodiment described herein.

[0855] Examples of bispecific antibody molecules that can be used in the present invention include (i) a single antibody having two arms comprising different antigen binding regions, (ii) a single chain antibody specific for two different epitopes, such as two scFvs linked in series by an additional peptide linker, (iii) a dual variable domain antibody (DVD-Ig TM ), wherein each light and heavy chain comprises two variable domains linked in series by a short peptide bond (

[40] ); (iv) chemically linked bispecific (Fab')2 fragments; (v) It is a fusion of two single-chain diabodies, resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) flexibodies, which are a combination of scFv and diabody, resulting in a multivalent molecule; (vii) so-called "dock and lock" molecules Based on the "dimerization and docking domain" in protein kinase A, when applied to Fab, it can produce trivalent bispecific binding proteins, which consist of two identical Fab fragments connected to different Fab fragments; (viii) so-called Scorpion molecules, which contain, for example, two scFvs fused to the two ends of a human Fab arm; and (ix) diabodies.

[0856] In one embodiment, the bispecific antibody of the present invention is a diabody, a cross-body, or a bispecific antibody obtained by controlled Fab arm exchange, for example (e.g. described in

[41] ), such as those described in the present invention.

[0857] Examples of different types of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules in which each side of the molecule comprises a Fab fragment or a portion of a Fab fragment of at least two different antibodies; (iii) IgG fusion molecules in which a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; (iv) Fc fusion molecules in which a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, Fc-region, or a portion thereof; (v) Fab fusion molecules in which different Fab fragments are fused together, to a heavy chain constant domain, Fc-region, or a portion thereof; and (vi) ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, ), wherein different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, ) are fused to each other, or to another protein or carrier molecule fused to the heavy chain constant domain, Fc-region or a portion thereof.

[0858] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to (TrionPharma / Fresenius Biotech,

[42] ), Knobs-into-Holes (Genentech,

[43] ), CrossMAbs (Roche,

[44] ) and electrostatic pairing (Amgen,

[45] -

[46] ; Chugai,

[47] ; Oncomed,

[48] ), LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52):43331-9, doi:10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-bodies and PIG-bodies (Pharmabcine, WO2010134666, WO2014081202), chain exchange engineered domain bodies (SEED-bodies) (EMD Serono,

[49] ), Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron,

[50] ), bispecific IgG1 and IgG2 (Pfizer / Rinat,

[51] ), Azymetric scaffold (Zymeworks / Merck,

[52] ), mAb-Fv (Xencor,

[53] ), bivalent bispecific antibodies (Roche, WO2009080254), and molecule (Genmab A / S,

[41] ).

[0859] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, dual targeting (DT)-Ig (GSK / Domantis, WO2009058383), two-in-one antibodies (Genentech, Bostrom et al. 2009. Science 323, 1610–1614), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star,

[54] ), Zybodies TM (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr; 5(2): 208-18), using a common light chain approach (Crucell / Merus,

[55] ), κλ Bodies (NovImmune, WO2012023053) and (CovX / Pfizer, Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17, 501–506).

[0860] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-IgTM (Abbott,

[56] ), dual-domain bispecific antibodies (Unilever; Sanofi Aventis,

[57] ), IgG-like bispecifics (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb; 32(2): 191-8), Ts2Ab (Med Immune / AZ, Dimasi et al. J Mol Biol. 2009 Oct 30; 393(3): 672-92) and BsAb (Zymogenetics, WO2010111625), HERCULES (Biogen Idec,

[58] ), scFv fusions (Novartis), scFv fusions (Changzhou Adam Biotech Inc,

[59] ) and TvAb (Roche,

[59] ,

[60] ).

[0861] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Academic Institution, Pearce et al Biochem Mol Biol Int. 1997 Sep; 42(6): 1179-88.), SCORPION (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics / BMS, WO2010111625), dual affinity redirecting technology (Fc-DART), and Fc-binding protein fusions. TM )(MacroGenics,

[62] ,

[63] ) and dual (ScFv)2-Fab (National Research Center for Antibody Medicine–China).

[0862] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), dual-acting or Bis-Fab (Genentech), (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0863] Examples of ScFv-based, diabody-based, and domain antibodies include, but are not limited to, bispecific T cell engagers (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Redirecting Technology (DART TM ) (MacroGenics), single-chain diabodies (Academic, Lawrence FEBS Lett. 1998 Apr 3; 425 (3): 479-84), TCR-like antibodies (AIT, Receptor Logics), human serum albumin ScFv fusions (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug; 88 (6): 667-75), dual-targeting (Ablynx, Hmila et al., FASEBJ. 2010), dual-targeting heavy chain domain-only antibody.

[0864] It is also contemplated that any monospecific antibody that meets the assay conditions described herein can form the basis of a bispecific antibody. That is, a bispecific antibody in which one of the binding regions binds to CD3 can be derived from any monospecific CD3 antibody that has been tested in a functional assay and meets the requirements described herein. Such bispecific antibodies can be provided by the methods described in

[41] , which is incorporated herein by reference.

[0865] In one aspect, the bispecific antibody of the present invention comprises a first Fc region comprising a first CH3 region and a second Fc region comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different, and the heterodimeric interaction between the first and second CH3 regions is made stronger than each homodimeric interaction of the first and second CH3 regions. More details about these interactions and how they are achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0866] Thus, in a specific embodiment, each of the first and second heavy chains comprises at least a hinge region, a CH2 region, and a CH3 region, wherein in the first heavy chain, at least one amino acid corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain is substituted, and in the second heavy chain, at least one amino acid corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain is substituted, and wherein the first and second heavy chains are not substituted at the same position. In this case, the term "substituted" refers to the replacement of an amino acid at a specific amino acid position by another naturally occurring or non-naturally occurring amino acid. Thus, a "substituted" amino acid at a position corresponding to a position in a human IgG1 heavy chain means that the amino acid at the specific position is different from the naturally occurring amino acid in the IgG1 heavy chain.

[0867] In one embodiment, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain in the first heavy chain is not K, L or M, and optionally the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is F, and the amino acid at at least one position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405 and Y407 in a human IgG1 heavy chain in the second heavy chain is substituted.

[0868] In one embodiment, the amino acid at the position corresponding to K409 of a human IgG1 heavy chain in the first heavy chain is not K, L or M, and the amino acid at the position corresponding to F405 of a human IgG1 heavy chain in the second heavy chain is not F and optionally the amino acid at the position corresponding to K409 of a human IgG1 heavy chain is K.

[0869] In one embodiment, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain in the first heavy chain is not F, R and G, and the amino acid at the position corresponding to a position selected from the group consisting of T366, L368, K370, D399, Y407 and K409 in a human IgG1 heavy chain in the second heavy chain is substituted.

[0870] In one embodiment, the amino acid in said first heavy chain at the position corresponding to K409 in a human IgG1 heavy chain is not K, L or M, and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is not F.

[0871] In a further embodiment, the amino acid at the position corresponding to F405 of human IgG1 heavy chain in said first heavy chain is L, and the amino acid at the position corresponding to K409 of human IgG1 heavy chain in said second heavy chain is R, or vice versa.

[0872] Thus, in one embodiment, the amino acid at the position corresponding to K409 of a human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to F405 of a human IgG1 heavy chain in the second heavy chain is L.

[0873] In a further embodiment, the humanized or chimeric CD3 antibodies of the invention comprise one or more inactive substitutions disclosed in any of the above embodiments, e.g., L234F, L235E, and D265A, in at least one of the first and second heavy chains; and the amino acid at the position corresponding to F405 is not F. In one embodiment, the humanized or chimeric CD3 antibodies of the invention comprise one or more inactive substitutions disclosed in any of the above embodiments, e.g., L234F, L235E, and D265A, in at least one of the first and second heavy chains; and an additional substitution at position K409, e.g., K409R. Specifically, in one embodiment, the humanized or chimeric CD3 antibodies of the invention comprise one or more inactive substitutions disclosed in any of the above embodiments, e.g., L234F, L235E, and D265A, in both the first and second heavy chains; and a substitution at position F405, e.g., F405L. In one embodiment, the humanized or chimeric CD3 antibodies of the invention comprise one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E, and D265A, in both the first and second heavy chains, and an additional substitution at position K409, such as K409R. Such antibodies can be used to generate bispecific antibodies.

[0874] Thus, in a further embodiment, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to K409 in a human IgG1 heavy chain in the second heavy chain is R.

[0875] In one embodiment, the amino acids at positions corresponding to L234, L235, D265, N297 and P331 of a human IgG1 heavy chain in at least one of the first and second heavy chains are F, E, A, N and P, respectively, the amino acid at position corresponding to F405 of a human IgG1 heavy chain in the first heavy chain is L, and the amino acid at position corresponding to K409 of a human IgG1 heavy chain in the second heavy chain is R.

[0876] In an alternative embodiment, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain in the second heavy chain is L.

[0877] In one embodiment, the amino acids at positions corresponding to L234, L235, D265, N297 and P331 of a human IgG1 heavy chain in at least one of the first and second heavy chains are F, E, A, N and P, respectively, the amino acid at position corresponding to K409 of a human IgG1 heavy chain in the first heavy chain is R, and the amino acid at position corresponding to F405 of a human IgG1 heavy chain in the second heavy chain is L.

[0878] In another embodiment, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain in both the first and the second heavy chains are F, E and A, respectively, the amino acid at position corresponding to F405 in a human IgG1 heavy chain in the first heavy chain is L, and the amino acid at position corresponding to K409 in a human IgG1 heavy chain in the second heavy chain is R.

[0879] In one embodiment, the amino acids at positions corresponding to L234, L235, D265, N297 and P331 of a human IgG1 heavy chain in both the first and the second heavy chain are F, E, A, N and P, respectively, the amino acid at position corresponding to F405 of a human IgG1 heavy chain in the first heavy chain is L, and the amino acid at position corresponding to K409 of a human IgG1 heavy chain in the second heavy chain is R.

[0880] In an alternative embodiment, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain in both the first and the second heavy chains are F, E and A, respectively, the amino acid at position corresponding to K409 in a human IgG1 heavy chain in the first heavy chain is R, and the amino acid at position corresponding to F405 in a human IgG1 heavy chain in the second heavy chain is L.

[0881] In one embodiment, the amino acids at positions corresponding to L234, L235, D265, N297 and P331 of a human IgG1 heavy chain in both the first and the second heavy chain are F, E, A, N and P, respectively, the amino acid at position corresponding to K409 of a human IgG1 heavy chain in the first heavy chain is R, and the amino acid at position corresponding to F405 of a human IgG1 heavy chain in the second heavy chain is L.

[0882] As described herein, T cells are recruited to specific target cells, such as cancer or tumor cells, providing a method for killing target cells. T cell-mediated killing can be achieved by bispecific antibodies in which a first binding region targets CD3 and a second binding region targets another target. Thus, in one embodiment, the first binding region is according to any of the embodiments described herein for humanized or chimeric CD3 antibodies, and the second binding region binds to a different target than the first binding region. It should be understood that when the antibody is a bispecific antibody, at least half of the antibody, i.e., one of the heavy and light chain pairs of the antibody, is a humanized or chimeric antibody as described herein. Thus, half of the bispecific antibody is a humanized or chimeric antibody that binds to CD3 according to the present invention and the other half can be a humanized, chimeric, fully non-human, or fully human antibody that binds to a second target. Thus, in one embodiment, the antibody comprises a first and a second heavy chain, a first and a second light chain, wherein the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human and are linked by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a different target. In one embodiment, the antibody comprises a first and a second heavy chain, a first and a second light chain, wherein the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are humanized or chimeric and are linked by a disulfide bridge to form a second binding region, wherein the first binding region is according to an aspect or embodiment described herein, and the second binding region binds to a different CD3 epitope than the first binding region.

[0883] As used herein, the term "disulfide bridge" refers to a covalent bond between two cysteine residues, ie, the interaction may also be referred to as a Cys-Cys interaction.

[0884] As used herein, the term "target" refers to a molecule to which the binding region of an antibody of the invention binds. When used in the context of antibody binding, the term includes any antigen against which an antibody is generated.

[0885] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human and are linked by a disulfide bridge to form a second binding region, wherein the first binding region binds to a different target according to any aspect or embodiment described herein; and wherein the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively.

[0886] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human and are linked by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a different CD3 epitope than the first binding region; and wherein the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain in at least one of the first and second heavy chains are F, E, and A, respectively.

[0887] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human and are linked by a disulfide bridge to form a second binding region, wherein the first binding region binds to a different target according to any aspect or embodiment described herein; and wherein the amino acids at positions L234, L235 and D265 in both the first and second heavy chains corresponding to positions in a human IgG1 heavy chain are F, E and A, respectively.

[0888] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human and are linked by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a different CD3 epitope than the first binding region; and wherein the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively.

[0889] In another aspect, the present invention relates to a method for reducing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 1, 2, and 3, the method comprising introducing a mutation into one of the three CDR sequences of the reference antibody, wherein the mutation is selected from a mutation at one of the following positions, wherein the positions are numbered according to the reference sequence of SEQ ID NO: 4.

[0890] The numbering of amino acids in the VH region and the positions to be mutated are based on the amino acid numbering in SEQ ID NO: 4. The numbering is based on a direct numbering scheme starting from the first amino acid to number 125 in the N-terminal to C-terminal direction. Figure 2 The numerical numbers corresponding to the positions in SEQ ID NO: 4 are shown in FIG. In addition, the CDR regions have been annotated according to the IMGT definition.

[0891] In one embodiment of the invention, the method comprises introducing a T31M or T31P mutation. Position T31 is according to SEQ ID NO:4.

[0892] In one embodiment of the invention, the method comprises introducing a mutation at position N57. Position N57 is according to SEQ ID NO: 4. In one embodiment, the mutation is N57E.

[0893] In one embodiment of the invention, the method comprises introducing a mutation at position H101. Position H101 is according to SEQ ID NO: 4. In one embodiment, the mutation is H101G or H101N.

[0894] In one embodiment of the invention, the method comprises introducing a mutation at position Y114. Position Y114 is according to SEQ ID NO: 4. In one embodiment, the mutation is Y114, Y114R or Y114V.

[0895] In one embodiment of the invention, the method comprises introducing a mutation in the VH CDR3 region corresponding to a position selected from the group consisting of H101, S110 and Y114.

[0896] In one embodiment of the invention, the method comprises introducing a mutation into the VH CDR3 region selected from the group consisting of H101G, H101N, S110A, S110G, Y114M, Y114R and Y114V.

[0897] In one embodiment of the invention, the method comprises introducing a mutation wherein the binding affinity of the antibody to the human CD3ε peptide having SEQ ID NO: 402 corresponds to 1.6×10 -8 M to 9.9x10 -8 M or 1.0x10 -7 to 9.9x10 -7 M's K D Values, as determined by biolayer interferometry.

[0898] In one embodiment of the invention, the method comprises introducing a mutation wherein the binding affinity of the antibody to the human CD3ε peptide having SEQ ID NO: 402 corresponds to 1.4×10 -8 M to 1.0x10 -8 M or 9.9x10 -9 to 1x10 -9 K D Values, as determined by biolayer interferometry.

[0899] In one embodiment of the invention, the binding affinity of the antibody to the human CD3ε peptide having SEQ ID NO: 402 corresponds to 1.6×10 -8 M to 9.9x10 -8 M or 1.0x10 -7 to 9.9x10 -7 M's K D Values, as determined by biolayer interferometry.

[0900] In another aspect, the invention relates to a method for increasing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable region (VH) region, wherein the VH region comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2, and 3, the method comprising introducing a mutation in VH CDR3 corresponding to position G105, wherein the positions are numbered according to the reference sequence of SEQ ID NO: 4.

[0901] In one embodiment, the method comprises introducing a mutation at position G105. Position G105 is according to SEQ ID NO: 4. In one embodiment, the mutation is G105P.

[0902] In one embodiment of the invention, the method comprises introducing up to 5 additional mutations, up to 4 additional mutations, up to 3 additional mutations, up to 2 additional mutations or up to 1 additional mutation into the CDRs of the VH region of the reference antibody shown in SEQ ID NO: 1, 2, 3.

[0903] In one embodiment of the invention, the method of increasing or decreasing binding affinity comprises comprising a binding region comprising a heavy chain variable region (VH), wherein the VH region comprises a CDR1, CDR2, and CDR3 sequence selected from the group consisting of:

[0904] a) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M];

[0905] b) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P];

[0906] c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,106,3[N57E];

[0907] d) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 176 [H101G];

[0908] e) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N];

[0909] f) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P];

[0910] g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A];

[0911] h) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G];

[0912] i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M];

[0913] j) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R]; and

[0914] k) CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V].

[0915] In another embodiment of the invention, the method comprises introducing a mutation in a VH region CDR2 region corresponding to N57E. In another embodiment of the invention, the method comprises introducing a mutation in a VH region CDR3 region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V. In another aspect, the invention relates to a method for reducing or increasing the binding affinity of an antibody to CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein the VH region comprises a mutation in one of the three CDR sequences of a reference antibody set forth in CDR1 SEQ ID: 1, CDR2 SEQ ID: 2, and CDR3 SEQ ID: 3, wherein the antibody comprises a mutation in one of the following positions: T31M, T31P, N57, H101, G105, S110, and Y114, wherein the position corresponds to the reference sequence of SEQ ID NO: 4.

[0916] In one embodiment of the invention, the method comprises introducing a mutation in a VH CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the invention, the method comprises introducing a mutation in a VH CDR2 region corresponding to N57E. In another embodiment of the invention, the method comprises introducing a mutation in a VH CDR3 region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V.

[0917] In another aspect, the invention relates to a method for reducing the binding affinity of an antibody that binds to CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2 and CDR3 having the CDR sequences shown in SEQ ID NOs: 1, 2 and 3, the method comprising introducing a mutation in one of the VH region CDR1, CDR2 or CDR3 sequences shown in SEQ ID NOs: 1, 2 or 3.

[0918] In one embodiment of the invention, the method comprises introducing a mutation in one of the three CDR regions of the VH region corresponding to one of the following positions: T31, N57, H101, S110 or Y114, wherein the position corresponds to the reference sequence of SEQ ID NO: 4.

[0919] In one embodiment of the present invention, the method comprises introducing a mutation into the CDR1 sequence of the VH region corresponding to position T31, wherein the CDR1 sequence is as shown in SEQ ID NO 1. When the mutation is represented by X, the resulting CDR1 sequence may be GFTFNXYA. In one embodiment, the three CDR sequences of the VH region may have the following sequences: CDR1 GFTFNXYA, CDR2 IRSKYNNYAT and CDR3 VRHGNFGNSYVSWFAY In one embodiment, the mutation at position T31 in CDR1 of the VH region is a T31M or T31P mutation.

[0920] In one embodiment of the present invention, the method comprises introducing a mutation into the CDR2 sequence of the VH region corresponding to position N57, wherein the CDR2 sequence is as shown in SEQ ID NO 2. When the mutation is represented by X, the resulting CDR2 sequence may be IRSKYNXYAT. In one embodiment, the three CDR sequences of the VH region may have the following sequences: CDR1 GFTFNTYA, CDR2 IRSKYNXYAT and CDR3 VRHGNFGNSYVSWFAY In one embodiment, the mutation at position N57 in VH region CDR2 is an N57E mutation.

[0921] In one embodiment of the present invention, the method comprises introducing a mutation into the CDR3 sequence of the VH region corresponding to position H101, wherein the CDR3 sequence is as shown in SEQ ID NO 3. When the mutation is represented by X, the resulting CDR3 sequence may be represented as VRXGNFGNSYVSWFAY In one embodiment, the three CDR sequences of the VH region may have the following sequences: CDR1GFTFNTYA, CDR2 IRSKYNNYAT and CDR3 VRXGNFGNSYVSWFAY In one embodiment, the mutation at position H101 in CDR3 of the VH region is an H101G or H101N mutation.

[0922] In one embodiment of the present invention, the method comprises introducing a mutation into the VH region CDR3 sequence corresponding to position S110, wherein the CDR3 sequence is as shown in SEQ ID NO 3. When the mutation is represented by X, the resulting CDR3 sequence may be represented as VRHGNFGNSYVXWFAY In one embodiment, the three CDR sequences of the VH region may have the following sequences: CDR1GFTFNTYA, CDR2 IRSKYNNYAT and CDR3 VRHGNFGNSYVXWFAY In one embodiment, the mutation at position H101 in the VH region CDR3 is an S110A or S110G mutation.

[0923] In one embodiment of the present invention, the method comprises introducing a mutation into the VH region CDR3 sequence corresponding to position Y114, wherein the CDR3 sequence is as shown in SEQ ID NO 3. When the mutation is represented by X, the resulting CDR3 sequence may be represented as VRHGNFGNSYVSWFAX In one embodiment, the three CDR sequences of the VH region may have the following sequences: CDR1GFTFNTYA, CDR2 IRSKYNNYAT and CDR3 VRHGNFGNSYVSWFAX In one embodiment, the mutation at position Y114 in the VH region CDR3 is a Y114M, Y114R or Y114V mutation.

[0924] In one embodiment of the invention, the method comprises introducing up to 3 mutations, up to 2 mutations or up to 1 mutation into one or more of the three CDRs of the VH region of the reference antibody shown in SEQ ID NO: 1, 2, 3.

[0925] In one embodiment of the invention, the method comprises introducing up to 10 mutations, up to 9 mutations, up to 8 mutations, up to 7 mutations, up to 6 mutations, up to 5 mutations, up to 4 mutations, up to 3 mutations, up to 2 mutations or up to 1 mutation into the variable heavy chain framework region of the antibody, wherein the mutations preferably do not alter the binding of the antibody to CD3 compared to the same antibody without the mutations.

[0926] In one embodiment of the invention, the method comprises introducing a mutation selected from T31M or T31P into a VH region CDR1 sequence. In another embodiment of the invention, the method comprises introducing a mutation into a VH region CDR2 sequence of N57E. In another embodiment of the invention, the method comprises introducing a mutation into a VH region CDR3 sequence selected from H101G, H101N, S110A, S110G, Y114M, Y114R, and Y114V.

[0927] In another aspect, the invention relates to a method for increasing the binding affinity of an antibody that binds to CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein the VH region comprises CDR sequences having CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1, 2 and 3, the method comprising introducing a mutation into one of the CDR1, CDR2 or CDR3 sequences of the VH region as shown in SEQ ID NO: 1, 2 or 3.

[0928] In one embodiment of the present invention, the method comprises introducing a mutation into the VH region CDR3 sequence corresponding to position G105, wherein the CDR3 sequence is shown in SEQ ID NO 3. When the mutation is represented by X, the resulting CDR3 sequence may be represented as VRHGNFXNSYVSWFAY In one embodiment, the three CDR sequences of the VH region may have the following sequences: CDR1GFTFNTYA, CDR2 IRSKYNNYAT and CDR3 VRXGNFGNSYVSWFAY In one embodiment, the mutation at position G105 in CDR3 of the VH region is a G105P mutation.

[0929] Nucleic acid constructs, expression vectors and host cells

[0930] In one aspect, the present invention relates to a nucleic acid construct encoding one or more of the sequences shown in Table 1. Therefore, the present invention relates to a nucleic acid construct encoding any one of the sequences shown in SEQ ID NOs: 107; 221; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293.

[0931] In a further aspect, the present invention relates to a nucleic acid construct encoding a sequence of a humanized or chimeric CD3 antibody of the invention, an expression vector comprising the nucleic acid construct of the invention, a host cell comprising the expression vector, and a method of producing the antibody by culturing the host cell under suitable conditions for producing and optionally recovering the antibody. The humanized CD3 antibody is also referred to as "huCD3."

[0932] In one embodiment, the present invention provides an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized or chimeric antibody of the present invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody of the present invention, or (iii) both (i) and (ii). Thus, the expression vector comprises one or more nucleic acid constructs or nucleic acid sequences of any aspect or embodiment described herein.

[0933] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the heavy and light chain CDR sequences, wherein the VH CDR sequence is selected from: SEQ ID NO.:12,2,3;14,2,3;16,2,3;18,2,3;20,2,3;22,2,3;24,2,3;26,2,3;28,2,3;30,2,3;32,2,3;34,2,3;36,2,3;38,2,3;:40,2,3;42,2,3; 44,2,3; :46,2,3; 48,2,3; 50,2,3; 52,2,3; 54,2,3; 56,2,3; 58,2,3; 6 0,2,3;62,2,3;64,2,3;66,2,3;68,2,3;70,2,3;72,2,3;74,2,3;76,2 ,3;78,2,3;80,2,3;82,2,3;84,2,3;86,2,3;88,2,3;90,2,3;92,2,3;94,2,3;96,2,3;98,2,3;1,100,3;1,102,3;1,104,3;1,106,3;1,108,3;1,110,3;1,112,3;1,114,3;1,116,3;1,118,3;1,120,3;1,122,3;1,124,3;1,126,3;1,128,3;1,130,3;1,132,3;1,134,3;1,136,3;1, 138,3;;1,140,3;1,142,3;1,144,3;1,146,3;1,148,3;1,150,3;1,152,3;1,154,3;1,156,3;1,158,3;1:1,2,176;1,2,178;1,2,180;1,2,182;1,2,184;1,2,186;1,2,188;1,2,190;:1,2,192;1,2,194;1,2,196;1,2,198;1,2,200;1,2,202;1,2,204;1,2,206;:1,2,208;1,2,210 ;1,2,212;1,2,214;1,2,216;1,2,218;1,2,220;:1,2,222;1,2,224;1,2,226;1,2,228;1,2,230;1,2,232;1,2,234;1,2,236;1,2,238;1,2,240;1,2,242;1,2,244;1,2,246;1,2,248;1,2,250;1,2,252;1,2,254;1,2,256;1,2,258;1,2,260;1,2,262;1,2,264;1,2,266;1,2,268;1,2,270; 1,2,272; 1,2,274; 1,2,276; 1,2,278; : 1,2,280; 1,2,282; 1,2,284; 1,2,286; : 1,2,288; 1,2,290; 1,2,292; 1,2,294; 1,2,296; 1,2,298 and 1,2,300. And wherein the VL CDR sequences are selected from SEQ ID NO: 6, GTN, 7; 302, GTN, 7; 304, GTN, 7; 306, GTN, 7; 308, GTN, 7; : 310, GTN, 7; 312, GTN, 7; 314, GTN, 7; 316, GTN, 7; 318, GTN, 7; 320, GTN, 7; 322, GTN, 7; 324, GTN, 7; 326, GTN, 7; 328, GTN, 7; 330, GTN, 7; : 6, GTN, 332; 6, GTN, 334; 6, GTN, 336; 6, GTN, 338; 6, GTN, 340; 6, GTN, 342; 6, GTN, 344; 6, GTN, 346; 6, GTN, 348; 6, GTN, 350; 6, GTN, 352; 6, GTN, 354; 6, GTN, 356; 6, GTN, 358; 6, GTN, 360; 6, GTN, 362; 6, GTN, 364; 6, GTN, 366; 6, GTN, 368; 6, GTN, 370; 6, GTN, 372; 6, GTN, 374; 6, GTN, 376; 6, GTN, 378; 6, GTN, 380; 6, GTN, 382; 6, GTN, 384; GTN, 386; : 6, GTN, 388; 6, GTN, 390;, GTN, 392; and 6, GTN, 394 as shown CDR sequences.;

[0934] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the heavy and light chain CDR sequences, wherein the VL region CDR1, CDR2, CDR3 region CDR sequences comprise the CDR sequences shown in SEQ ID NOs.: 6, GTN, 7 and the VH region CDR1, CDR2, CDR3 region CDR sequences are selected from: CDR1, CDR2, CDR3 shown in SEQ ID NOs.: 54, 2, 3; CDR1, CDR2, CDR3 shown in SEQ ID NOs.: 58, 2, 3; CDR1, CDR2, CDR3 shown in SEQ ID NOs: 1, 106, 3; CDR1, CDR2, CDR3 shown in SEQ ID NOs: 1, 2, 176; CDR1, CDR2, CDR3 shown in SEQ ID NOs: 1, 2, 184; CDR1, CDR2, CDR3 shown in SEQ ID NOs: 1, 2, 220; CDR1, CDR2, CDR3 shown in SEQ ID NOs: 1, 2, 236; CDR1, CDR2, CDR3 shown in SEQ ID NO 1, 2,244; CDR1, CDR2, CDR3 shown in SEQ ID NO 1, 2,284; CDR1, CDR2, CDR3 shown in SEQ ID NO 1, 2,292 and CDR1, CDR2, CDR3 shown in SEQ ID NO 1, 2,298.

[0935] In a specific embodiment, the expression vector comprises a nucleic acid sequence encoding a variant of one or more of the above amino acid sequences, the variant having up to 25 amino acid modifications, such as up to 20, such as up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative or non-conservative substitutions, or at least 80% identity to any of the sequences, such as at least 85% identity or 90% identity or 95% identity to any of the above amino acid sequences, such as 96% identity or 97% identity or 98% identity or 99% identity. The present invention also relates to nucleic acid sequences that are different from the above nucleic acid sequences but encode the same amino acid sequence as the antibody of the present invention due to differences in the genetic code. For example, the nucleic acid sequence may be different but produce the same amino acid sequence as any of the amino acid sequences described herein. How to identify such additional nucleic acid sequences based on the genetic code is well known to the skilled person.

[0936] In a further embodiment, the expression vector further comprises a nucleic acid sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of an antibody (eg, a human antibody).

[0937] Such expression vectors described above can be used to recombinantly produce the antibodies of the present invention.

[0938] In the context of the present invention, the expression vector may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (comprising a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the humanized or chimeric CD3 antibody-encoding nucleic acid is contained in a naked DNA or RNA vector, including, for example, a linear expression element (as described, for example, in

[64] ), a compact nucleic acid vector (as described, for example, in

[65] and / or

[66] ), a plasmid vector such as pBR322, pUC 19 / 18 or pUC 118 / 119, a "midge" minimal size nucleic acid vector (as described, for example, in

[67] ), or as a precipitated nucleic acid vector construct, such as CaPO4. - - Precipitated constructs (as described in, for example,

[68] ,

[69] ,

[70] and

[71] ). Such nucleic acid vectors and their uses are well known in the art (see, for example,

[72] and

[73] ).

[0939] In one embodiment, the vector is suitable for expressing a humanized or chimeric CD3 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (

[74] ), pET vectors (Novagen, Madison WI), and the like.

[0940] The expression vector may additionally or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in

[75] and

[76] ).

[0941] The nucleic acid construct and / or vector may further comprise a nucleic acid sequence encoding a secretion / localization sequence that can target a polypeptide (e.g., a nascent polypeptide chain) to the cytoplasmic space or cell culture medium. Such sequences are known in the art and include secretion leaders or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial trafficking sequences, chloroplast trafficking sequences), membrane localization / anchor sequences (e.g., terminator transfer sequences, GPI anchor sequences), and the like, which are well known in the art.

[0942] In the expression vectors of the present invention, the humanized or chimeric CD3 antibody-encoding nucleic acid may comprise or be associated with any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication for plasmid products in E. coli, antibiotic resistance genes as selective markers, and / or suitable cloning sites (e.g., polylinkers). The nucleic acid construct and / or vector may also comprise an inducible promoter, such as CMV IE, as opposed to a constitutive promoter (the skilled artisan will appreciate that these terms are actually descriptors of the degree of gene expression under certain conditions).

[0943] In one embodiment, the humanized or chimeric CD3 antibody-encoding expression vector is located in a host cell or host animal and / or is delivered to the host cell or host animal via a viral vector.

[0944] Such expression vectors can be used to recombinantly produce humanized or chimeric CD3 antibodies.

[0945] In one aspect, the present invention provides a host cell comprising an expression vector of the present invention.

[0946] In one aspect, the humanized or chimeric CD3 antibodies of any aspect or embodiment described herein are provided by using a recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cell that produces the antibody. Thus, the present invention provides recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cells that produce humanized or chimeric CD3 antibodies or immunoglobulins as defined herein. Examples of host cells include yeast, bacteria and mammalian cells, such as CHO or HEK-293 cells. For example, in one embodiment, the host cell comprises a nucleic acid sequence stably integrated into the cell genome, which comprises a sequence encoding the expression of a humanized or chimeric CD3 antibody as described herein. In another embodiment, the host cell comprises a non-integrated nucleic acid sequence, such as a plasmid, cosmid, phagemid or linear expression element, which comprises a sequence encoding the expression of a humanized or chimeric CD3 antibody as described herein.

[0947] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which an expression vector or nucleic acid construct or sequence has been introduced. It should be understood that the term refers not only to the specific subject cell, but also to the progeny of the cell. Because certain modifications may occur during passage due to mutations or environmental influences, the progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, eukaryotic host cells such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytes; and prokaryotic cells such as Escherichia coli; and other eukaryotic hosts such as plant cells and fungi.

[0948] In a further aspect, the present invention relates to a method for producing a humanized or chimeric CD3 antibody of the present invention, said method comprising the steps of:

[0949] a) cultivating the host cell of the present invention as described above, and

[0950] b) recovering and / or purifying the antibody of the present invention from the culture medium.

[0951] In a further aspect, the nucleotide sequence encoding the sequence of a humanized or chimeric CD3 antibody further encodes a second moiety, such as a therapeutic polypeptide. Exemplary therapeutic polypeptides are described elsewhere herein. In one embodiment, the present invention relates to a method for producing a humanized or chimeric CD3 antibody fusion protein, the method comprising the steps of:

[0952] a) culturing a host cell comprising an expression vector comprising the nucleotide sequence, and

[0953] b) recovering and / or purifying the humanized or chimeric CD3 antibody fusion protein from the culture medium.

[0954] Composition

[0955] In one aspect, the invention provides a composition comprising an antibody or bispecific antibody according to any aspect and embodiment described herein.

[0956] In one aspect, the present invention provides a pharmaceutical composition comprising an antibody or bispecific antibody as defined in any one of the aspects and embodiments described herein and a pharmaceutically acceptable carrier.

[0957] Pharmaceutical compositions may be formulated according to conventional techniques, such as those described in

[77] , using a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients.

[0958] The pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients should be suitable for the humanized or chimeric antibodies of the present invention and the selected mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined by the absence of a significant negative effect on the biological properties required for antigen binding of the selected compound or pharmaceutical composition of the present invention (e.g., less than a substantial effect (10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0959] The pharmaceutical compositions of the present invention may also include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers and / or other materials suitable for inclusion in pharmaceutical compositions.

[0960] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, and that is non-toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors well known in the medical art, including the activity of the specific composition of the present invention or its amide used, the route of administration, the number of doses, the rate of excretion of the specific compound used, the duration of treatment; other drugs, compounds, and / or materials used in combination with the specific composition used; the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and other factors.

[0961] The pharmaceutical composition can be administered by any suitable route and mode. Suitable routes for administering the humanized or chimeric antibodies of the present invention in vivo and in vitro are well known in the art and can be selected by one of ordinary skill in the art.

[0962] In one embodiment, the pharmaceutical compositions of the present invention are administered parenterally.

[0963] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0964] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0965] In a preferred embodiment, the pharmaceutical composition is administered subcutaneously.

[0966] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, absorption delaying agents, and the like that are physiologically compatible with the humanized or chimeric antibodies of the invention.

[0967] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, glucose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil; carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical art.

[0968] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated. Reference to an "active compound" is also contemplated to encompass humanized or chimeric antibodies of the present invention.

[0969] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0970] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable antioxidant, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0971] The pharmaceutical compositions of the present invention may also comprise isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0972] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which may increase the half-life or efficacy of the pharmaceutical composition. The humanized or chimeric antibodies of the present invention may be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid alone or with wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art (see, for example,

[78] ).

[0973] In one embodiment, the humanized or chimeric antibodies of the present invention can be formulated to ensure appropriate in vivo distribution. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For pharmaceutically active substances, the use of such media and agents is known in the art. Unless any conventional media or agents are incompatible with the active compound, it is contemplated that they will be used in the pharmaceutical compositions of the present invention. Other active or therapeutic compounds may also be incorporated into the compositions.

[0974] Pharmaceutical compositions for injection must generally be sterile and stable under the conditions of preparation and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be an aqueous or non-aqueous solvent or dispersion medium, including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersant, and by using a surfactant. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, such as monostearate and gelatin, in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent together with one or a combination of ingredients, such as those listed above, as needed, followed by sterile microfiltration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle comprising a basic dispersion medium and the desired other ingredients, such as those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution thereof.

[0975] Sterile injection solutions can be prepared by mixing the required amount of the active compound with a composition or combination of compositions listed above as needed into a suitable solvent, followed by aseptic microfiltration. Generally speaking, dispersions are prepared by mixing the active compound into a sterile solvent comprising a basic dispersion medium and other components of the needs listed above. In the case of sterile powders for the preparation of sterile injection solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which obtain a powder of the active ingredient plus any other required components from the sterile filtered solution before it.

[0976] Therapeutic applications

[0977] In another aspect, the present invention relates to a humanized or chimeric antibody or pharmaceutical composition of the invention as defined in any aspect or embodiment described herein for use as a medicament.

[0978] In another aspect, the present invention relates to a humanized or chimeric antibody or pharmaceutical composition of the invention as defined in any aspect or embodiment described herein for use in treating a disease.

[0979] In one embodiment of the present invention, the bispecific antibodies, compositions, and pharmaceutical compositions are used to treat diseases.

[0980] In one embodiment of the present invention, the bispecific antibody, composition, or pharmaceutical composition is used to treat a disease, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

[0981] The humanized or chimeric antibodies or pharmaceutical compositions of the present invention can be used to treat any cancer in which the effector mechanism of cytotoxic T cells is required. For example, the humanized or chimeric antibodies can be administered to cells cultured in vitro or ex vivo, or to human subjects in vivo, for example, to treat or prevent conditions such as cancer, inflammatory or autoimmune conditions. The term "subject" as used herein is generally a person who responds to the humanized or chimeric antibodies or pharmaceutical compositions. Subjects can, for example, include human patients with conditions that can be corrected or improved by modulating target function or by directly or indirectly causing cell killing.

[0982] In another aspect, the present invention provides a method for treating or preventing a condition, such as cancer, in which recruitment of T cells will aid in treatment or prevention, the method comprising administering a therapeutically effective amount of a humanized or chimeric antibody or pharmaceutical composition of the present invention to a subject in need thereof. The method generally comprises administering to a subject an amount of a humanized or chimeric antibody effective to treat or prevent the condition.

[0983] In a specific aspect, the present invention relates to a method of treating cancer comprising administering to a subject in need thereof a humanized or chimeric antibody or pharmaceutical composition of the invention as defined in any aspect and embodiment described herein.

[0984] In another aspect, the invention relates to the use or method as defined in any aspect or embodiment described herein, wherein the humanized or chimeric antibody is a bispecific antibody that specifically binds both CD3 and a cancer-specific target or a target that is overexpressed in or associated with cancer, such as HER2, CD19, EpCAM, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2 or MCSP (or HMW-MAA), CD20 and wherein the disease is cancer, such as breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer and squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, soft tissue cancer (such as synovial sarcoma), indolent or aggressive forms of B-cell lymphoma, chronic lymphocytic leukemia or acute lymphocytic leukemia.

[0985] The effective dosage and dosage regimen of the humanized or chimeric antibody depends on the disease or condition to be treated and can be determined by one skilled in the art.

[0986] A physician with ordinary skill in the art can readily determine and prescribe the desired effective amount of a pharmaceutical composition. For example, a physician may start the dosage of the humanized or chimeric antibody used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable dosage of the composition of the present invention is the amount of the humanized or chimeric antibody that is the lowest dose effective to produce a therapeutic effect according to a specific dosage regimen. Such an effective dose generally depends on the factors described above.

[0987] For example, an "effective amount" for therapeutic use can be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated, for example, in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by examining the ability of humanized or chimeric antibodies to inhibit cell growth or induce cytotoxicity using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound, i.e., a therapeutic humanized or chimeric antibody or pharmaceutical composition of the present invention, can reduce tumor size or otherwise improve the subject's symptoms. One of ordinary skill in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or selected route of administration.

[0988] An exemplary, non-limiting range for a therapeutically effective amount of a humanized or chimeric antibody of the invention is about 0.001-30 mg / kg, e.g., about 0.001-20 mg / kg, e.g., about 0.001-10 mg / kg, e.g., about 0.001-5 mg / kg, e.g., about 0.001-2 mg / kg, e.g., about 0.001-1 mg / kg, e.g., about 0.001, about 0.01, about 0.1, about 1, about 5, about 8, about 10, about 12, about 15, about 18 mg / kg.

[0989] Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and, for example, administered in the vicinity of the target site.

[0990] The dosage regimen in the above-described methods of treatment and uses is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be given, several divided doses may be given over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0991] In one embodiment, the efficacy of the treatment is monitored during the treatment period, eg, at predetermined time points.

[0992] If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In another embodiment, the humanized or chimeric antibody or pharmaceutical composition is administered by slow continuous infusion over a long period of time, for example, more than 24 hours, to minimize unwanted side effects.

[0993] While it is possible to administer the humanized or chimeric antibody of the present invention alone, it is preferred to administer the humanized or chimeric antibody as a pharmaceutical composition as described above.

[0994] An effective dose of the humanized or chimeric antibody of the present invention can also be administered using a weekly, biweekly, or triweekly dosing period. The dosing period can be limited to, for example, 8 weeks, 12 weeks, or until clinical progression is confirmed. Alternatively, an effective dose of the humanized or chimeric antibody of the present invention can be administered every other week, two weeks, or three weeks.

[0995] In one embodiment, the humanized or chimeric antibody can be administered by infusion at a dose of mg / m 2The calculated weekly dose is given. Such dosage can be, for example, based on the mg / kg dosage provided above, according to the following: dosage (mg / kg) x70:1.8. Such administration can be repeated for example 1-8 times, for example 3-5 times. Administration can be carried out for a period of time of 2-24 hours, for example 2-12 hours by continuous infusion. In one embodiment, humanized or chimeric antibodies can be given for a long time by slow continuous infusion, for example, more than 24 hours, to reduce toxic side effects.

[0996] In one embodiment, when administered once a week, the humanized or chimeric antibody can be administered up to 8 times, e.g., 4-6 times, as a weekly dose calculated as a fixed dose. Such a regimen can be repeated one or more times as needed, e.g., after 6 or 12 months. Such a fixed dose can be, for example, based on the mg / kg dose provided above, with a body weight estimate of 70 kg. The dose can be determined or adjusted by measuring the amount of the humanized or chimeric antibody of the invention in the blood after administration, e.g., by obtaining a biological sample and using an anti-idiotypic antibody targeting the binding region of the humanized or chimeric antibody of the invention.

[0997] In one embodiment, the humanized or chimeric antibody can be administered by maintenance therapy, eg, once a week for a period of 6 months or more.

[0998] Humanized or chimeric antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when cancer is in remission.

[0999] For ease of administration and uniformity of dosage, parenteral compositions can be formulated in dosage unit form. As used herein, dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by and directly dependent on: (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for treatment of individual sensitivities.

[1000] Humanized or chimeric antibodies can also be given prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when the cancer is in remission. This may be particularly useful in patients in whom it is difficult to locate a tumor that is known to be present due to other biological factors.

[1001] Diagnostic applications

[1002] The humanized or chimeric antibodies of the present invention can also be used for diagnostic purposes using compositions comprising the humanized or chimeric antibodies described herein. Thus, the present invention provides diagnostic methods and compositions using the humanized or chimeric antibodies described herein. Such methods and compositions can be used for purely diagnostic purposes, such as detecting or identifying a disease, as well as for monitoring the progress of therapeutic treatment, monitoring disease progression, evaluating post-treatment status, monitoring disease recurrence, evaluating the risk of developing a disease, and the like.

[1003] In one aspect, the invention relates to a method for diagnosing a disease characterized by involvement or accumulation of CD3-expressing cells, comprising administering to a subject a humanized or chimeric antibody of the invention, a composition of the invention, or a pharmaceutical composition of the invention, optionally wherein the humanized or chimeric antibody is labeled with a detectable agent.

[1004] In one aspect, humanization or chimeric antibodies of the present invention are used in vitro, for example, for detecting the level of target in a sample obtained from a patient or the level of cells expressing target of interest on its cell surface, diagnosing a cell indicating disease in which a specific target of interest is expressed and bound by humanization or chimeric antibodies or a disease related to pathogenesis. This can be achieved, for example, by contacting a humanization or chimeric antibody of the present invention under conditions allowing antibody to bind to the target, optionally together with a control sample. Complex formation (e.g., using ELISA) can then be detected. When a control sample is used together with a test sample, the level of humanization or chimeric antibodies or antibody-target complexes is analyzed in two samples, and statistically significant higher levels of humanization or chimeric antibodies or antibody-target complexes in the test sample indicate higher levels of target in the test sample compared to the control sample.

[1005] Examples of conventional immunoassays in which the humanized or chimeric antibodies of the invention can be used include, without limitation, ELISA, RIA, FACS analysis, plasmon resonance analysis, chromatography, tissue immunohistochemistry, Western blotting, and / or immunoprecipitation.

[1006] Thus, in one embodiment, the invention relates to a method of diagnosing a disease characterized by involvement or accumulation of CD3-expressing cells, comprising administering to a subject the antibody, bispecific antibody, composition or pharmaceutical composition of any aspect or embodiment described herein, optionally wherein the antibody is labeled with a detectable label.

[1007] In one embodiment, the present invention relates to a method for detecting the presence of a target or a cell expressing a target in a sample, comprising:

[1008] - contacting the sample with a humanized or chimeric antibody of the present invention under conditions that allow the humanized or chimeric antibody to bind to the target in the sample; and

[1009] - Analyzing whether a complex is formed. Typically, the sample is a biological sample.

[1010] In one embodiment, the sample is a tissue sample of cells known or suspected to contain a specific target and / or express a target. For example, in situ detection of target expression can be achieved by removing a histological sample from the patient and providing a humanized or chimeric antibody of the present invention to such a sample. Humanized or chimeric antibodies can be provided by applying or applying a humanized or chimeric antibody to a sample, which is then detected using a suitable tool. It is then possible not only to detect the presence of cells that express the target, but also to detect the distribution of cells that detect the target or express the target in the examined tissue (for example, in the case of evaluating the spread of cancer cells). Using the present invention, it is readily understood by those of ordinary skill that any one of the various histological methods (for example, staining procedures) can be improved to achieve such in situ detection.

[1011] In the above-mentioned determination method, humanized or chimeric antibodies can be labeled with detectable substances to allow the antibody of combination to be detected. Alternatively, the (first) specific humanized or chimeric antibody of combination can be detected by labeling with a detectable substance and combining the antibody detection of the first specific humanized or chimeric antibody. In addition, in the above-mentioned determination method, the diagnostic composition of the antibody or bispecific antibody comprising any aspect or embodiment as described herein can be used. Therefore, in one aspect, the present invention relates to the diagnostic composition of the antibody or bispecific antibody comprising any aspect or embodiment as described herein.

[1012] The target level in the sample can also be evaluated by competitive immunoassay using a target standard labeled with a detectable substance and an unlabeled target-specific humanized or chimeric antibody. In this type of assay, a biological sample, a labeled target standard, and a target-specific humanized or chimeric antibody are combined, and the amount of the labeled target standard bound to the unlabeled target-specific humanized or chimeric antibody is measured. The amount of target in the biological sample is inversely proportional to the amount of the labeled target standard bound to the target-specific humanized or chimeric antibody.

[1013] Suitable labels for target-specific humanized or chimeric antibodies, secondary antibodies, and / or target standards for use in in vitro diagnostic techniques include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; examples of luminescent materials include luminol; and examples of suitable radioactive materials include 125 I. 131 I.35 S and 3 H.

[1014] In one aspect, the target-specific humanized or chimeric antibodies of the invention are used for in vivo imaging of target-expressing tissues, such as tumors.For in vivo methods, antibody fragments, such as (Fab')2, Fab and Fab' fragments, are particularly advantageous because of their rapid distribution kinetics.

[1015] In vivo imaging can be performed by any suitable technique. For example, 99 Tc, 131 I. 111 Target-specific humanized or chimeric antibodies (e.g., antibodies or fragments) labeled with In or other gamma-emitting isotopes can be used to image target-specific antibody accumulation or distribution in target-expressing tissues, such as tumors, using a gamma scintillation camera (e.g., the Elscint Apex 409 ECT device), typically using a low-energy high-resolution collimator or a low-energy universal collimator. Alternatively, 89 Zr, 76 Br, 18 F or other positron-emitting radionuclide labels can be used to image the distribution of target-specific humanized or chimeric antibodies or antibody fragments using positron emission tomography (PET) in tumors. Images obtained using such techniques can be used to evaluate the biodistribution of a target in a patient, mammal, or tissue, for example, where the target is used as a biomarker for the presence of cancer / tumor cells. Variations on this technique can include the use of magnetic resonance imaging (MRI) to improve imaging via gamma camera technology. Conventional immunoscintigraphy methods and principles are described, for example, in

[79] ,

[80] , and

[81] . In addition, such images can also or alternatively be used as the basis for surgical techniques to remove the tumor. In addition, such in vivo imaging techniques can allow identification and localization of tumors in cases where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.) but the tumor cannot be identified by traditional analytical techniques. All of these methods are features of the present invention.

[1016] The in vivo imaging and other diagnostic methods provided by the present invention are particularly useful for detecting micrometastases in human patients (eg, patients not previously diagnosed with cancer or patients in recovery / remission from cancer).

[1017] In one embodiment, the present invention provides an in vivo imaging method, wherein a target-specific humanized or chimeric antibody of the present invention is conjugated to a radiopaque agent that facilitates detection, the conjugated humanized or chimeric antibody is administered to a host, for example by injection into the bloodstream, and the presence and location of the labeled humanized or chimeric antibody in the host is analyzed. Through this technology and any other diagnostic methods provided herein, the present invention provides methods for screening for the presence of disease-associated cells in a biological sample obtained from a human patient or from a human patient and / or methods for evaluating the distribution of a target-specific humanized or chimeric antibody prior to target-specific ADC therapy.

[1018] For diagnostic imaging, radioisotopes can be conjugated to target-specific humanized or chimeric antibodies directly or indirectly through the use of intermediate functional groups. Useful intermediate functional groups include chelating agents such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example,

[82] ).

[1019] In addition to radioisotopes and radiopaque agents, diagnostic methods can be performed using target-specific antibodies conjugated to dyes (e.g., biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers for magnetic resonance imaging (MRI), such as paramagnetic ions (see, for example,

[83] , which describes MRI techniques and the preparation of antibodies conjugated to MRI enhancers). Such diagnostic / detection agents can be selected from agents for MRI and fluorescent compounds. In order to load the target-specific humanized or chimeric antibody with a radioactive metal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail to which multiple chelating groups are attached to bind the ions. Such a tail can be a polymer such as polylysine, a polysaccharide, or another derivatized or derivatized chain with pendant groups to which the chelating groups can be attached, such as porphyrins, polyamines, crown ethers, dithiosemicarbazones, polyoximes, etc., which are known for this purpose. The chelating agent can be coupled to the target-specific humanized or chimeric antibody using standard chemistry.

[1020] Thus, the present invention provides diagnostic target-specific humanized or chimeric antibodies, wherein the target-specific humanized or chimeric antibody is conjugated to a contrast agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound contrast enhancement agent) or a radionuclide (which can be, for example, a γ-, β-, α-, Auger electron-, or positron-emitting isotope).

[1021] In one aspect, the invention relates to a diagnostic composition comprising an antibody or bispecific antibody of the invention.

[1022] In a further aspect, the present invention relates to a kit for detecting the presence of a target antigen or a cell expressing the target in a sample, comprising:

[1023] - a target-specific humanized or chimeric antibody of the invention; and

[1024] -Instructions for use of the kit.

[1025] Therefore, in one aspect, the present invention provides a kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising the steps of:

[1026] a) contacting the sample with the antibody or bispecific antibody of the invention under conditions that allow formation of a complex between the antibody or bispecific antibody and CD3; and

[1027] b) Analyze whether a complex is formed.

[1028] In one embodiment, the present invention provides a test kit for diagnosing cancer, which comprises a container comprising a target-specific humanized or chimeric antibody, and one or more reagents for detecting the binding of target-specific humanized or chimeric antibodies to the target. Reagents may include, for example, fluorescent labels, enzyme labels or other detectable labels. Reagents may also include a second or third antibody or a reagent for an enzyme reaction, wherein the enzyme reaction produces a product that can be visualized. In one embodiment, the present invention provides a diagnostic kit, which comprises one or more target-specific humanized or chimeric antibodies of the present invention in a labeled or unlabeled form in a suitable container, a reagent for indirect assay incubation, and a substrate or a derivatizing agent (depending on the nature of the label) for detecting in such an assay. Control reagents and instructions for use may also be included.

[1029] Diagnostic kits for use with target-specific humanized or chimeric antibodies, such as labeled target-specific antibodies, can also be provided for detecting the presence of a target in a tissue sample or host. In such diagnostic kits, as well as in kits for therapeutic use described elsewhere herein, target-specific humanized or chimeric antibodies can typically be provided in a lyophilized form in a container alone or in combination with another antibody specific for a target cell or peptide. Typically, a pharmaceutically acceptable carrier (e.g., an inert diluent) and / or its components, such as Tris, phosphate or carbonate buffer, stabilizers, preservatives, biocides, inert proteins such as serum albumin, etc. (typically in separate containers for mixing) and additional reagents (also typically in separate containers) can also be included. In some kits, a second antibody capable of binding to the target-specific humanized or chimeric antibody is also included, typically in a separate container. The second antibody is typically conjugated to a label and formulated in a manner similar to the target-specific humanized or chimeric antibodies of the present invention. Using the methods described above and elsewhere herein, target-specific humanized or chimeric antibodies can be used to define subsets of cancer / tumor cells and characterize such cells and associated tumor tissue.

[1030] Anti-idiotypic antibodies

[1031] In a further aspect, the invention relates to anti-idiotypic antibodies in combination with the humanized or chimeric antibodies of the invention as described herein.

[1032] In one embodiment, the invention relates to an anti-idiotypic antibody that binds to an antibody or bispecific antibody according to any one of the claims of the invention.

[1033] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants that are typically associated with the antigen binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genotype as the source of the CD3 monoclonal antibody with an anti-Id monoclonal antibody for which the anti-Id monoclonal antibody is prepared. Immune animals can typically recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibodies (anti-Id antibodies) to these idiotypic determinants. Such antibodies are described, for example, in US 4,699,880. Such antibodies are another feature of the present invention.

[1034] The anti-Id antibody can also be used as an "immunogen" to induce an immune response in another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id antibody can be epitopically identical to the original monoclonal antibody that induced the anti-Id antibody. Thus, by using antibodies directed against the idiotypic determinants of the monoclonal antibody, it is possible to identify other clones expressing antibodies of the same specificity. The anti-Id antibody can be altered (thereby generating anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein for the CD3-specific antibodies of the invention. For example, the monoclonal anti-Id antibody can be coupled to a carrier such as a keyhole limpet antibody. Hemocyanin (KLH) is coupled and used to immunize BALB / c mice. Sera from these mice typically contain anti-anti-Id antibodies with binding properties similar to (if not identical to) the original / parental CD3 antibody.

[1035] sequence

[1036] Table 1

[1037]

[1038]

[1039]

[1040]

[1041]

[1042]

[1043]

[1044]

[1045]

[1046]

[1047]

[1048]

[1049]

[1050]

[1051]

[1052]

[1053]

[1054]

[1055]

[1056]

[1057]

[1058]

[1059]

[1060]

[1061]

[1062]

[1063]

[1064]

[1065]

[1066]

[1067]

[1068]

[1069]

[1070]

[1071]

[1072]

[1073]

[1074]

[1075]

[1076]

[1077]

[1078]

[1079] The CDR regions were annotated according to the IMGT definition. Example

[1080] Example 1 - Generation of Humanized CD3 Antibodies and Non-Activating Antibody Variants

[1081] Humanization of CD3 antibodies

[1082] The humanization of the murine CD3 antibody (US 8,236,308, described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK) using an improved form of its germline humanization (CDR-grafting) technology (EP 0 629 240). Using this technology, one different VH chain (SEQ ID NO: 4) and two different VL chains (SEQ ID NOs: 8, 10) were designed. By combining these one VH with two VL chains, two different antibodies were generated. The humanized variants are described herein as huCD3. Thus, a humanized variant of the invention comprising a VH and a VL is described, for example, as IgG1-huCD3-H1L1, which means that the particular variant has an IgG1 isotype, is a humanized CD3, and comprises a VH amino acid sequence designated "H1" and defined according to SEQ ID NO: 4 and a VL amino acid sequence designated "L1" and defined according to SEQ ID NO: 8. Thus, H1 refers to the variable heavy chain region VH1, L1 refers to the variable light chain region VL1, and so on.

[1083] Specifically, the variants IgG1-huCD3-H1L1 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO: 4 and the VL1 sequence shown in SEQ ID NO: 8) and IgG1-huCD3-L1-T41K (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO: 4 and the VL sequence shown in SEQ ID NO: 10).

[1084] b12 antibody

[1085] In some embodiments, antibody b12, an HIV-1 gpl20-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812-23.) was used as a negative control and is referred to as "IgG1-b12."

[1086] Express

[1087] Antibodies were expressed as IgG1, κ or IgG1, λ, with or without the non-activating mutations described below and with mutations in the CH3 domain, which were used to generate bispecific antibodies by the methods described below. A mixture of plasmid DNA encoding both the heavy and light chains of the antibodies was transiently transfected into Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer.

[1088] Antibody purification

[1089] The culture supernatant was filtered through a 0.2 μm dead-end filter loaded onto a 5 mL MabSelect SuRe column (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9, and dialyzed overnight to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun). Alternatively, after purification, the eluate was loaded onto a HiPrep desalting column and the antibody was exchanged to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun) buffer. After dialysis or buffer exchange, the sample was sterile filtered through a 0.2 μm dead-end filter. Purity was determined by SDS-PAGE, and concentration was measured by absorbance at 280 nm. The purified antibody was stored at 2-8 ° C.

[1090] Example 2: Generation of mutant libraries

[1091] Random mutagenesis was performed to generate point mutations using the Quick Change mutagenesis kit (Stratagene, according to the manufacturer's instructions) and HC (p33HGTE-huCD3-H1) and LC (p33L-huCD3-L1-T41K) expression plasmids as templates. The HC plasmid encodes the monovalent UniBody-TE format as described in WO2011110642. Each selected position was randomized using primers containing NNS codons at the selected position (N = G, A, T, or C and S = G or C). The mutant library was transformed into OneShot DH5α (Invitrogen) according to the manufacturer's instructions.

[1092] Colony picking and LEE PCR

[1093] For each mutation position, 96 clones were picked into 50 μL LEE (Linear Expression Element) PCR buffer (5 μL 10x AccuPrime PCR buffer 1, 44.6 μL water (B. Braun), 0.1 μL CMV P f (MAR5) and 0.1 μL TkpAr (MAR1) primers (100 μM stock), 0.2 μL AccuPrime Taq (Invitrogen) to amplify the expression cassette (promoter to poly A) from the expression plasmid. LEE PCR was performed by incubating the mixture for 2'94°C, [30"94°C, 30"55°C, 5'68°C] 35x, 10'72°C and stored at 4°C until further use.

[1094] Each library (12) of 96 colonies was sequenced using Sanger sequencing (Beckman Coulter Genomics, UK).

[1095] Table 2: Primer sequences used for LEE PCR

[1096]

[1097] Example 3: Expression of mutant library and IgG quantification

[1098] For each mutant (12 x 96 total), 1.11 μL HC and 1.11 μL LC LEE PCR products were diluted in 2.78 μL water. 5 μL of the DNA dilution was used to transfect a single well of a 96-well plate.

[1099] 0.4 μL ExpiFectamine TM293 (Invitrogen, US) and 4.6 μL Opti-MEM (Gibco, US) were mixed and incubated at room temperature for 5 minutes. Next, the Fectin / Opti-MEM mixture was added to 5 μL DNA dilution solution and incubated at room temperature for 30 minutes. Finally, 8.3 μL of the Fectin / Opti-MEM / DNA mixture was added to 117.5 μL Expi293F TM In all procedures, cells were shaken with Expi293F TM After transfection, the cells were incubated at 37°C / 8% CO2 for 5 days.

[1100] Five days after transfection, the supernatant was collected and the antibody concentration in the supernatant was measured by biolayer interferometry using Octet RED (ForteBio, US).

[1101] Example 4: Generation of CD3 / TCR-LC13 screening library

[1102] Freestyle 293-F cells (Invitrogen, US) were co-transfected with expression constructs encoding human α and β chains of the TCR (SEQ ID NO: 396 and SEQ ID NO: 397, respectively), human CD3δ (SEQ ID NO: 398), human CD3ε (SEQ ID NO: 399), human CD3γ (SEQ ID NO: 400), and human CD3ζ (SEQ ID NO: 401). Signal peptide sequences were excluded from these sequences. Transfection was performed according to the manufacturer's instructions (Invitrogen, US). One day after transfection, cells were frozen until further use.

[1103] Example 5: Screening of affinity mutants

[1104] Homogeneous assay (dose response)

[1105] Based on the sequence data, mutants were selected where the sequence traces showed high PHRED scores, indicating the absence of multiple mutations. For each mutation, multiple redundant clones were selected when available.

[1106] The binding of recombinantly produced UniBody molecules in cell culture supernatants was determined by homogeneous antigen-specific binding assays using Fluorometric Micro volume Assay Technology (FMAT; Applied Biosystems, Foster City, CA, USA). In the assay, the binding of the designed samples or monovalent antibody molecules to CD3 / TCR-LC13 (Freestyle 293-F cells transiently expressing human CD3 and human T cell receptor (TCR), generated as described above) and Freestyle 293-F wild-type cells (negative control that does not express human TCR) was analyzed in a dose response. The IgG levels of the samples were normalized before the dose response binding.

[1107] Dilution series samples were added to the cells to allow binding to CD3. Subsequently, the binding of the monovalent antibody molecules was detected using a fluorescent conjugate (goat anti-human IgG Fcγ-Alexa647; Jackson ImmunoResearch). CD3-specific humanized mouse antibody IgG1-HuM291-F405L (produced in Freestyle 293-F cells) and the monovalent antibody UniTE-huCD3-H1L1-LT41K were used as positive controls, and ChromPure Human IgG whole molecule (Jackson ImmunoResearch) was used as a negative control. The samples were scanned using the Applied Biosystems 8200 Cell Detection System (8200CDS), and the total fluorescence within the sample concentration range was used as a readout. When the count was above 50, the sample was positive, and the count x fluorescence (total fluorescence) was at least 3 times that of the negative control.

[1108] Heatmap

[1109] From the homogeneous dose response screen, a 4-parameter sigmoidal model was used to fit the binding curves. From the fit, the maximum binding of each mutant was determined. For each mutant, the average maximum binding was calculated and plotted as Figure 1 Shown are the ratios between the mean maximum and wt binding.

[1110] Comparison

[1111] The selected HC mutants generated from these libraries were aligned and Figure 2 Depicted in. The CDR area has been Defines annotations. Sequence numbers are annotated according to the direct numbering scheme.

[1112] Example 6: Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange

[1113] The bispecific antibodies according to the present invention can be produced by using the methods disclosed in WO2011131746 and WO2013060867 (Genmab).

[1114] For example, a mutation in position F405L may be introduced into one parent antibody of the IgG1 isotype, and a mutation in position K409R may be introduced into another parent antibody of the IgG1 isotype.

[1115] The two parent antibodies at a final concentration of 0.5 mg / mL (equimolar concentration) can be incubated with 25 mM 2-mercaptoethylamine-HCl (2-MEA) at 37° C. for 90 minutes in a total volume of 100 μL Tris-EDTA (TE) under reducing conditions. The reduction reaction is stopped when the reducing agent 2-MEA is removed by using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol.

[1116] The bispecific antibody can be filtered on a 0.2 μm dead-end filter and the absorbance of the bispecific antibody at 280 nm (A280) can be measured to determine its final concentration.

[1117] Example 7: Combining Data

[1118] For all binding assays below, a selected panel of heavy chain variants of huCD3-H1L1 were tested in different formats:

[1119] Table 3: Selected affinity variants of monovalent antibody-TE format huCD3-H1L1

[1120]

[1121]

[1122] Octet binding affinity determination of CD3 affinity mutants in monovalent antibody-TE format

[1123] The affinity of the affinity VH variants (Table 3) of the selected group was determined on ForteBio OctetHTX using biolayer interferometry. CD3 affinity mutants in the form of monovalent antibody-TE (2 μg / mL) were loaded for 600 s to an anti-human Fc capture (AHC) biosensor (ForteBio, Portsmouth, UK; Catalog No. 18-5060) for a loading response of 0.4 nm. The antibody in the form of UniBody-TE was used to specifically measure the monovalent interaction affinity between CD3 affinity mutants and CD3ε27-GSKα ligands. After baseline (150 s), the binding (1000 s) and dissociation (1000 s) of CD3ε27-GSKα (100 and 1000 nM) were determined. The CD3ε27-GSKα protein consists of a human CD3ε peptide (aa1-27) fused to the N-terminus of κLC (SEQ ID NO: 402). For calculations, the theoretical molecular weight of CD3ε27-GSKα based on the amino acid sequence, ie, 27.1 kDa, was used. The experiment was performed while shaking at 1000 rpm and 30°C.

[1124] Data were analyzed with ForteBio data analysis software v8.1 using a 1:1 model and global perfect fit, an association time of 1000 s, and a dissociation time of 200 s. Data traces were corrected by subtracting a reference curve (a CD3 affinity mutant without CD3ε27-GSKα), aligning the Y axis to the last 5 s of baseline, and applying interstep correction and Savitzky-Golay filtering.

[1125] Table 4: Equilibrium dissociation constants (KD) of selected variants

[1126]

[1127]

[1128] nd = not determined

[1129] T cell binding of affinity variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K) in flow cytometry (FACS)

[1130] T cell binding of purified VH affinity variants of humanized CD3 (IgG1-huCD3-H1L1) antibodies was determined using fluorescence-activated cell sorting on a FACSCanto 752 (BD Biosciences). T cells were isolated from the buffy coat fraction of anticoagulated human donor blood samples and resuspended in PBS / 0.1% BSA / 0.02% azide at 1.8×10E6 cells / mL. 50 μL of T cell suspension and 50 μL of antibody dilution were combined on ice in a 96-well plate, incubated at 4°C for 30 min, and washed twice with PBS / 0.1% BSA / 0.02% azide. Next, 50 μL of a secondary antibody, R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1 / 200 in PBS / 0.1% BSA / 0.02% azide, was added for staining, and the mixture was incubated at 4°C for 30 minutes, followed by washing twice with PBS / 0.1% BSA / 0.02% azide. The cells were resuspended in 120 μL of PBS / 0.1% BSA / 0.02% azide, and the PE geometric mean fluorescence intensity was measured. Binding curves were analyzed using GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA) using nonlinear regression (sigmoidal dose-response with variable slope), and the apparent affinity (KD) was derived from the concentration of half-maximal binding. Figure 4 Binding curves of affinity variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K) are shown. Figure 5 Binding curves for a low-affinity variant of humanized CD3 (UniTE-huCD3-H1L1-LT41K) are shown.

[1131] Table 5: Summary of binding data for CD3 affinity mutants in monovalent antibody-TE format

[1132]

[1133]

[1134] For all following binding assays, a selected panel of preferred heavy chain variants was tested (see Table 5).

[1135] Octet Binding Affinity Assay of IgG1-huCD3-H1L1-FEAL Affinity Mutants The affinity of selected CD3 affinity variants in the IgG1-huCD3-H1L1-FEAL format was determined using biolayer interferometry on a ForteBio Octet HTX (ForteBio, UK) (Table 6). Anti-human Fc capture biosensors (cat: 18-5060, ForteBio, UK) were loaded with hIgG (1 μg / mL) for 600 s. After a baseline (200 s), association (1000 s) and dissociation (2000 s) of CD3 E27-GSKa were determined using a CD3 E27-GSKa concentration range of 27.11 μg / mL to 0.04 μg / mL (1000 nM to 1.4 nM) and a three-fold dilution step (sample diluent, cat: 18-5028, ForteBio, UK). For calculations, the theoretical molecular weight of CD3E27-GSKα based on the amino acid sequence, ie 27.11 kDa, was used. The experiments were performed while shaking at 1000 rpm and 30° C. Each antibody was tested in at least two independent experiments (Table 6).

[1136] Data were analyzed using ForteBio Data Analysis Software v8.1 using a 1:1 model and global perfect fit, with an association time of 1000 s and a dissociation time of 100 s. Data traces were corrected by subtracting a reference curve (without CD3E27-GSKα antibody), aligning the Y axis to the last 10 s of baseline, and applying an interstep correction and Savitzky-Golay filter. Data traces with responses < 0.05 nm were excluded from the analysis.

[1137] Table 6

[1138] average <kd>(nM) < / kd> SDEV SEM CV <kon> < / kon> SDEV SEM CV <kdis> < / kdis> SDEV SEM CV ikB n IgG1-huCD3-G105P-FEAL 5 2 1 45 4.7E+05 9.7E+04 5.6E+04 21 2.5E-03 1.0E-03 5.8E-04 40 8.3 3 IgG1-huCD3-FEAL 15 6 3 37 2.7E+05 5.1E+04 2.9E+04 19 4.0E-03 1.6E-03 9.1E-04 39 7.8 3 IgG1-huCD3-Y114V-FEAL 29 8 4 26 2.2E+05 3.3E+04 1.9E+04 15 6.3E-03 9.7E-04 5.6E-04 15 7.5 3 IgG1-huCD3-T31P-FEAL 42 9 4 21 1.9E+05 3.8E+04 1.6E+04 20 7.8E-03 1.3E-03 5.3E-04 17 7.4 6 IgG1-huCD3-Y114M-FEAL 42 14 8 33 2.6E+05 6.2E+04 3.6E+04 24 1.0E-02 1.5E-03 8.7E-04 15 7.4 3 IgG1-huCD3-H101N-FEAL 45 13 7 29 4.8E+05 2.2E+05 1.2E+05 45 2.0E-02 3.1E-03 1.8E-03 16 7.3 3 IgG1-huCD3-Y114R-FEAL 46 10 6 22 1.5E+05 4.1E+04 2.4E+04 27 6.8E-03 4.1E-04 2.4E-04 6 7.3 3 IgG1-huCD3-S110A-FEAL 72 15 6 21 1.8E+05 2.5E+04 1.0E+04 14 1.3E-02 1.6E-03 6.4E-04 12 7.1 6 IgG1-huCD3-N57E-FEAL 91 30 17 33 2.1E+05 2.8E+04 1.6E+04 13 1.9E-02 4.0E-03 2.3E-03 21 7.0 3 IgG1-huCD3-T31M-FEAL 99 23 13 23 1.9E+05 2.5E+04 1.5E+04 14 1.8E-02 2.6E-03 1.5E-03 14 7.0 3 IgG1-huCD3-Y32A-FEAL 105 31 22 29 2.2E+05 1.1E+05 7.5E+04 48 2.2E-02 4.4E-03 3.1E-03 20 7.0 2 IgG1-huCD3-H101L-FEAL 107 39 23 37 2.7E+05 4.3E+04 2.5E+04 16 2.8E-02 7.5E-03 4.4E-03 27 7.0 3 IgG1-huCD3-H101K-FEAL 120 94 55 79 2.2E+05 1.9E+05 1.1E+05 84 1.7E-02 9.8E-03 .7E-03 58 6.9 3 IgG1-huCD3-S110G-FEAL 153 120 70 79 3.8E+05 4.2E+05 2.4E+05 112 2.6E-02 8.3E-03 4.8E-03 32 6.8 3 IgG1-huCD3-H101G-FEAL 683 169 97 25 3.0E+04 9.2E+03 5.3E+03 30 2.0E-02 8.5E-04 4.9E-04 4 6.2 3 IgG1-huCD3-H1011-FEAL nd 0 IgG1-huCD3-H101F-FEAL nd 0

[1139] T cell binding affinity determination of IgG1-huCD3-H1L1-FEAL affinity mutants

[1140] By using RosetteSep human T cell enrichment mixture (Cat: 15021C.1, Stemcell Technologies, France) according to the manufacturer's instructions to separate the T cells from donor buffy coat (Sanquin, Amsterdam, The Netherlands). In brief, 50 μ L of T cell separation mixture is added to 1 mL of buffy coat and incubated at room temperature for 20 minutes. Next, buffy coat (1: 3, v / v) is diluted with PBS (cat: 3623140, B.Braun, Germany) and gently transferred to a 50 mL falcon tube (cat: 227261, Greiner bio-one, The Netherlands) equipped with 15 mL of lymphocyte separation culture medium (cat: 17-829E, Lonza, Switzerland). Without braking, the tube is centrifuged at room temperature for 20 minutes at 1200 x g. T cells are collected from density culture medium and washed twice with PBS.

[1141] 2×10E6 T cells / mL were resuspended in FACS buffer and 50 μL were transferred to a round-bottom 96-well plate (cat: 650101, Greiner bio-one, The Netherlands). Starting at 5 μg / mL, 50 μL of 5-fold diluted antibody solution was added and incubated at 4°C for 30 min. The 96-well plate was centrifuged at 300xg for 5 minutes at 4°C and the supernatant was discarded. The cells were washed twice on ice with ice-cold FACS buffer and a 1:200 diluted secondary antibody (anti-IgG Fcγ-PE(fab)′2, cat: 109-116-098, Jackson Immuno Research, UK) was added to 100 μL / well and incubated for 30 minutes and washed twice with FACS buffer. Fluorescence intensity was measured on a FACS Canto and the geometric mean was calculated by FlowJo V10 software. Figures were prepared by GraphPad (V6.04). See Figure 5 .

[1142] Example 9: In vitro cytotoxicity screening of CD3 affinity mutants

[1143] Cytotoxicity of CD3 affinity mutants against solid tumor cell lines (Alamy Blue assay)

[1144] T cells were isolated from donor buffy coats (Sanquin, Amsterdam, The Netherlands) using RosetteSep human T cell enrichment cocktail (Cat: 15021C.1, Stemcell Technologies, France) according to the manufacturer's instructions. NCI-N87 (25.000 cells / well) ( Figure 6A ), SKOV3 (16.000 cells / well) ( Figure 6B ) and MDA-MB-231 (16.000 cells / well) ( Figure 6C ) cells were seeded into flat-bottom 96-well plates (cat: 655180, Greiner-bio-one, The Netherlands) and allowed to adhere at 37°C for 3-5 hours. T cells were added to the tumor cells at the following ratios: NCI-N87 cells:T cells, 1:3; SKOV3 cells:T cells, 1:4; MDA-MB-231 cells:T cells, 1:8. Antibody solution was then added at a 10-fold dilution, and the plates were incubated at 37°C for 2 days. Next, the supernatant was discarded and the adhered cells were washed twice with PBS. 150 μL of a 10% Alamar Blue (cat: DAL1100, Life Technologies, The Netherlands) solution prepared in RPMI-1640 (cat: BE12-115F, Lonza, Switzerland) medium supplemented with 10% donor bovine serum and iron (cat: 10371-029, Life Technologies, The Netherlands) was added to the wells and incubated at 37°C for 3-5 hours. Absorbance was measured using an Envision multilabel plate reader (PerkinElmer, US). Staurosporine (cat: S6942, Sigma-Aldrich, US)-treated cells were set to 100% kill, and untreated cells were set to 0% kill. Viable cells were calculated by subtracting staurosporine-treated cells from all groups, and the percentages were plotted against the untreated group. Graphs were generated using GraphPad (V6.04). See Figure 6.

[1145] Cytotoxicity of CD3 affinity mutants against hematological cell lines (chromium release assay)

[1146] 5x10E6 Daudi cells / mL were incubated in complete medium with 100 μCi of chromium for 1 h at 37°C under shaking conditions. Next, the cells were washed twice in PBS and resuspended in 5 mL of complete cell culture medium (RPMI 1640 with 10% donor bovine serum and iron). 5.000 Daudi cells were seeded into a round-bottom 96-well plate. T cells from donor buffy coats (purchased from Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep Human T Cell Enrichment Mix (Cat: 15021C.1, Stemcell Technologies, France) according to the manufacturer's instructions. T cells were added to the Daudi cells at a ratio of 1:10 (tumor cells: T cells), followed by the addition of two-fold diluted antibody solution. The plates were incubated at 37°C for 24 h. After 24 h, the plates were centrifuged at 300xg for 3 minutes, the supernatant was collected and the radioactivity was measured. See Figure 7 .

[1147] Table 7

[1148]

[1149] Example 10: Tumor efficacy of CD3xHER2 bispecific antibody in a co-transplantation model (human PBMC + NCI-N87 cells) in NOD-SCID mice

[1150] The in vivo anti-tumor efficacy of several CD3xHER2 bispecific antibodies was evaluated in the subcutaneous NCI-N87 co-transplant model (Figure 8). As the CD3 arm of the bispecific antibody, humanized WT CD3 (huCD3-FEAL) and 4 different CD3 affinity variants (N57E, H101K, S110A, Y114M) were used. In all cases, the HER2-targeting arm was the same (Herceptin FEAR).

[1151] BisG1-huCD3-FEALx1014-Herceptin-FEAR

[1152] BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR

[1153] BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR

[1154] BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR

[1155] BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR

[1156] In this model, HLA-A-matched human naive PBMCs, as a source of human T cells, were co-inoculated with NCI-N87 tumor cells at two different dose levels (0.5 and 0.05 mg / kg).

[1157] Mice were sorted into treatment groups (n=4 per treatment group). On day 0, a mixture of HLA-A matched hPBMCs (5x10E6, Sanquin) and NCI-N87 (5x10E6) cells in 200 μL PBS / 0.1% BSA was subcutaneously (sc) inoculated into each female NOD-SCID mouse (NOD.CB-17-Prkdc scid The right flank of 6-11 weeks old (Charles-River) mice was injected with 5 different CD3xHER2 antibodies at two different concentrations (0.5 and 0.05 mg / kg) directly after tumor cell injection. For all bispecific antibodies, 5 different CD3xHER2 antibodies were administered intravenously (150 μL) at two different concentrations (0.5 and 0.05 mg / kg). Tumor volume was measured at least twice a week. Tumor volume (mm) was measured by caliper (PLEXX). 3 ) is calculated as: 0.52×(length)×(width) 2 .

[1158] NCI-N87 cells (ATCC #CRL-5822, gastric carcinoma derived from the stomach) were thawed and cultured in RPMI 1640 (Lonza, BE12-115F) supplemented with 10% donor bovine serum with iron (Gibco, catalog number 10371-029), penicillin / streptomycin, 0.45% glucose (Sigma, G8769), sodium pyruvate (Cambrex, BE13-115E), and 0.075% sodium bicarbonate (Cambrex, BE17-613E). Cells were grown in a CellSTACK culture chamber and harvested at logarithmic phase and counted by trypan blue exclusion.

[1159] For each study, hPBMCs were isolated from buffy coats (Sanquin) by Ficoll density centrifugation from human HLA-A matched donors for NCI-N87 (HLA-A-01,23). The isolated cells were frozen in nitrogen and thawed before use. All cells were washed in PBS / 0.1% BSA, filtered through a cell strainer, and resuspended to a concentration of 50 x 10E6 cells / mL in PBS / 0.1% BSA.

[1160] The results are shown in Figure 8. Figures 8A-B show the mean tumor volume over time after treatment. Figures 8C-D show a dot plot representation of the mean NCI-N87 tumor volume at day 44. Statistical analysis (Mann-Whitney) of tumor volumes on day 44 (the last day all groups remained intact) showed significant tumor growth inhibition (p<0.05) for BisG1-huCD3-FEALx1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR, and BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR, but not for BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR and BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR, compared to control (PBMC) at a dose of 0.05 mg / kg. At a dose of 0.5 mg / kg, BisG1-huCD3-FEALx1014-Herceptin-FEAR and all CD3-arm affinity variants, except BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR, showed significant inhibition of tumor growth (p<0.05) compared to the PBS (PBMC) control group.

[1161] At doses of 0.05 and 0.5 mg / kg, BisG1-huCD3-FEALx1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR, and BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR significantly (p < 0.05) reduced NCI-N87 tumor volume. BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR significantly reduced NCI-N87 tumor volume only at the 0.5 mg / kg dose (p < 0.05). BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR had no effect on NCI-N87 tumor growth at either dose tested.

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[86] Bostrom et al., PLos One. 2011;6(4)e17887. <110> Genmabu Joint Stock Company <120> Humanized or chimeric CD3 antibodies <130> P / 0091-WO <160> 411 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 1 Gly Phe Thr Phe Asn Thr Tyr Ala 1 5 <2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe[[ID=I3]] 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 5 <211> 375 <212> PRT <213> Synthetic Sequence <220> <223> N / A <400> 5 Gly Ala Ala Gly Thr Gly Ala Ala Gly Cys Thr Gly Gly Thr Gly Gly 1 5 10 15 Ala Ala Thr Cys Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala Cys Thr 20 25 30 Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Cys Gly Gly Ala 35 40 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Gly Ala Gly Cys Thr 50 55 60 Gly Thr Gly Cys Cys Gly Cys Cys Ala Gly Cys Gly Gly Cys Thr Thr 65 70 75 80 Cys Ala Cys Cys Thr Thr Cys Ala Ala Cys Ala Cys Cys Thr Ala Cys 85 90 95 Gly Cys Cys Ala Thr Gly Ala Ala Cys Thr Gly Gly Gly Thr Gly Cys 100 105 110 Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys Thr Gly Gly Cys Ala Ala 115 120 125 Ala Gly Gly Cys Cys Thr Gly Gly Ala Ala Thr Gly Gly Gly Thr Gly 130 135 140 Gly Cys Cys Cys Gly Gly Ala Thr Cys Ala Gly Ala Ala Gly Cys Ala 145 150 155 160 Ala Gly Thr Ala Cys Ala Ala Cys Ala Ala Thr Thr Ala Cys Gly Cys 165 170 175 Cys Ala Cys Cys Thr Ala Cys Thr Ala Cys Gly Cys Cys Gly Ala Cys 180 185 190 Ala Gly Cys Gly Thr Gly Ala Ala Gly Gly Ala Cys Cys Gly Gly Thr 195 200 205 Thr Cys Ala Cys Cys Ala Thr Cys Ala Gly Cys Cys Gly Gly Gly Ala 210 215 220 Cys Gly Ala Cys Ala Gly Cys Ala Ala Gly Ala Gly Cys Ala Gly Cys 225 230 235 240 Cys Thr Gly Thr Ala Cys Cys Thr Gly Cys Ala Gly Ala Thr Gly Ala 245 250 255 Ala Cys Ala Ala Cys Cys Thr Gly Ala Ala Ala Ala Cys Cys Gly Ala 260 265 270 Gly Gly Ala Cys Ala Cys Cys Gly Cys Cys Ala Thr Gly Thr Ala Cys 275 280 285 Thr Ala Cys Thr Gly Cys Gly Thr Gly Cys Gly Gly Cys Ala Cys Gly 290 295 300 Gly Cys Ala Ala Cys Thr Thr Cys Gly Gly Cys Ala Ala Cys Ala Gly 305 310 315 320 Cys Thr Ala Thr Gly Thr Gly Thr Cys Thr Thr Gly Gly Thr Thr Thr 325 330 335 Gly Cys Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly 340 345 350 Gly Cys Ala Cys Cys Cys Thr Cys Gly Thr Gly Ala Cys Ala Gly Thr 355 360 365 Gly Thr Cys Thr Ala Gly Cys 370 375 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 6 Thr Gly Ala Val Thr Thr Ser Asn Tyr 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 7 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 8 <211> 109 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 8 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 9 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 9 Cys Ala Gly Gly Cys Cys Gly Thr Cys Gly Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Gly Ala Ala Cys Cys Cys Ala Gly Cys Thr Thr Thr Thr Cys 20 25 30 Cys Gly Thr Gly Thr Cys Thr Cys Cys Thr Gly Gly Cys Gly Gly Cys 35 40 45 Ala Cys Cys Gly Thr Gly Ala Cys Cys Cys Thr Gly Ala Cys Cys Thr 50 55 60 Gly Cys Ala Gly Ala Thr Cys Thr Thr Cys Thr Ala Cys Ala Gly Gly 65 70 75 80 Cys Gly Cys Cys Gly Thr Gly Ala Cys Cys Ala Cys Cys Ala Gly Cys 85 90 95 Ala Ala Cys Thr Ala Cys Gly Cys Cys Ala Ala Cys Thr Gly Gly Gly 100 105 110 Thr Gly Cys Ala Gly Cys Ala Gly Ala Cys Ala Cys Cys Cys Gly Gly 115 120 125 Cys Cys Ala Gly Gly Cys Cys Thr Thr Thr Ala Gly Ala Gly Gly Ala 130 135 140 Cys Thr Gly Ala Thr Cys Gly Gly Cys Gly Gly Cys Ala Cys Cys Ala 145 150 155 160 Ala Cys Ala Ala Gly Ala Gly Gly Gly Cys Ala Cys Cys Thr Gly Gly 165 170 175 Cys Gly Thr Gly Cys Cys Ala Gly Cys Cys Ala Gly Ala Thr Thr Cys 180 185 190 Ala Gly Cys Gly Gly Cys Ala Gly Cys Cys Thr Gly Ala Thr Cys Gly 195 200 205 Gly Ala Gly Ala Thr Ala Ala Gly Gly Cys Cys Gly Cys Cys Cys Thr 210 215 220 Gly Ala Cys Ala Ala Thr Cys Ala Cys Thr Gly Gly Cys Gly Cys Cys 225 230 235 240 Cys Ala Gly Gly Cys Thr Gly Ala Cys Gly Ala Cys Gly Ala Gly Ala 245 250 255 Gly Cys Ala Thr Cys Thr Ala Cys Thr Thr Thr Thr Gly Cys Gly Cys 260 265 270 Cys Cys Thr Gly Thr Gly Gly Thr Ala Cys Ala Gly Cys Ala Ala Cys 275 280 285 Cys Thr Gly Thr Gly Gly Gly Thr Gly Thr Thr Cys Gly Gly Cys Gly 290 295 300 Gly Ala Gly Gly Cys Ala Cys Cys Ala Ala Gly Cys Thr Gly Ala Cys 305 310 315 320 Ala Gly Thr Gly Cys Thr Gly 325 <210> 10 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 10 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 11 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 11 Cys Ala Gly Gly Cys Cys Gly Thr Cys Gly Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Gly Ala Ala Cys Cys Cys Ala Gly Cys Thr Thr Thr Thr Cys 20 25 30 Cys Gly Thr Gly Thr Cys Thr Cys Cys Thr Gly Gly Cys Gly Gly Cys 35 40 45 Ala Cys Cys Gly Thr Gly Ala Cys Cys Cys Thr Gly Ala Cys Cys Thr 50 55 60 Gly Cys Ala Gly Ala Thr Cys Thr Thr Cys Thr Ala Cys Ala Gly Gly 65 70 75 80 Cys Gly Cys Cys Gly Thr Gly Ala Cys Cys Ala Cys Cys Ala Gly Cys 85 90 95 Ala Ala Cys Thr Ala Cys Gly Cys Cys Ala Ala Cys Thr Gly Gly Gly 100 105 110 Thr Gly Cys Ala Gly Cys Ala Gly Ala Ala Gly Cys Cys Cys Gly Gly 115 120 125 Cys Cys Ala Gly Gly Cys Cys Thr Thr Thr Ala Gly Ala Gly Gly Ala 130 135 140 Cys Thr Gly Ala Thr Cys Gly Gly Cys Gly Gly Cys Ala Cys Cys Ala 145 150 155 160 Ala Cys Ala Ala Gly Ala Gly Gly Gly Cys Ala Cys Cys Thr Gly Gly 165 170 175 Cys Gly Thr Gly Cys Cys Ala Gly Cys Cys Ala Gly Ala Thr Thr Cys 180 185 190 Ala Gly Cys Gly Gly Cys Ala Gly Cys Cys Thr Gly Ala Thr Cys Gly 195 200 205 Gly Ala Gly Ala Thr Ala Ala Gly Gly Cys Cys Gly Cys Cys Cys Thr 210 215 220 Gly Ala Cys Ala Ala Thr Cys Ala Cys Thr Gly Gly Cys Gly Cys Cys 225 230 235 240 Cys Ala Gly Gly Cys Thr Gly Ala Cys Gly Ala Cys Gly Ala Gly Ala 245 250 255 Gly Cys Ala Thr Cys Thr Ala Cys Thr Thr Thr Thr Gly Cys Gly Cys 260 265 270 Cys Cys Thr Gly Thr Gly Gly Thr Ala Cys Ala Gly Cys Ala Ala Cys 275 280 285 Cys Thr Gly Thr Gly Gly Gly Thr Gly Thr Thr Cys Gly Gly Cys Gly 290 295 300 Gly Ala Gly Gly Cys Ala Cys Cys Ala Ala Gly Cys Thr Gly Ala Cys 305 310 315 320 Cys Gly Thr Cys Cys Thr Ala 325 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 12 Gly Phe Thr Phe Ala Thr Tyr Ala 1 5 <210> 13 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 13 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 14 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 14 Gly Phe Thr Phe Cys Thr Tyr Ala 1 5 <210> 15 <211> 125 <212> PRT <213> artificial sequence <220> <223> N / A <400> 15 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Cys Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 16 <211> 8 <212> PRT <213> Synthetic sequence <220> <223> N / A <400> 16 Gly Phe Thr Phe Asp Thr Tyr Ala 1 5 <210> 17 <211> 125 <212> PRT <213> Synthetic sequence <220> <223> N / A <400> 17 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 18 Gly Phe Thr Phe Phe Thr Tyr Ala 1 5 <210> 19 <211> 125<0003​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 20 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 20 Gly Phe Thr Phe Gly Thr Tyr Ala 1 5 <210> 21 <211> 125 <212> PRT <213> artificial sequence <220> <223> NOW <400> 21 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 22 Gly Phe Thr Phe His Thr Tyr Ala 1 5 <210> 23 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 23 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 [[ID= 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 24 [[ID=^]]<211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 24 Gly Phe Thr Phe Lys Thr Tyr Ala 1 5 <210> 25 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 25 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 26 Gly Phe Thr Phe Leu Thr Tyr Ala 1 5 <210> 27 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 27 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 28 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 28 Gly Phe Thr Phe Pro Thr Tyr Ala 1 5 <210> 29 <211> 125 <212> PRT <213> artificial sequence <220> <223> N / A <400> 29 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Pro Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 10​​​​​​​​​​​​​​​​​​​Gly Phe Thr Phe Gln Thr Tyr Ala 1 5 <210> 31 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 31 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gln Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 32 Gly Phe Thr Phe Arg Thr Tyr Ala 1 5 <210> 33 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 33 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 34 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 34 Gly Phe Thr Phe Thr Thr Tyr Ala 1 5 <210> 35 <211> 125 <212> PRT <213> artificial sequence <220> <223> NOW <400> 35 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 8 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 36 Gly Phe Thr Phe Val Thr Tyr Ala 1 5 <210> 37 <211> 125 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 37 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Val Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 38 <211> 8 <212> PRT <213> Synthetic Sequence <220> <223> N / A <400> 38 Gly Phe Thr Phe Trp Thr Tyr Ala 1 5 <210> 39 <211> 125 <212> PRT <213> Synthetic Sequence <220> <223> N / A <400> 39 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Trp Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 40 Gly Phe Thr Phe Asn Ala Tyr Ala 1 5 <210> 41 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 41 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ala Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 42 Gly Phe Thr Phe Asn Cys Tyr Ala 1 5 <210> 43 <211> 125 <212> PRT <213> artificial sequence <220> <223> N / A <400> 43 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Cys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 44 <211> 8 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 44 Gly Phe Thr Phe Asn Asp Tyr Ala 1 5 <210> 45 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 45 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 46 Gly Phe Thr Phe Asn Glu Tyr Ala 1 5 <210> 47 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 47 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Glu Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 48 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 48 Gly Phe Thr Phe Asn Phe Tyr Ala 1 5 <210> 49 <211> 125 <212> PRT <213> artificial sequence <220> <223> NOW <400> 49 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Phe Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 50 Gly Phe Thr Phe Asn His Tyr Ala 1 5 <210> 51 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 51 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn His Tyr ` `20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 52 Gly Phe Thr Phe Asn Leu Tyr Ala 1 5 <210> 53 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 53 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Leu Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 54 Gly Phe Thr Phe Asn Met Tyr Ala 1 5 <210> 55 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 55 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 56 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 56 Gly Phe Thr Phe Asn Asn Tyr Ala 1 5 <210> 57 <211> 125 <212> PRT <213> artificial sequence <220> <223> N / A <400> 57 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 58 <211> 8 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 58 Gly Phe Thr Phe Asn Pro Tyr Ala 1 5 <210> 59 <211> 125 <212> PRT <213> Synthetic sequence <220> <223> N / A <400> 59 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Pro Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 60 <211> 8 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 60 Gly Phe Thr Phe Asn Gln Tyr Ala 1 5 <210> 61 <211> 125 <212> PRT <213> Artificial sequence <220> <223> N / A <400> 61 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Gln Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 62 <211> 8 <212> PRT <213> artificial sequence <220> <223> NOW <400> 62 Gly Phe Thr Phe Asn Trp Tyr Ala 1 5 <210> 63 <211> 125 <212> PRT <213> artificial sequence <220> <223> NOW <400> 63 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Trp Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 64 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 64 Gly Phe Thr Phe Asn Tyr Tyr Ala 1 5 <210> 65 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 65 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Tyr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 66 Gly Phe Thr Phe Asn Thr Ala Ala 1 5 <210> 67 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 67 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Ala 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 68 <211> 8 <212> PRT <213> Synthetic Sequence <220> <223> N / A <400> 68 Gly Phe Thr Phe Asn Thr Cys Ala 1 5 <210> 69 <211> 125 <212> PRT <213> Synthetic Sequence <220> <223> N / A <400> 69 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Cys 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser...

Claims

1. A humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1, 106, and 3, respectively, and wherein the VL region comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 412, and 7, respectively.

2. The antibody of claim 1, wherein the VH region has the VH sequence shown in SEQ ID NO:

107.

3. The antibody of claim 1 or 2, wherein the VL region has the VL sequence shown in SEQ ID NO:

8.

4. The antibody of claim 1 or 2, wherein the VL region has the VL sequence shown in SEQ ID NO:

10.

5. The antibody of claim 1 or 2, wherein the human CD3 is human CD3ε as shown in SEQ ID NO:

399.

6. The antibody of claim 1 or 2, wherein the binding affinity of the antibody to the human CD3ε peptide having SEQ ID NO: 402 as determined by biolayer interferometry corresponds to 1.0×10 -7 to 9.9x10 -7 M's K D value.

7. The antibody of claim 1 or 2, wherein the antibody is a humanized antibody.

8. The antibody of claim 1 or 2, wherein the antibody is a full-length antibody.

9. The antibody of claim 1 or 2, wherein the antibody has an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

10. The antibody of claim 1 or 2, wherein the antibody is monovalent, bivalent or multivalent.

11. The antibody of claim 1 or 2, wherein the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

12. The antibody of claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are F and E; or A and A, respectively.

13. The antibody of claim 12, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are F and E, respectively.

14. The antibody of claim 12, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are A and A, respectively.

15. The antibody of claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively.

16. The antibody of claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain are F, E, and A, respectively.

17. The antibody of claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain are A, A, and A, respectively.

18. The antibody of claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 of a human IgG1 heavy chain are F, E, A, Q, and S, respectively.

19. The antibody of claim 1, wherein the antibody has reduced binding affinity for human CD3 compared to a reference antibody having the VH CDR sequences shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO:

3.

20. A bispecific antibody comprising a first binding region of the antibody of any one of claims 1 to 19, and a second binding region that binds to a different target than the first binding region.

21. The bispecific antibody of claim 20, wherein said antibody comprises a first and a second heavy chain.

22. The bispecific antibody of claim 21, wherein the first and second heavy chains each comprise at least a hinge region, a CH2 region, and a CH3 region, wherein in the first heavy chain at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain has been substituted, and in the second heavy chain at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain has been substituted, and wherein the first and second heavy chains are not substituted at the same position.

23. The bispecific antibody of claim 22, wherein (i) the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the second heavy chain is R; or (ii) the amino acid at the position corresponding to F409 of the human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to K405 of the human IgG1 heavy chain in the second heavy chain is L.

24. The bispecific antibody of any one of claims 20-23, wherein the first binding region is the binding region of the antibody of any one of claims 1-11, and the second binding region binds to a different target than the first binding region.

25. A method of reducing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, and wherein the VL region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 412, and 7, the method comprising introducing an N57E mutation into the CDR2 sequence of the VH region of the reference antibody, wherein positions are numbered according to the reference sequence of SEQ ID NO:

4.

26. The method of claim 25, wherein the antibody with reduced binding affinity comprises a binding region comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 106, and 3, and the VL region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 412, and 7.

27. The method of claim 25 or 26, wherein the binding affinity of the antibody to the human CD3ε peptide having SEQ ID NO: 402 as determined by biolayer interferometry corresponds to 1.0 x 10 -7 to 9.9x10 -7 M's K D value.

28. The method of claim 25 or 26, wherein the human CD3 is expressed on T cells.

29. The method of claim 25 or 26, wherein the human CD3 is in isolated form.

30. The method of claim 25 or 26, wherein the human CD3 is isolated human CD3 epsilon-peptide.

31. A nucleic acid construct encoding the antibody of any one of claims 1-24.

32. An expression vector comprising (i) a nucleic acid sequence encoding the heavy chain sequence of the humanized or chimeric antibody of claim 1 or 2; and (ii) a nucleic acid sequence encoding the light chain sequence of the humanized or chimeric antibody of claim 3 or 4.

33. A set of expression vectors comprising (i) an expression vector comprising a nucleic acid sequence encoding the heavy chain sequence of the humanized or chimeric antibody of claim 1 or 2; and (ii) an expression vector comprising a nucleic acid sequence encoding the light chain sequence of the humanized or chimeric antibody of claim 3 or 4.

34. A host cell comprising the expression vector of claim 32 or a set of expression vectors of claim 33.

35. The host cell of claim 34, wherein the host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.

36. A composition comprising the antibody of any one of claims 1-19 or the bispecific antibody of any one of claims 20-24.

37. A pharmaceutical composition comprising the antibody of any one of claims 1 to 19 or the bispecific antibody of any one of claims 20 to 24 and a pharmaceutically acceptable carrier.

38. Use of the antibody of any one of claims 1 to 19 or the bispecific antibody of any one of claims 20 to 24 for the preparation of a medicament for treating a disease characterized by involvement or accumulation of cells expressing CD3, the disease being selected from breast cancer, gastric cancer, and ovarian cancer.

39. Use of the antibody of any one of claims 1 to 19, the composition of claim 36, or the pharmaceutical composition of claim 37 for the preparation of a medicament for diagnosing a disease characterized by involvement or accumulation of cells expressing CD3, the disease being selected from breast cancer, gastric cancer, and ovarian cancer.

40. The use of claim 39, wherein the antibody is labeled with a detectable agent.

41. A method for producing an antibody according to any one of claims 1 to 19, comprising the steps of a) cultivating the host cell according to any one of claims 34-35; and b) purifying the antibody from the culture medium.

42. A diagnostic composition comprising the antibody of any one of claims 1 to 19 or the bispecific antibody of any one of claims 20 to 24.

43. Use of the antibody of any one of claims 1 to 19 or the bispecific antibody of any one of claims 20 to 24 for the preparation of a kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample.

44. A kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising i) the antibody of any one of claims 1 to 19 or the bispecific antibody of any one of claims 20 to 24; and ii) instructions for use of the kit.

Images