Humanized or chimeric CD3 antibodies

Abstract

The present invention relates to humanized or chimeric antibodies that bind CD3. The invention further relates to bispecific antibodies, compositions, pharmaceutical compositions, uses of said antibodies in the treatment of disease and methods of treatment.

Description

[0001] This application is a continuation divisional application of the invention application filed on July 14, 2016, with Chinese national application number 201680054638.0 and invention title "Humanized or Chimeric CD3 Antibody" (application number 202210585379.1). Invention Field

[0002] This invention relates to humanized or chimeric antibodies that bind to human CD3, compositions comprising said humanized or chimeric antibodies, and the use of said humanized or chimeric antibodies in the treatment of diseases. Background of the Invention

[0003] Differentiation cluster 3 (CD3) has been known for many years and is therefore of interest in many respects. In particular, antibodies against CD3 or the T-cell receptor complex (of which CD3 is a component) are known. In vitro characterization of antibodies against recombinant chimeric CD3 allotype variants and various humanized OKT3 effector functional variants has been described [1].

[0004] CD3 antibodies, such as muromonab-CD3, have been widely used to treat acute allogeneic transplant rejection. In addition, in the absence of sustained immunosuppressive therapy, treatment with the anti-CD3 monoclonal antibody hOKT3γ1 (Ala-Ala) for at least 2 years after the onset of type 1 diabetes resulted in improved C-peptide response and clinical parameters [2].

[0005] A promising approach to improving targeted antibody therapy is through the delivery of cytotoxic cells specifically targeting cancer cells expressing antigens. The concept of using T cells to effectively kill tumor cells has been described[3]. However, initial clinical studies were rather disappointing, mainly due to low efficacy, severe side effects (cytokine bursts), and the immunogenicity of bispecific antibodies[4]. Advances in the design and application of bispecific antibodies have partially overcome the initial hurdle of cytokine bursts and improved clinical efficacy without dose-limiting toxicity[5].

[0006] For example, certain bispecific antibodies that target antigens on tumor cells with one arm and CD3 on, for example, 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 antibody Fc region is potentially formed, leading to the killing of tumor cells [4]. Caputsuzumab is composed of a mouse IgG2a / rat IgG2b heavy chain heterodimer and has been found to be successfully used to treat cancer-associated ascites after intraperitoneal application [6]. However, the mouse / rat heterozygote is immunogenic [7] and cannot be used for long-term treatment in humans. Frequent treatment-related adverse events are attributed to symptoms associated with caputsuzumab-induced cytokine release (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) [8]-[9], which involve potent polyclonal T cell activation caused by caputsuzumab due to its active Fc fragment. Another antibody is ertumaxomab (HER2xCD3), which induces cytotoxicity in cell lines expressing HER2. Ertumaxomab is currently in phase II clinical development for metastatic breast cancer.

[10] -

[11]

[0007] The efficacy of CD3 bispecific antibodies and other forms of CD3-based bispecific antibodies 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. Some CD3 bispecific antibodies exhibit high potency when CD3 affinity is low (EpCamxCD3-Bortoletto 2002 PMID12385030, MT103 / Blinatumomab vs TandAb-Molhoj 2007 PMID 17083975), while others show high potency using high CD3 affinity (Reusch 2015, Mabs, PMID 25875246). High CD3 affinity is required in certain situations, such as when bispecific antibodies containing an anti-CD3 targeting arm and a second arm targeting a selected tumor-associated antigen are administered to ex vivo expanded activated T cells from a patient. In the latter case, when the product is infused back into the patient to mediate the cytolysis of tumor cells, CD3 affinity should be high to maintain interaction with the expanded T cells (Reusch 2006 Clin CancerRes PMID 16397041). However, high-affinity anti-CD3 antibodies are much less potent in TCR triggering at low copy numbers compared to low-affinity ligands, as they exhibit approximately 1:1 stoichiometry and linear dose-response curves, indicating a single-cycle rather than a sequential triggering pattern of T cell responses (Viola 1996 Science, PMID 8658175). In other words, the low affinity of the CD3 arm allows T cells to move flexibly from one target and / or target cell to another (Hoffman 2005, PMID: 15688411).

[0008] Low CD3 affinity can potentially prevent biased targeting of bispecific antibodies to T cells (due to first encounter in circulation) and thus improve biodistribution and minimize interference with normal T-cell immune responses. The desired CD3 affinity can be tailored to maximize product efficacy based on the target and target copy number of the second arm, indication, and / or route of administration. A set of CD3 variants covering a range of CD3 affinities can be essential for antibody products to meet these specific customized needs.

[0009] CD3 antibodies that cross-react with CD3 in cynomolgus monkeys and / or rhesus monkeys have been described

[12] -

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

[0010] The objective of this invention is to provide humanized or chimeric CD3 antibodies with optimized affinity for CD3. Therefore, an object of this invention is to provide humanized or chimeric CD3 antibodies optimized compared to reference antibodies, such as those specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. Thus, such antibodies may have reduced or increased affinity for CD3 compared to reference antibodies specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. Another object of this invention is to provide antibodies with lower binding affinity for CD3 compared to antibodies specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. The inventors have found that antibodies with reduced binding affinity for the CD3 peptide shown in SEQ ID NO:402 compared to reference antibodies having the VH region sequence shown 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 with reduced binding affinity for CD3 compared to the reference antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence 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 with higher binding affinity for CD3 compared to the antibodies specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8.

[0011] In one aspect, the present invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region contains a mutation in one of 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, said mutation being located at one of the following positions: T31M, T31P, N57, H101, G105, S110, and Y114, wherein said position is numbered according to the reference sequence of SEQ ID NO:4. The amino acids in SEQ ID NO:4 are numbered according to a direct numerical numbering scheme from the first amino acid to the number 125 in the direction from the N-terminus to the C-terminus. Figure 2 The numerical designation corresponding to the position of SEQ ID NO:4 is shown. Furthermore, the VH CDR area has been annotated according to the IMGT definition.

[0012] In one embodiment of the invention, the antibody has a decreased or increased 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.

[0013] In some embodiments of the present invention, antibodies with reduced binding affinity to human CD3 molecules, such as CD3 peptides, such as SEQ ID NO:402, compared to reference antibodies, can have the same cytolytic activity against target cells as reference antibodies.

[0014] In one embodiment of the invention, the antibody contains a mutation at the N57 position corresponding to SEQ ID NO:4. In one embodiment, the mutation is N57E.

[0015] In one embodiment of the invention, the antibody contains a mutation at the position corresponding to H101G in SEQ ID NO:4. In one embodiment, the mutation is either H101G or H101N.

[0016] In one embodiment of the invention, the antibody contains a mutation at the position corresponding to G105 in SEQ ID NO:4. In one embodiment, the mutation is G105P.

[0017] In one embodiment of the invention, the antibody contains a mutation at the position corresponding to Y114 in SEQ ID NO:4. In one embodiment, the mutation is Y114M, Y114R, or Y114V.

[0018] In one embodiment, the present invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises a CDR1, CDR2, and CDR3 region having a CDR sequence selected from:

[0019] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:12,2,3;

[0020] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:14,2,3;

[0021] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:16,2,3;

[0022] d) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:18,2,3;

[0023] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:20,2,3;

[0024] f) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:22,2,3;

[0025] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:24,2,3;

[0026] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:26,2,3;

[0027] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:28,2,3;

[0028] j) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:30,2,3;

[0029] k) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:32,2,3;

[0030] l) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:34,2,3;

[0031] m) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:36,2,3;

[0032] n) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:38,2,3;

[0033] o) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:40,2,3;

[0034] p) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:42,2,3;

[0035] q) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:44,2,3;

[0036] r) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:46,2,3;

[0037] s) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:48,2,3;

[0038] t) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:50,2,3;

[0039] u) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:52,2,3;

[0040] v) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3;

[0041] w) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:56,2,3;

[0042] x) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3;

[0043] y) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:60,2,3;

[0044] z) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:62,2,3;

[0045] aa) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:64,2,3;

[0046] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:66,2,3;

[0047] cc) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:68,2,3;

[0048] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:70,2,3;

[0049] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:72,2,3 are as follows:

[0050] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:74,2,3 are shown in the image.

[0051] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:76,2,3;

[0052] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:78,2,3;

[0053] ii) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:80,2,3;

[0054] jj) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:82,2,3;

[0055] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:84,2,3;

[0056] ll) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:86,2,3;

[0057] The CDR1, CDR2 and (mm) shown in SEQ ID NO:88,2,3 are shown in the image.

[0058] CDR3 sequence;

[0059] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:90,2,3;

[0060] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:92,2,3 are as follows;

[0061] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:94,2,3 (pp)

[0062] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 96,2,3 are as follows;

[0063] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:98,2,3;

[0064] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,100,3;

[0065] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,102,3 are tt.

[0066] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,104,3;

[0067] vv) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3;

[0068] ww)SEQ ID NO:1,108,3 shows CDR1, CDR2 and

[0069] CDR3 sequence;

[0070] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,110,3 are xx).

[0071] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,112,3 are as follows:

[0072] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,114,3;

[0073] aaa) SEQ ID NO:1,116,3 shows CDR1, CDR2 and

[0074] CDR3 sequence;

[0075] bbb) SEQ ID NO:1,118,3 shows CDR1, CDR2 and

[0076] CDR3 sequence;

[0077] ccc) SEQ ID NO:1,120,3 shows CDR1, CDR2 and

[0078] CDR3 sequence;

[0079] ddd) SEQ ID NO:1,122,3 shows CDR1, CDR2 and

[0080] CDR3 sequence;

[0081] The CDR1, CDR2 and CDR2 shown in SEQ ID NO:1,124,3 are eee)

[0082] CDR3 sequence;

[0083] fff)The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,126,3;

[0084] ggg) SEQ ID NO:1,128,3 shows CDR1, CDR2 and

[0085] CDR3 sequence;

[0086] hhh) SEQ ID NO:1,130,3 shows CDR1, CDR2 and

[0087] CDR3 sequence;

[0088] iii) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,132,3;

[0089] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,134,3;

[0090] kkk)SEQ ID NO:1,136,3 shows CDR1, CDR2 and

[0091] CDR3 sequence;

[0092] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,138,3;

[0093] The CDR1, CDR2 and CDR3 shown in SEQ ID NO:1,140,3 are mmm)

[0094] CDR3 sequence;

[0095] nnn) SEQ ID NO:1,142,3 shows CDR1, CDR2 and

[0096] CDR3 sequence;

[0097] ooo)SEQ ID NO:1,144,3 shows CDR1, CDR2 and

[0098] CDR3 sequence;

[0099] ppp) SEQ ID NO:1,146,3 shows CDR1, CDR2 and

[0100] CDR3 sequence;

[0101] The CDR1, CDR2 and qqq) SEQ ID NO:1,148,3 shown are CDR1, CDR2 and

[0102] CDR3 sequence;

[0103] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,150,3;

[0104] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,152,3;

[0105] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,154,3;

[0106] uuu)SEQ ID NO:1,156,3 shows CDR1, CDR2 and

[0107] CDR3 sequence;

[0108] The CDR1, CDR2 and CDR3 shown in SEQ ID NO:1,158,3 are vvv)

[0109] CDR3 sequence;

[0110] www) SEQ ID NO:1,160,3 shows CDR1, CDR2 and

[0111] CDR3 sequence;

[0112] The CDR1, CDR2 and SEQ ID NO:1,162,3 shown in xxx)

[0113] CDR3 sequence;

[0114] yyy)SEQ ID NO:1,164,3 shows CDR1, CDR2 and

[0115] CDR3 sequence;

[0116] zzz) SEQ ID NO:1,166,3 shows CDR1, CDR2 and

[0117] CDR3 sequence;

[0118] aaaa) SEQ ID NO:1,168,3 shows CDR1, CDR2 and

[0119] CDR3 sequence;

[0120] The CDR1, CDR2 and bbbb) SEQ ID NO:1,2,170 shown are CDR1, CDR2 and

[0121] CDR3 sequence;

[0122] CDR1, CDR2 and cccc)SEQ ID NO:1,2,172 shown

[0123] CDR3 sequence;

[0124] dddd)SEQ ID NO:1,2,174 shows CDR1, CDR2 and

[0125] CDR3 sequence;

[0126] The CDR1, CDR2 and eeee) SEQ ID NO:1,2,176 shown

[0127] CDR3 sequence;

[0128] The CDR1, CDR2 and SEQ ID NO:1,2,178 shown in ffff)

[0129] CDR3 sequence;

[0130] gggg) SEQ ID NO:1,2,180 shows CDR1, CDR2 and

[0131] CDR3 sequence;

[0132] hhhh) SEQ ID NO:1,2,182 shows CDR1, CDR2 and

[0133] CDR3 sequence;

[0134] iiii) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184;

[0135] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,186;

[0136] kkkk)SEQ ID NO:1,2,188 shows CDR1, CDR2 and

[0137] CDR3 sequence;

[0138] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,190;

[0139] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,192 (mmmm)

[0140] nnnn) SEQ ID NO:1,2,194 shows CDR1, CDR2 and

[0141] CDR3 sequence;

[0142] oooo) SEQ ID NO:1,2,196 shows CDR1, CDR2 and

[0143] CDR3 sequence;

[0144] CDR1, CDR2 and pppp) SEQ ID NO:1,2,198 shown

[0145] CDR3 sequence;

[0146] The CDR1, CDR2 and qqqq) SEQ ID NO:1,2,200 shown are CDR1, CDR2 and

[0147] CDR3 sequence;

[0148] rrrr) SEQ ID NO:1,2,202 shows CDR1, CDR2 and

[0149] CDR3 sequence;

[0150] The CDR1, CDR2 and SEQ ID NO:1,2,204 shown in ssss)

[0151] CDR3 sequence;

[0152] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,206;

[0153] uuuu)SEQ ID NO:1,2,208 shows CDR1, CDR2 and

[0154] CDR3 sequence;

[0155] The CDR1, CDR2 and CDR2 shown in SEQ ID NO:1,2,210 are vvvv)

[0156] CDR3 sequence;

[0157] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,212 are wwww)

[0158] The CDR1, CDR2 and (xxxx) SEQ ID NO:1,2,214 shown are CDR1, CDR2 and

[0159] CDR3 sequence;

[0160] yyyy)SEQ ID NO:1,2,216 shows CDR1, CDR2 and

[0161] CDR3 sequence;

[0162] zzzz) SEQ ID NO:1,2,218 shows CDR1, CDR2 and

[0163] CDR3 sequence;

[0164] aaaaa)SEQ ID NO:1,2,220 shows CDR1, CDR2 and

[0165] CDR3 sequence;

[0166] bbbbb)SEQ ID NO:1,2,222 shows CDR1, CDR2 and

[0167] CDR3 sequence;

[0168] ccccc)SEQ ID NO:1,2,224 shows CDR1, CDR2 and

[0169] CDR3 sequence;

[0170] ddddd)SEQ ID NO:1,2,226 shows CDR1, CDR2 and

[0171] CDR3 sequence;

[0172] eeeee) SEQ ID NO:1,2,228 shows CDR1, CDR2 and

[0173] CDR3 sequence;

[0174] fffff)SEQ ID NO:1,2,230 shows CDR1, CDR2 and

[0175] CDR3 sequence;

[0176] ggggg)SEQ ID NO:1,2,232 shows CDR1, CDR2 and

[0177] CDR3 sequence;

[0178] hhhhh) SEQ ID NO:1,2,234 shows CDR1, CDR2 and

[0179] CDR3 sequence;

[0180] iiiii) CDR1, CDR2 and shown in SEQ ID NO:1,2,236

[0181] CDR3 sequence;

[0182] jjjjj)SEQ ID NO:1,2,238 shows CDR1, CDR2 and

[0183] CDR3 sequence;

[0184] The CDR1, CDR2 and CDR2 shown in SEQ ID NO:1,2,240 are kkkkk)

[0185] CDR3 sequence;

[0186] The CDR1, CDR2 and CDR2 shown in SEQ ID NO:1,2,242 are...

[0187] CDR3 sequence;

[0188] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244 (mmmmm)

[0189] The CDR1, CDR2 and SEQ ID NO:1,2,246 shown are nnnnn)

[0190] CDR3 sequence;

[0191] ooooo) SEQ ID NO:1,2,248 shows CDR1, CDR2 and

[0192] CDR3 sequence;

[0193] The CDR1, CDR2 and CDR2 shown in SEQ ID NO:1,2,250 (pppppp)

[0194] CDR3 sequence;

[0195] The CDR1, CDR2 and SEQ ID NO:1,2,252 shown in qqqqq)

[0196] CDR3 sequence;

[0197] (rrrrr) SEQ ID NO:1,2,254 shows CDR1, CDR2 and

[0198] CDR3 sequence;

[0199] The CDR1, CDR2 and SEQ ID NO:1,2,256 shown in ssssss)

[0200] CDR3 sequence;

[0201] ttttt) SEQ ID NO:1,2,258 shows CDR1, CDR2 and

[0202] CDR3 sequence;

[0203] uuuuu) SEQ ID NO:1,2,260 shows CDR1, CDR2 and

[0204] CDR3 sequence;

[0205] The CDR1, CDR2 and CDR2 shown in SEQ ID NO:1,2,262 are vvvvv)

[0206] CDR3 sequence;

[0207] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,264;

[0208] The CDR1, CDR2 and SEQ ID NO:1,2,266 shown in xxxxx)

[0209] CDR3 sequence;

[0210] yyyyy)SEQ ID NO:1,2,268 shows CDR1, CDR2 and

[0211] CDR3 sequence;

[0212] zzzzz) SEQ ID NO:1,2,270 shows CDR1, CDR2 and

[0213] CDR3 sequence;

[0214] aaaaaa)SEQ ID NO:1,2,272 shows CDR1, CDR2 and

[0215] CDR3 sequence;

[0216] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,274;

[0217] CDR1, CDR2 and cccccc)SEQ ID NO:1,2,276 shown

[0218] CDR3 sequence;

[0219] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,278;

[0220] eeeeee) SEQ ID NO:1,2,280 shows CDR1, CDR2 and

[0221] CDR3 sequence;

[0222] The CDR1, CDR2 and SEQ ID NO:1,2,282 shown in ffffff)SEQ ID NO:1,2,282

[0223] CDR3 sequence;

[0224] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284;

[0225] (hhhhhh) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,286;

[0226] iiiiii) CDR1, CDR2 and shown in SEQ ID NO:1,2,288

[0227] CDR3 sequence;

[0228] jjjjjj)SEQ ID NO:1,2,290 shows CDR1, CDR2 and

[0229] CDR3 sequence;

[0230] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,292 (kkkkkk) are kkkkkkk)

[0231] llllll)SEQ ID NO:1,2,294 shows CDR1, CDR2 and

[0232] CDR3 sequence;

[0233] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,296 (mmmmmm) are mmmmmm)

[0234] nnnnnn)SEQ ID NO:1,2,298 shows CDR1, CDR2 and

[0235] CDR3 sequence; and

[0236] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1, 2 and 300 (oooooo)

[0237] That is, in a first aspect of the invention, the inventors have found that humanized or chimeric antibodies to the CD3 peptide SEQ ID NO:402 have optimized binding affinity to the reference antibody (e.g., the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8) compared to a reference antibody. As shown in Example 7, the reference antibody specified by the VL sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8 has a binding affinity of 1.5 x 10⁻⁶ for the CD3 peptide of SEQ ID NO:402. -8 The binding affinity of M. In some embodiments of the invention, the antibody has a binding affinity of less than 1.5 × 10⁻⁶ for the CD3 peptide of SEQ ID NO:402. -8 M, for example, 1.6 × 10 -8 M to 9.9×10 -8 The binding affinity of M, or for example, 1.0 × 10⁻⁶. -7 Up to 9.9×10 -7 The binding affinity of M was determined by the biolayer interferometry method described in Table 6 of Example 7. In some embodiments of the invention, the binding affinity of the antibody to the CD3 peptide of SEQ ID NO:402 is greater than 1.5 × 10⁻⁶. -8 M, for example, 1.4 × 10 -8 Up to 1.0×10 -8M, for example, 9.9 × 10 -9 Up to 1×10 -9 M or, for example, 9.9 × 10 -9 Up to 1×10 -9 M. Binding affinity corresponds to K D value.

[0238] In one aspect of the invention, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises a CDR1, CDR2, and CDR3 region having a CDR sequence selected from:

[0239] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3[T31M];

[0240] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0241] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0242] d) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,176[H101G];

[0243] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184[H101N];

[0244] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0245] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236[S110A];

[0246] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244[S110G];

[0247] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284[Y114M];

[0248] j) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,292[Y114R];

[0249] k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,298[Y114V]; and

[0250] l) CDR1, CDR2, and CDR3 sequences that have at least 90% or at least 95% amino acid sequence identity with any one of the three CDR sequences shown in a) to k), provided that CDR1, CDR2, and CDR3 sequences do not have the sequences shown in SEQ ID NO: 1, 2, and 3.

[0251] On the other hand, the present invention relates to humanized or chimeric antibodies, 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.

[0252] On the other hand, 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 containing a heavy chain variable region (VH) region, wherein the VH region comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1, 2, and 3, the method comprising introducing a mutation in one of the three CDR sequences of the reference antibody, the mutation being selected from mutations at one of the following positions: T31M, T31P, N57, H101, S110, S110, and Y114, wherein the positions are numbered according to the reference sequence of SEQ ID NO:4.

[0253] In one embodiment of the invention, the method includes introducing a mutation in the VH region CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the invention, the method includes introducing a mutation in the VH region CDR2 region corresponding to N57E. In yet another embodiment of the invention, the method includes introducing a mutation in the VH region CDR3 region selected from H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V.

[0254] In one implementation, CD3 is human CD3ε.

[0255] In another aspect, the present invention relates to bispecific antibodies comprising a first binding region of the antibody of the present invention and a second binding region that binds to a different target than the first antigen binding region.

[0256] In another aspect, the present invention relates to nucleic acid constructs encoding one or more amino acid sequences of the present invention.

[0257] 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 of (i) and (ii).

[0258] In another respect, the present invention relates to host cells containing the expression vector of the present invention.

[0259] In another respect, the present invention relates to compositions comprising the antibodies or bispecific antibodies of the present invention.

[0260] In another respect, the present invention relates to pharmaceutical compositions comprising the antibody or bispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0261] In another aspect, the present invention relates to the antibody or bispecific antibody, composition or pharmaceutical composition of the present invention, which is used as a drug.

[0262] In another aspect, the present invention relates to the antibody or bispecific antibody, composition or pharmaceutical composition of the present invention for the treatment of diseases.

[0263] In another aspect, the present invention relates to methods for treating diseases, including administering the antibodies or bispecific antibodies, compositions or pharmaceutical compositions of the present invention to a subject in need.

[0264] On the other hand, the present invention relates to a method of administering antibodies or bispecific antibodies, wherein the antibodies or bispecific antibodies are administered subcutaneously or locally.

[0265] In one aspect, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, comprising administering a subject a humanized or chimeric antibody, composition, or pharmaceutical composition of the present invention, optionally wherein the humanized or chimeric antibody is labeled with a detectable reagent.

[0266] In another aspect, the present invention relates to a method for generating 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.

[0267] In another aspect, the present invention relates to diagnostic compositions comprising antibodies or bispecific antibodies according to any embodiment disclosed herein.

[0268] In one embodiment, the diagnostic composition is a companion diagnostic used to screen and select patients who will benefit from bispecific antibody therapy.

[0269] In another aspect, the present invention relates to a method for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising the steps of: a) contacting the sample with an antibody or bispecific antibody of the present invention under conditions that allow the formation of a complex between the antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

[0270] In another respect, the present invention relates to a kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising i) the antibody or bispecific antibody of the present invention, and ii) instructions for use of the kit.

[0271] In another aspect, the present invention relates to anti-individual genotype antibodies or anti-individual genotype antibody pairs that combine with the antibodies of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0272] Figure 1 Heatmap showing the binding ratio of the mutant to the wtUniTE-huCD3-H1L1-T41K molecule. A ratio higher than 1 indicates a stronger binding than wt, while a ratio lower than 1 indicates a weaker binding than wt. Binding was measured on Freestyle 293-F cells transfected with CD3 / TCR-LC13.

[0273] 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.

[0274] Figure 3 T cell binding profiles of selected VH affinity variants of the humanized CD3 (UniTE-huCD3-H1L1-T41K) antibody, as determined by flow cytometry. The described affinity variants cover a broad range of T cell binding capabilities between wild-type and undetectable responses.

[0275] Figure 4 T cell binding curves of selected VH affinity variants of the humanized CD3 (UniTE-huCD3-H1L1-T41K) antibody, as determined by flow cytometry, showed very low, undetectable T cell binding.

[0276] Figure 5 T cell binding profiles of selected VH affinity variants of the humanized CD3 (BisG1-huCD3-H1L1-X-FEAL / 1014-Herceptin-FEAR) antibody, as determined by flow cytometry. The described affinity variants cover a broad range of T cell binding capabilities between wild-type and undetectable responses.

[0277] Figure 6: Cytotoxicity of CD3 affinity variants to solid tumor cell lines as measured by the Almar 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 tested affinity variants depicted cover a broad spectrum of cytotoxicity between wild-type responses and no observed cytotoxicity in any of the tested tumor cell lines.

[0278] Figure 7 Cytotoxicity of CD3 affinity variants to hematologic (Daudi) cell lines, measured by chromium release assay. T cell:Daudi cell ratio = 10:1, incubation for 24 hours, 1 donor. The tested affinity variants depicted cover a wide range of cytotoxicity between wild-type responses and cytotoxicity not observed in the tested tumor cell lines.

[0279] Figure 8: Cytotoxicity of CD3xHER2 bispecific antibody in the NCI-N87 human PBMC co-implantation model in NOD-SCID mice. HLA-A-matched unstimulated human PBMCs were co-inoculated with NCI-N87 tumor cells in NOD-SCID mice as a source of human T cells at two different dose levels of CD3 affinity antibody (0.5 and 0.05 mg / kg). Humanized WTCD3 (huCD3) and four different CD3 affinity variants (N57E, H101K, S110A, Y114M) were tested. (A) Mean tumor volume over time after treatment with 0.05 mg / kg antibody (n=4 per group). (B) Mean tumor volume over time after treatment with 0.5 mg / kg antibody (n=4 per group). (C) Mean tumor volume on day 44 after treatment with 0.05 mg / kg antibody on day 0 (n=4 per group). (D) Mean tumor volume on day 44 after treatment with 0.5 mg / kg antibody on day 0 (n=4 per group). Data for C and D have been statistically analyzed. Invention Details

[0280] On one hand, this invention relates to humanized or chimeric antibodies that bind to human CD3 and have optimized affinity for CD3. Therefore, an object of the invention is to provide humanized or chimeric CD3 antibodies that are optimized compared to reference antibodies, such as those specified by VH sequence SEQ ID NO:4 and VL sequence SEQ ID NO:8. Another object of the invention is to provide antibodies that have optimized in vivo efficacy compared to reference antibodies, such as those specified by VH sequence SEQ ID NO:4 and VL sequence SEQ ID NO:8. Another object of the invention is to provide antibodies with lower binding affinity for CD3 compared to those specified by VH sequence SEQ ID NO:4 and VL sequence SEQ ID NO:8. Another object of the invention is to provide antibodies with higher binding affinity for CD3 compared to those specified by VH sequence SEQ ID NO:4 and VL sequence SEQ ID NO:8.

[0281] In one aspect, the present invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region contains a mutation in one of 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, said mutation being located at one of the following positions: T31M, T31P, N57, H101, G105, S110, and Y114, wherein said position is numbered according to the reference sequence of SEQ ID NO:4. The amino acids in SEQ ID NO:4 are numbered according to a direct numerical numbering scheme from the first amino acid to the number 125 in the direction from the N-terminus to the C-terminus. Figure 2 The numerical designation corresponding to the position of SEQ ID NO:4 is shown. Furthermore, the CDR area has been annotated according to the IMGT definition.

[0282] In one embodiment of the invention, the antibody has a decreased or increased 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.

[0283] In some embodiments of the present invention, antibodies with reduced binding affinity to CD3 molecules, such as CD3 peptides, such as SEQ ID NO:402, may have the same cytolytic activity against target cells as reference antibodies.

[0284] In one embodiment of the invention, the antibody comprises a T31M or T31P mutation. Position T31 is specified according to SEQ ID NO:4.

[0285] In one embodiment of the invention, the antibody contains a mutation at position N57. Position N57 is defined in SEQ ID NO:4. In one embodiment, the mutation is N57E.

[0286] In one embodiment of the invention, the antibody contains a mutation at position H101. Position H101 is defined in SEQ ID NO:4. In one embodiment, the mutation is H101G or H101N.

[0287] In one embodiment of the invention, the antibody contains a mutation at position G105. Position G105 is defined in SEQ ID NO:4. In one embodiment, the mutation is G105P.

[0288] In one embodiment of the invention, the antibody contains a mutation at position Y114. Position Y114 is defined in SEQ ID NO:4. In one embodiment, the mutation is Y114M, Y114R, or Y114V.

[0289] As shown in Example 7, the reference antibody specified by SEQ ID NO:4 and VL sequence SEQ ID NO:8 has a KD value corresponding to 1.5 × 10⁻⁶. -8 The binding affinity of M for the CD3 peptide of SEQ ID NO:402.

[0290] 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 × 10⁻⁶. -8 M, for example, 1.6 × 10 -8 M to 9.9×10 -8 The binding affinity of M, or for example, 1.0 × 10⁻⁶. -7 Up to 9.9×10 -7 The binding affinity of M was determined, as described in Example 7, by the biolayer interferometry method. In some embodiments of the invention, the antibody has a binding affinity greater than 1.5 × 10⁻⁶ for the CD3 peptide of SEQ ID NO: 402. -8 M, for example, 1.4 × 10 -8 Up to 1.0×10 -8 M, for example, 9.9 × 10 -9 Up to 1×10 -9 M.

[0291] In one embodiment, the present invention provides a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises a CDR1, CDR2, and CDR3 region having a CDR sequence selected from:

[0292] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:12,2,3;

[0293] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:14,2,3;

[0294] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:16,2,3;

[0295] d) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:18,2,3;

[0296] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:20,2,3;

[0297] f) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:22,2,3;

[0298] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:24,2,3;

[0299] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:26,2,3;

[0300] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:28,2,3;

[0301] j) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:30,2,3;

[0302] k) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:32,2,3;

[0303] l) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:34,2,3;

[0304] m) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:36,2,3;

[0305] n) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:38,2,3;

[0306] o) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:40,2,3;

[0307] p) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:42,2,3;

[0308] q) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:44,2,3;

[0309] r) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:46,2,3;

[0310] s) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:48,2,3;

[0311] t) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:50,2,3;

[0312] u) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:52,2,3;

[0313] v) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3;

[0314] w) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:56,2,3;

[0315] x) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3;

[0316] y) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:60,2,3;

[0317] z) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:62,2,3;

[0318] aa) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:64,2,3;

[0319] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:66,2,3;

[0320] cc) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:68,2,3;

[0321] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:70,2,3;

[0322] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:72,2,3 are as follows:

[0323] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:74,2,3 are shown in the image.

[0324] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:76,2,3;

[0325] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:78,2,3;

[0326] ii) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:80,2,3;

[0327] jj) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:82,2,3;

[0328] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:84,2,3;

[0329] ll) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:86,2,3;

[0330] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:88,2,3 (mm)

[0331] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:90,2,3;

[0332] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:92,2,3 are as follows;

[0333] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:94,2,3 (pp)

[0334] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 96,2,3 are as follows;

[0335] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:98,2,3;

[0336] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,100,3;

[0337] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,102,3 are tt.

[0338] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,104,3;

[0339] vv) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3;

[0340] ww)The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,108,3;

[0341] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,110,3 are xx).

[0342] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,112,3 are as follows:

[0343] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,114,3;

[0344] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,116,3;

[0345] bbb)The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,118,3;

[0346] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,120,3;

[0347] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,122,3;

[0348] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,124,3 are eee)

[0349] fff)The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,126,3;

[0350] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,128,3 are ggg)

[0351] hhh) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,130,3;

[0352] iii) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,132,3;

[0353] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,134,3;

[0354] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,136,3 (kkk) are kkk)

[0355] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,138,3;

[0356] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,140,3 (mmm)

[0357] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,142,3;

[0358] ooo) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,144,3;

[0359] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,146,3 (ppp)

[0360] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,148,3;

[0361] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,150,3;

[0362] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,152,3;

[0363] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,154,3;

[0364] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,156,3;

[0365] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,158,3 (vvv)

[0366] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,160,3 (www);

[0367] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,162,3 (xxx)

[0368] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,164,3;

[0369] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,166,3 are zzz.

[0370] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,168,3 (aaaa)

[0371] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,170;

[0372] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,172;

[0373] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,174;

[0374] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,176;

[0375] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,178;

[0376] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,180;

[0377] hhhh) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,182;

[0378] iiii) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184;

[0379] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,186;

[0380] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,188 (kkkk) are:

[0381] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,190;

[0382] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,192 (mmmm)

[0383] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,194;

[0384] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,196;

[0385] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,198;

[0386] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,200;

[0387] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,202;

[0388] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,204;

[0389] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,206;

[0390] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,208;

[0391] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,210 (vvvv) are vvvv)

[0392] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,212 are wwww)

[0393] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,214 (xxxx) are:

[0394] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,216 are as follows:

[0395] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,218;

[0396] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,220 (aaaaa) are:

[0397] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,222 are shown in bbbbb).

[0398] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,224 are ccccc.

[0399] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,226;

[0400] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,228 (eeeee) are:

[0401] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,230;

[0402] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,232 (ggggg) are the sequences shown in ggggg)

[0403] (hhhhh) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,234;

[0404] iiiii) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236;

[0405] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,238 (jjjjj) are:

[0406] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,240 (kkkkk) are kkkkk)

[0407] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,242;

[0408] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244 (mmmmm)

[0409] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,246;

[0410] ooooo) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,248;

[0411] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,250 (pppppp) are ppppp)

[0412] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,252;

[0413] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,254;

[0414] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,256;

[0415] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,258;

[0416] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,260;

[0417] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,262 (vvvvv) are vvvvv)

[0418] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,264;

[0419] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,266 (xxxxx)

[0420] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,268;

[0421] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,270 (zzzzz) are zzzzz)

[0422] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,272 (aaaaaaa) are aaaaaaa)

[0423] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,274;

[0424] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,276;

[0425] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,278;

[0426] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1,2,280;

[0427] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,282;

[0428] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284;

[0429] (hhhhhh) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,286;

[0430] iiiiii) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,288;

[0431] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,290 are jjjjjjj.

[0432] The CDR1, CDR2 and CDR2 shown in SEQ ID NO:1,2,292 are kkkkkkk)

[0433] CDR3 sequence;

[0434] llllll)SEQ ID NO:1,2,294 shows CDR1, CDR2 and

[0435] CDR3 sequence;

[0436] (mmmmmm) CDR1 and CDR2 shown in SEQ ID NO:1,2,296

[0437] and CDR3 sequence;

[0438] nnnnnn)SEQ ID NO:1,2,298 shows CDR1, CDR2 and

[0439] CDR3 sequence; and

[0440] oooooo) SEQ ID NO:1, 2, 300 shows CDR1, CDR2

[0441] And CDR3 sequence.

[0442] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises a CDR1, CDR2, and CDR3 region having a CDR sequence selected from:

[0443] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3[T31M];

[0444] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0445] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0446] d) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,176[H101G];

[0447] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184[H101N];

[0448] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0449] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236[S110A];

[0450] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244[S110G];

[0451] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284[Y114M];

[0452] j) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,292[Y114R];

[0453] k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,298[Y114V]; and

[0454] l) CDR1, CDR2, and CDR3 sequences that have at least 90% or at least 95% amino acid sequence identity with any one of the three CDR sequences shown in a) to k), provided that CDR1, CDR2, and CDR3 sequences do not have the sequences shown in SEQ ID NO: 1, 2, and 3.

[0455] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises a CDR1, CDR2, and CDR3 region having a CDR sequence selected from:

[0456] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3[T31M];

[0457] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0458] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0459] d) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,176[H101G];

[0460] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184[H101N];

[0461] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0462] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236[S110A];

[0463] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244[S110G];

[0464] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284[Y114M];

[0465] j) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,292[Y114R];

[0466] k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1,2,298[Y114V], and

[0467] The CDR1, CDR2, and CDR3 sequences described in l)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 change the binding affinity to human CD3.

[0468] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises a VH sequence selected from:

[0469] a) The VH sequence shown in SEQ ID NO:13;

[0470] b) The VH sequence shown in SEQ ID NO:15;

[0471] c) The VH sequence shown in SEQ ID NO:17;

[0472] d) The VH sequence shown in SEQ ID NO:19;

[0473] e) The VH sequence shown in SEQ ID NO:21;

[0474] f) The VH sequence shown in SEQ ID NO:23;

[0475] g) The VH sequence shown in SEQ ID NO:25;

[0476] h) The VH sequence shown in SEQ ID NO:27;

[0477] i) The VH sequence shown in SEQ ID NO:29;

[0478] j) The VH sequence shown in SEQ ID NO:31;

[0479] k) The VH sequence shown in SEQ ID NO:33;

[0480] l) The VH sequence shown in SEQ ID NO:35;

[0481] m) The VH sequence shown in SEQ ID NO:37;

[0482] n) The VH sequence shown in SEQ ID NO:39;

[0483] o) The VH sequence shown in SEQ ID NO:41;

[0484] p) The VH sequence shown in SEQ ID NO:43;

[0485] q) The VH sequence shown in SEQ ID NO:45;

[0486] r) The VH sequence shown in SEQ ID NO:47;

[0487] s) The VH sequence shown in SEQ ID NO:49;

[0488] t) The VH sequence shown in SEQ ID NO:51;

[0489] u) The VH sequence shown in SEQ ID NO:53;

[0490] v) The VH sequence shown in SEQ ID NO:55;

[0491] w) The VH sequence shown in SEQ ID NO:57;

[0492] x) The VH sequence shown in SEQ ID NO:59;

[0493] y) The VH sequence shown in SEQ ID NO:61;

[0494] z) The VH sequence shown in SEQ ID NO:63;

[0495] aa) The VH sequence shown in SEQ ID NO:65;

[0496] bb) The VH sequence shown in SEQ ID NO:67;

[0497] cc) The VH sequence shown in SEQ ID NO:69;

[0498] (dd) The VH sequence shown in SEQ ID NO:71;

[0499] The VH sequence shown in SEQ ID NO:73 (ee)

[0500] The VH sequence shown in SEQ ID NO:75;

[0501] The VH sequence shown in SEQ ID NO:77 (gg)

[0502] The VH sequence shown in SEQ ID NO:79;

[0503] ii) The VH sequence shown in SEQ ID NO:81;

[0504] jj) The VH sequence shown in SEQ ID NO:83;

[0505] The VH sequence shown in SEQ ID NO:85;

[0506] ll) The VH sequence shown in SEQ ID NO:87;

[0507] The VH sequence shown in SEQ ID NO:89 (mm);

[0508] The VH sequence shown in SEQ ID NO:91;

[0509] oo) The VH sequence shown in SEQ ID NO:93;

[0510] (pp) The VH sequence shown in SEQ ID NO:95;

[0511] The VH sequence shown in SEQ ID NO: 97 (qq)

[0512] (rr) The VH sequence shown in SEQ ID NO:99;

[0513] The VH sequence shown in SEQ ID NO:101;

[0514] The VH sequence shown in SEQ ID NO:103;

[0515] The VH sequence shown in SEQ ID NO:105;

[0516] The VH sequence shown in SEQ ID NO:107;

[0517] ww)The VH sequence shown in SEQ ID NO:109;

[0518] The VH sequence shown in SEQ ID NO: 111;

[0519] The VH sequence shown in SEQ ID NO:113 (yy)

[0520] (zz) The VH sequence shown in SEQ ID NO:115;

[0521] The VH sequence shown in SEQ ID NO: 117 (aaa)

[0522] bbb) The VH sequence shown in SEQ ID NO:119;

[0523] ccc) The VH sequence shown in SEQ ID NO:121;

[0524] The VH sequence shown in SEQ ID NO:123 (ddd)

[0525] The VH sequence shown in SEQ ID NO:125;

[0526] fff) The VH sequence shown in SEQ ID NO:127;

[0527] The VH sequence shown in SEQ ID NO:129 (ggg)

[0528] The VH sequence shown in SEQ ID NO: 131 (hhh)

[0529] iii) The VH sequence shown in SEQ ID NO:133;

[0530] The VH sequence shown in SEQ ID NO: 135;

[0531] The VH sequence shown in SEQ ID NO:137 (kkk)

[0532] The VH sequence shown in SEQ ID NO: 139;

[0533] The VH sequence shown in SEQ ID NO:141 (mmm)

[0534] The VH sequence shown in SEQ ID NO:143;

[0535] ooo) The VH sequence shown in SEQ ID NO:145;

[0536] The VH sequence shown in SEQ ID NO: 147 (ppp)

[0537] The VH sequence shown in SEQ ID NO:149 (qqq)

[0538] rrr) The VH sequence shown in SEQ ID NO:151;

[0539] The VH sequence shown in SEQ ID NO:153 (sss)

[0540] The VH sequence shown in SEQ ID NO:155 (ttt)

[0541] The VH sequence shown in SEQ ID NO:157;

[0542] The VH sequence shown in SEQ ID NO:159 (vvv)

[0543] The VH sequence shown in SEQ ID NO:161 (www);

[0544] The VH sequence shown in SEQ ID NO:163 (xxx);

[0545] The VH sequence shown in SEQ ID NO:165 (yyy)

[0546] zzz) The VH sequence shown in SEQ ID NO:167;

[0547] The VH sequence shown in SEQ ID NO: 169 (aaaa)

[0548] The VH sequence shown in SEQ ID NO:171 (bbbb)

[0549] The VH sequence shown in SEQ ID NO:173 (cccc)

[0550] The VH sequence shown in SEQ ID NO:175;

[0551] The VH sequence shown in SEQ ID NO:177 (eeee)

[0552] The VH sequence shown in SEQ ID NO:179;

[0553] The VH sequence shown in SEQ ID NO:181 (gggg)

[0554] The VH sequence shown in SEQ ID NO: 183 (hhhh)

[0555] iiii) The VH sequence shown in SEQ ID NO:185;

[0556] The VH sequence shown in SEQ ID NO: 187 (jjjj)

[0557] The VH sequence shown in SEQ ID NO:189 (kkkk)

[0558] The VH sequence shown in SEQ ID NO:191;

[0559] The VH sequence shown in SEQ ID NO:193 (mmmm)

[0560] The VH sequence shown in SEQ ID NO:195;

[0561] The VH sequence shown in SEQ ID NO:197 (oooo)

[0562] The VH sequence shown in SEQ ID NO:199 (pppp)

[0563] The VH sequence shown in SEQ ID NO:201 (qqqq)

[0564] The VH sequence shown in SEQ ID NO:203;

[0565] The VH sequence shown in SEQ ID NO:205 (ssss)

[0566] The VH sequence shown in SEQ ID NO:207 (tttt)

[0567] The VH sequence shown in SEQ ID NO:209;

[0568] The VH sequence shown in SEQ ID NO:211 (vvvv)

[0569] The VH sequence shown in SEQ ID NO:213 (wwww)

[0570] The VH sequence shown in SEQ ID NO:215 (xxxx);

[0571] The VH sequence shown in SEQ ID NO:217 (yyyy)

[0572] The VH sequence shown in SEQ ID NO:219 (zzzz)

[0573] The VH sequence shown in SEQ ID NO:221 (aaaaa)

[0574] bbbbb) The VH sequence shown in SEQ ID NO:223;

[0575] The VH sequence shown in SEQ ID NO:225 (ccccc)

[0576] The VH sequence shown in SEQ ID NO:227 (ddddd)

[0577] The VH sequence shown in SEQ ID NO:229 (eeeee)

[0578] The VH sequence shown in SEQ ID NO:221;

[0579] The VH sequence shown in SEQ ID NO:223 (ggggg)

[0580] The VH sequence shown in SEQ ID NO:225 (hhhhh)

[0581] iiiii) The VH sequence shown in SEQ ID NO:227;

[0582] The VH sequence shown in SEQ ID NO:229 (jjjjj)

[0583] The VH sequence shown in SEQ ID NO:231 (kkkkk)

[0584] The VH sequence shown in SEQ ID NO:233;

[0585] The VH sequence shown in SEQ ID NO:235 (mmmmmm)

[0586] The VH sequence shown in SEQ ID NO:237;

[0587] ooooo) The VH sequence shown in SEQ ID NO:239;

[0588] The VH sequence shown in SEQ ID NO:241 (pppppp)

[0589] The VH sequence shown in SEQ ID NO:243 (qqqqq)

[0590] The VH sequence shown in SEQ ID NO:245;

[0591] The VH sequence shown in SEQ ID NO:247 (sssss)

[0592] The VH sequence shown in SEQ ID NO:249 (ttttt)

[0593] The VH sequence shown in SEQ ID NO:251;

[0594] The VH sequence shown in SEQ ID NO:253 (vvvvv)

[0595] The VH sequence shown in SEQ ID NO:255 (wwwww)

[0596] The VH sequence shown in SEQ ID NO:257 (xxxxx)

[0597] The VH sequence shown in SEQ ID NO:259 (yyyyy)

[0598] The VH sequence shown in SEQ ID NO:261 (zzzzz)

[0599] The VH sequence shown in SEQ ID NO:263 (aaaaaaa)

[0600] The VH sequence shown in SEQ ID NO:265 (bbbbbb)

[0601] The VH sequence shown in SEQ ID NO:267 (cccccc)

[0602] The VH sequence shown in SEQ ID NO:269;

[0603] The VH sequence shown in SEQ ID NO:271 (eeeeee)

[0604] The VH sequence shown in SEQ ID NO:273 (ffffff)

[0605] The VH sequence shown in SEQ ID NO:275 (gggggg)

[0606] The VH sequence shown in SEQ ID NO:277 (hhhhhh)

[0607] iiiiii) The VH sequence shown in SEQ ID NO:279;

[0608] The VH sequence shown in SEQ ID NO:281 (jjjjjjj)

[0609] The VH sequence shown in SEQ ID NO:283 (kkkkkk)

[0610] The VH sequence shown in SEQ ID NO:285;

[0611] The VH sequence shown in SEQ ID NO:287 (mmmmmm)

[0612] The VH sequence shown in SEQ ID NO:289;

[0613] The VH sequence shown in SEQ ID NO:291 (oooooo)

[0614] The VH sequence shown in SEQ ID NO:293 (pppppp)

[0615] The VH sequence shown in SEQ ID NO:295 (qqqqqq)

[0616] The VH sequence shown in SEQ ID NO:297;

[0617] The VH sequence shown in SEQ ID NO:299 (ssssss) and

[0618] The VH sequence shown in SEQ ID NO:301 (tttttt)

[0619] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises a VH sequence selected from:

[0620] a) The VH sequence shown in SEQ ID NO:55[T31M]

[0621] b) The VH sequence shown in SEQ ID NO:59[T31P]

[0622] c) The VH sequence shown in SEQ ID NO:107[N57E]

[0623] d) The VH sequence shown in SEQ ID NO:177[H101G]

[0624] e) The VH sequence shown in SEQ ID NO:185[H101N]

[0625] f) The VH sequence shown in SEQ ID NO:221[G105P]

[0626] g) The VH sequence shown in SEQ ID NO:237[S110A]

[0627] h) The VH sequence shown in SEQ ID NO:245[S110G]

[0628] i) The VH sequence shown in SEQ ID NO:285[Y114M]

[0629] j) The VH sequence shown in SEQ ID NO:293[Y114R], and

[0630] k) The VH sequence shown in SEQ ID NO:299[Y114V].

[0631] In one embodiment of the invention, the humanized or chimeric antibody includes a binding region, wherein the binding region includes a light chain variable (VL) region, wherein the VL region includes CDR1, CDR2, and CDR3 having a CDR sequence selected from the following:

[0632] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,7;

[0633] b) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:302, GTN,7; c) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:304, GTN,7; d) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:306, GTN,7; e) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:308, GTN,7; f) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:310, GTN,7; g) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:312, GTN,7; h) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:314, GTN,7; i) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:316, GTN,7; j) SEQ ID The sequences shown in SEQ ID NO:318,GTN,7 are CDR1, CDR2, and CDR3; k) the sequences shown in SEQ ID NO:320,GTN,7 are CDR1, CDR2, and CDR3; l) the sequences shown in SEQ ID NO:322,GTN,7 are CDR1, CDR2, and CDR3; m) the sequences shown in SEQ ID NO:324,GTN,7 are CDR1, CDR2, and CDR3; n) the sequences shown in SEQ ID NO:326,GTN,7 are CDR1, CDR2, and CDR3; o) the sequences shown in SEQ ID NO:328,GTN,7 are CDR1, CDR2, and CDR3; p) the sequences shown in SEQ ID NO:330,GTN,7 are CDR1, CDR2, and CDR3; q) the sequences shown in SEQ ID NO:6,GTN,332 are CDR1, CDR2, and CDR3; r) the sequences shown in SEQ ID NO:318,GTN,7 are CDR1, CDR2, and CDR3. The sequences shown in SEQ ID NO:6,GTN,334 are CDR1, CDR2, and CDR3; s) are CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,336; t) are CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,338; u) are CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,340; v) are CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,342; w) are CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,344; x) are CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,346; y) are CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,348.z) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 350; aa) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 352; bb) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 354; cc) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 356; dd) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 358; ee) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 360; ff) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 362;

[0634] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,364;

[0635] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,366;

[0636] ii) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,368;

[0637] jj) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,370;

[0638] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,372;

[0639] ll) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,374;

[0640] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,376 (mm)

[0642] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,378;

[0643] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,380;

[0644] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,382 (pp)

[0645] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,384;

[0646] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,386;

[0647] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,388;

[0648] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,390;

[0649] The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6,GTN,392;

[0650] and

[0651] The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:6,GTN,394 (vv)

[0652] In another embodiment of the invention, the humanized or chimeric antibody comprises a binding region containing a light chain variable (VL) region, wherein the VL region comprises one of the VL sequences selected from:

[0653] a) The VL sequence shown in SEQ ID NO:8; and

[0654] b) The VL sequence shown in SEQ ID NO:10.

[0655] As used herein, the term "antibody" means an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which, under typical physiological conditions, has the ability to specifically bind to an antigen and has a half-life of a significant time period, 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 time defined by the relevant function (e.g., sufficient time to induce, promote, enhance, and / or regulate the physiological response associated with the antibody binding to the antigen and / or sufficient time for the antibody to recruit effector activity). The binding region (or binding domain, which may also be used herein, both terms having 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 an antibody (Ab) mediates 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 in the classical complement activation pathway). As stated above, the term "antibody" as used herein, unless otherwise stated or explicitly contradicted by the context, includes fragments of an antibody that retain the ability to specifically interact with (e.g., bind) an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments included within the term "antibody" include (i) Fab' or Fab fragments, a type of antibody composed of V... L 、V H C L and C H (i) a monovalent fragment consisting of a domain, or a monovalent antibody described in WO2007059782 (Genmab A / S); (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) essentially composed of V H and C H (iv) The Fd fragment consisting of a single-arm antibody; and (iv) the V fragment consisting primarily of a single-arm antibody. L and V H The Fv segment consists of two structural domains. Furthermore, although the Fv segment has two structural domains V... L and V H Encoded by individual genes, but they can be linked together using recombination methods via synthetic linkers that allow them to be made into a single protein chain, wherein V LPairing with the VH region forms a monovalent molecule (referred to as 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 specified or clearly indicated by the context. Although such fragments are generally included in the meaning of antibody, they are collectively and independently distinctive 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 antigens, provided by any known technology, such as enzyme cleavage, peptide synthesis, and recombinant techniques. The resulting antibodies may have any isotype.

[0656] As used herein, the terms “immunoglobulin heavy chain,” “heavy chain of immunoglobulin,” or “heavy chain” refer to one of the chains of an immunoglobulin. A heavy chain typically consists of a heavy chain variable region (hereinafter referred to as VH) and a heavy chain constant region (hereinafter referred to as CH) that defines the isotype of the immunoglobulin. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chain constant region may further contain 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 of which may be 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), two heavy chains are interconnected by disulfide bonds in what are called “hinge regions.” Similar to the heavy chain, each light chain typically consists of several regions: a light chain variable region (hereinafter referred to as VL) and a light chain constant region (hereinafter referred to as CL). The light chain constant region typically contains a CL domain. Furthermore, the VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions whose sequences can be highly variable and / or form structure-defined rings), also known as complement-determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

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

[16] -

[17] .

[0657] 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) encoded by the heavy chain constant region gene [SEQ ID NO:407]). Therefore, in one embodiment, the antibody comprises the heavy chain of an immunoglobulin of type IgG1 or any allotype thereof. Furthermore, each heavy chain isotype may be combined with a kappa(κ) or lambda(λ) light chain.

[0658] As used herein, the term “chimeric antibody” refers to an antibody in which the variable region is derived from a non-human species (e.g., rodents) and the constant region is derived from a different species, such as humans. Chimeric antibodies can be produced by antibody engineering. “Antibody engineering” is a general term used for different types of antibody modification, and it is a method well known to those skilled in the art. Specifically, 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 by both genetic or enzymatic methods. The production of chimeric antibodies is within the knowledge of those skilled in the art, and therefore the production of the 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 may typically contain a non-human (e.g., mouse) variable region that is specific to the target antigen, and human constant antibody heavy and light chain domains. The term “variable region” or “variable domain” as used in the context of chimeric antibodies refers to a region containing both the CDR and framework regions of the heavy and light chains of an immunoglobulin.

[0659] As used herein, the term “humanized antibody” refers to a genetically engineered nonhuman antibody containing a human antibody constant domain and a nonhuman variable domain modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six nonhuman antibody complementarity-determining regions (CDRs) that together form the antigen-binding site onto the homologous human receptor frame region (FR) (see

[19] -

[20] ). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the frame residues of the parent antibody (i.e., the nonhuman antibody) into the human frame region (reversion mutation). Structural homology modeling can help identify amino acid residues in the frame region that are important for the antibody's binding properties. Thus, a humanized antibody may contain a nonhuman CDR sequence, optionally a substantially human frame region containing one or more amino acid reversion mutations mutated into a nonhuman amino acid sequence, and a fully human constant region. Optionally, additional amino acid modifications, which are not necessarily reversion mutations, may be applied to obtain humanized antibodies with preferred characteristics, such as affinity and biochemical properties.

[0660] Humanized or chimeric antibodies according to any aspect or embodiment of the invention may be referred to as “humanized or chimeric CD3 antibody”, “humanized or chimeric antibody of the invention”, “CD3 antibody” or “CD3 antibody of the invention”, all of which have the same meaning and purpose, unless the context otherwise contradicts.

[0661] Non-human antibodies have amino acid sequences that differ from human antibodies, and therefore, non-human antibodies may be immunogenic when administered to human patients. However, despite their non-human origin, the CDR region of the antibody is responsible for its ability to bind to its target antigen, and humanization aims to maintain the antibody's specificity and binding affinity. Therefore, non-human therapeutic antibodies are humanized to minimize their immunogenicity in humans, while simultaneously maintaining the specificity and binding affinity of the non-human antibody.

[0662] The term "binding region" as used in this article refers to a region that can bind to any molecule, such as a polypeptide (e.g., an antibody present on a cell, bacterium, or virus).

[0663] As used in this article, the term "binding" refers to the binding of an antibody to a predetermined antigen or target. When measured on a BIAcore 3000 instrument using, for example, surface plasmon resonance (SPR) technology, with antigens as ligands and antibodies as analytes, the binding typically corresponds to approximately 10. -6 M or smaller, such as 10 -7 M or smaller, for example, about 10 -8 M or smaller, for example, about 10 -9 M or smaller, approximately 10 -10 M or smaller or about 10 -11 M or even smaller K D The affinity of K is at least 10 times, for example at least 100 times, for example at least 1,000 times, for example at least 10,000 times, for example at least 10000 times, or for example at least 100,000 times, or for example at least 100,000 times, compared to the affinity of K for non-specific antigens that are not the intended antigen or closely related antigens (e.g., BSA, casein). D The affinity of the antibody for the predetermined antigen. The degree of lower affinity depends on the antibody's K... D So that when the antibody K D At extremely low levels (i.e., when the antibody is highly specific), the affinity of the antigen can be at least 10,000 times lower than that of the nonspecific antigen. The term "K" as used herein... D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction."

[0664] As used in this article, the term "human CD3" refers to human differentiation cluster 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 in this article and is not limited to human CD3 unless the context contradicts it. In mammals, this complex comprises one CD3γ (gamma) chain (human CD3γ 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 (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TK0). These chains associate with molecules called T-cell receptors (TCRs) and generate activation signals in T lymphocytes. TCR and CD3 molecules together form the TCR complex.

[0665] To the best of the knowledge of those skilled in the art, the amino acid sequences referred to by Swissprot numbers include a signal peptide, which is removed post-translational. Therefore, proteins present on cell surfaces, such as CD3, do not contain a signal peptide. Specifically, 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 CD3δ (delta), SEQ ID NO:399 shows the amino acid sequence of mature human CD3ε (epsilon), SEQ ID NO:403 shows the amino acid sequence of mature cynomolgus monkey CD3ε, and SEQ ID NO:404 shows the amino acid sequence of mature rhesus monkey CD3ε. Therefore, the term “mature” as used herein refers to a protein that does not contain any signal or leader sequence.

[0666] It is well known that signal peptide sequence homology, length, and cleavage site location vary significantly between different proteins. Signal peptides can be determined by various methods, such as SEQ ID NO:399 of this invention, which has been determined according to the SignalP application (available at http: / / www.cbs.dtu.dk / services / SignalP / ).

[0667] In a specific embodiment, the humanized or chimeric antibody of the present invention binds to the ε chain of CD3, such as the ε chain of human CD3 (SEQ ID NO:399). In yet another specific embodiment, the humanized or chimeric antibody binds to an epitope within amino acids 1-27 of the N-terminal portion of human CD3ε (epsilon) (SEQ ID NO:402). In such specific embodiments, the antibody may even further cross-react with other non-human primate species such as cynomolgus monkeys (cynomolgus monkey CD3ε SEQ ID NO:403) and / or rhesus monkeys (rhesus monkey CD3ε SEQ ID NO:404).

[0668] Compared to the original antibody, the antibody of the present invention, which includes the CDR sequence as defined herein and further includes a frame region, may differ in sequences other than the CDR sequence, but still retains full binding capacity. Therefore, the present invention also relates to antibodies comprising amino acid sequences of variable regions having some sequence identity with any sequence described herein.

[0669] The term “sequence identity” as used in the context of this invention refers to the percentage of identity between two sequences as a function of the number of common positions shared by the sequences (i.e., % homology = number of common positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which is introduced for optimal alignment of the two sequences. The percentage of 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 of 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 VectorNTI). Software version 11.5; used in Invitrogen Inc.

[0670] Therefore, in one embodiment of the invention, the antibody comprises a binding region containing 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:

[0671] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3[T31M];

[0672] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0673] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0674] d) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,176[H101G];

[0675] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184[H101N];

[0676] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0677] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236[S110A];

[0678] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244[S110G];

[0679] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284[Y114M];

[0680] j) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,292[Y114R];

[0681] k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,298[Y114V]; and

[0682] l) CDR1, CDR2, and CDR3 sequences that have at least 90% or at least 95% amino acid sequence identity with any one of the three CDR sequences shown in a) to k), provided that CDR1, CDR2, and CDR3 sequences do not have the sequences shown in SEQ ID NO: 1, 2, and 3.

[0683] As shown in Sequence Listing 1 of this document, the VH region consists of a 125-amino acid sequence. Therefore, the second VH sequence, consisting of 125 amino acids whose 124 amino acid positions are identical to one of the first VH sequences listed above, has 99.2% sequence identity with the first VH sequence. The second sequence, consisting of 125 amino acids whose 120 amino acid positions are identical to one of the first VH sequences listed above, has 96% sequence identity with the first VH sequence. The second sequence, consisting of 125 amino acids whose 115 amino acid positions are identical to one of the first VH sequences listed above, has 92% sequence identity with the first VH sequence.

[0684] In its specific implementation, the VH region has at least 96% amino acid sequence identity with at least one VH sequence specified in the group.

[0685] In one embodiment of the invention, the mutation is located in the frame region of the VH region. Therefore, in some embodiments, the three CDR sequences in the VH region are 100% identical to the antibody of the present invention, but amino acid variations may occur in the frame region of the VH region. When the CDR is included in the reference frame of SEQ ID NO:407, such amino acid variations in the frame region preferably do not alter the antibody's binding affinity to CD3.

[0686] Mutations in the VH sequence that cause changes in sequence identity are preferably conserved, physical, or functional amino acids. Substituting amino acids with similar amino acids can increase the likelihood of maintaining the functionality of the parental antibody.

[0687] In one embodiment of the present invention, the antibody is a humanized antibody.

[0688] In one embodiment of the present invention, the antibody is a full-length antibody.

[0689] The humanized antibodies of this invention can be generated by comparing the amino acid sequences of the heavy and light chain variable regions with a database of human germline variable region sequences to identify heavy and light chain human sequences with appropriate homology for use as human variable frame regions. A series of humanized heavy and light chain variable regions can be designed by transplanting, for example, mouse CDRs onto the frame region (as identified above) and, if desired, by reversing mutations (mutating one or more human amino acid residues at a specified position in the frame region back to non-human amino acids) to specific mouse residue sequences that may be crucial for restoring antibody binding efficacy. Then, according to the applied computer technology: iTope TM and TCED TM The variant sequences of the possible T cell epitopes determined by (

[24] ,

[25] and

[26] ) were selected with the lowest incidence.

[0690] Furthermore, the humanized antibodies of the present invention can also be "deimmunized". Deimmunization may be necessary because the presence of human T cell epitopes in protein sequences such as the humanized antibodies of the present invention can 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 the T cell epitopes without significantly reducing the binding affinity of the antibody.

[0691] Therefore, in one embodiment of the present invention, a humanized antibody can be generated by a method comprising the following steps: (i) comparing a database of non-human fully variable heavy chain sequences and / or fully variable light chain sequences with human germline sequences, (ii) selecting a human germline sequence having the highest homology with the non-human sequence to obtain a humanized sequence, (iii) optimizing the humanized sequence by reverse mutation if necessary, and (iv) expressing the sequence in a suitable expression system.

[0692] Therefore, the full-length antibody of the present invention can be generated by a method comprising the following steps: (i) comparing a database of non-human variable heavy chain sequences and variable light chain sequences with human ancestral sequences; (ii) selecting a human ancestral sequence with the highest homology to the non-human sequence; (iii) transplanting a non-human CDR into the selected human ancestral line to obtain a humanized sequence; (iv) optimizing the humanized sequence by reverse mutation if necessary; (v) identifying constant heavy chain 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 antibody of the present invention can therefore be generated as described in Example 1. It is within the knowledge of those skilled in the art that the full-length antibody is generated starting from a CDR sequence or a completely variable region sequence. Therefore, those skilled in the art will know how to generate the full-length antibody of the present invention.

[0693] The term "complete heavy chain sequence" used in this article refers to a sequence consisting of variable heavy chain and constant heavy chain sequences.

[0694] The term “complete light chain sequence” used in this paper refers to a sequence consisting of variable light chain and constant light chain sequences.

[0695] Reversion mutations can be introduced via standard DNA mutagenesis. Such standard techniques for DNA mutagenesis are described in

[18] . Alternatively, commercially available kits such as Quickchange can be used. TM Site-directed mutagenesis kits (Stratagene), or the desired reversion mutations, can be introduced through de novo DNA synthesis.

[0696] Therefore, in one implementation, the antibody is a humanized antibody.

[0697] Chimeric antibodies can be generated by replacing all the constant region sequences of a non-human (e.g., mouse) antibody with a human-derived constant region sequence. Thus, the fully non-human variable region sequence is retained in the chimeric antibody. Therefore, the chimeric antibody of the present invention can be generated by a method comprising the steps of expressing a non-human variable heavy chain (SEQ ID NO:405), a non-human variable light chain sequence (SEQ ID NO:406), a human constant heavy chain, and a human constant light chain sequence in a suitable expression system, thereby generating a full-length chimeric antibody. Alternative methods may be used. Such methods for generating chimeric antibodies are within the knowledge of those skilled in the art, and therefore those skilled in the art will know how to generate the chimeric antibody of the present invention. Therefore, in order to prepare the chimeric antibody of the present invention, the mutation of the present invention will be introduced into a non-human (e.g., mouse) VH or VL sequence.

[0698] Therefore, in one implementation, the antibody is a chimeric antibody.

[0699] In one implementation, the antibody is a full-length antibody. As used herein, the term "full-length antibody" refers to an antibody (e.g., a parent or variant antibody) that contains all the constant and variable heavy and light chain domains corresponding to those naturally present in wild-type antibodies of that isotype.

[0700] In one embodiment, the antibody comprises an Fc region containing first and second immunoglobulin heavy chains.

[0701] As used herein, the term "Fc region" refers to a region that includes at least the hinge region, the CH2 region, and the CH3 region in the direction from the N-end to the C-end. The Fc region may further include the CH1 region at the N-end of the hinge region.

[0702] As used herein, the term "hinge region" refers to the hinge region of the immunoglobulin heavy chain. Therefore, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 of the Eu number as described in Kabat.

[0703] Unless otherwise stated or contradicted in the context, the amino acids in the constant region sequence are numbered in this document according to the Eu-number 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”.

[0704] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of the 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 can also be any other subtype described herein.

[0705] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of the 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 can also be any other subtype described herein.

[0706] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of the 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 can also be any other subtype described herein.

[0707] 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 type, 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).

[0708] When targeting the antigen CD3, which is part of the T-cell receptor (TCR), the T-cell-specific mechanism of cell killing is ideal. Other effector functions, such as complement activation, may be undesirable, and therefore, reduced effector function is desirable. C1q binding is the first step in the complement cascade and is therefore used as an indicator of the antibody's complement-dependent cytotoxic (CDC) capacity. If C1q binding to the antibody can be avoided, activation of the complement cascade can also be avoided.

[0709] Therefore, in one embodiment, the antibody comprises a modified Fc region such that the 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 the wild-type antibody, wherein C1q binding is determined by ELISA. In a preferred embodiment, the antibody comprises a modified Fc region such that the binding of C1q to the antibody is reduced by at least 99% to 100% compared to the wild-type antibody, wherein C1q binding is determined by ELISA.

[0710] As used herein, the term "modification" refers to an amino acid sequence in the Fc region that differs from the amino acid sequence of the wild-type Fc region. That is, amino acid residues at designated positions in the wild-type Fc region have been substituted, deleted, or inserted to alter, for example, the binding site to C1q, the binding site to other effector molecules, or the binding to the Fc receptor (FcR). Such modifications to the amino acid sequence can be prepared by substituting one or more amino acids with conserved amino acids, or by substituting one or more amino acids with alternative amino acids that are physically and / or functionally similar to those present in the wild type. Substitution can also be prepared by substituting non-conserved amino acids.

[0711] In the context of this invention, amino acids can be described as conserved or non-conserved amino acids, and can therefore be classified accordingly. Amino acid residues can also be classified into types defined by selectable physical and functional properties. Therefore, the types of amino acids can be reflected in one or two of the following tables:

[0712] Conserved amino acid residues

[0713] 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 Aroma residues F, Y and W

[0714] Selective physical and functional classification of amino acid residues

[0715]

[0716] In the context of this invention, the substitution in antibodies, such as humanized or chimeric antibodies, is represented as follows:

[0717] Original amino acid – position – replaced amino acid;

[0718] Following accepted amino acid nomenclature, three-letter or single-letter codes are used, including the codes Xaa and X, to indicate any amino acid residue. Therefore, the symbol "L234F" or "Leu234Phe" means that the antibody contains leucine replaced by phenylalanine at amino acid position 234.

[0719] The substitution of an amino acid at a given position with any other amino acid is represented as follows:

[0720] Original amino acid – position; or for example, “L234”.

[0721] The original amino acid and / or the replaced amino acid may contain more than one, but not all, amino acids, separated by commas or slashes. For example, leucine at position 234 may be replaced with phenylalanine, arginine, lysine, or tryptophan.

[0722] "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".

[0723] In the context of this invention, such naming can be used interchangeably, but have the same meaning and purpose.

[0724] Furthermore, the term "substitution" includes substitution with 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 substitution site 234 includes various substitutions such as 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, where X indicates any amino acid other than the original amino acid. These substitutions may also be referred to as L234A, L234C, etc., or L234A,C, etc., or L234A / C / , etc. The naming similarly applies to all and all positions mentioned herein, and this document particularly includes any of such substitutions.

[0725] The antibodies of the present invention may also contain deletions of amino acid residues. Such deletions may be represented as "del", and include, for example, written as L234del. Thus, in such an embodiment, the leucine at position 234 is deleted from the amino acid sequence.

[0726] The terms “amino acid” and “amino acid residue” are used interchangeably in this article.

[0727] In one embodiment of the invention, the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 regions having three CDR sequences selected from the group consisting of:

[0728] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3[T31M];

[0729] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0730] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0731] d) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,176[H101G];

[0732] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184[H101N];

[0733] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0734] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236[S110A];

[0735] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244[S110G];

[0736] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284[Y114M];

[0737] j) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,292[Y114R];

[0738] k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,298[Y114V]; and

[0739] The CDR1, CDR2, and CDR3 sequences described in l)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 change the binding affinity to human CD3.

[0740] In one embodiment of the invention, the additional mutations or substitutions are conserved, physical, or functional amino acids.

[0741] In some embodiments, binding to CD3 can be with full-length CD3, such as CD3 present on T cells. In other embodiments, binding to CD3 can be with a CD3 peptide, such as that shown in SEQ ID NO:402. Binding to the CD3 peptide and whether there are any additional mutations that could modify the binding to CD3 can be determined by a biolayer interferometry as disclosed in Example 7.

[0742] In one embodiment, the antibody comprises an Fc region containing first and second immunoglobulin heavy chains.

[0743] As used herein, the term "C1q binding" refers to the binding of C1q to an antibody when the antibody binds to its antigen. The term "binding to its antigen" as used herein refers to the binding of an antibody to its antigen both in vivo and in vitro.

[0744] The term “reduced” as used herein, when referring to C1q binding, means that the antibodies of the present invention reduce, minimize, or even completely inhibit the binding of C1q to the antibody compared to the binding of C1q to wild-type antibodies.

[0745] The terms “reduced” or “reduced” or any variations thereof, as used herein, when used in relation to the binding affinity of antibodies binding 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 specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8 when binding to the CD3 peptide of SEQ ID NO:402, and determined by the biolayer interferometry as described in Example 7.

[0746] As used herein, the term "binding affinity" refers to the binding of an antibody to a predetermined antigen or target, which typically corresponds to K. D Affinity. The term "K" used in this article. D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction."

[0747] As used herein, the term "wild-type antibody" refers to an antibody identical to the antibody being tested, except that it is not inactive, when used in the comparative assay of the antibodies of the present invention. In this context, the term "inactive" means that the modified Fc region has reduced or no C1q binding, i.e., reduced or no Fc-mediated T-cell proliferation as measured by ELISA in PBMC-based functional assays (i.e., T-cell proliferation as measured in peripheral blood mononuclear cell (PBMC) functional assays); and / or reduced or no Fc-mediated CD69 expression as measured in PBMC-based functional assays. Therefore, wild-type antibodies contain amino acids naturally present in the immunoglobulin heavy chain, i.e., antibodies without any amino acid modifications that could alter or reduce the antibody's ability to interact with, for example, C1q, Fc receptors, etc. Thus, such wild-type antibodies will remain activated antibodies capable of binding, for example, C1q. Wild-type antibodies and the antibodies of the present invention may contain amino acid modifications that do not affect the antibody's ability to induce effector function, in order to prepare the antibodies as bispecific antibodies, etc.

[0748] As used herein, the term "ELISA" refers to enzyme-linked immunosorbent assay, an assay that uses antibodies and color changes to identify substances. A first specific antibody is attached to the surface of a plate. A protein from the sample is then added, where binding to the first specific antibody is tested. A second antibody, which binds to the antibody from the sample, is added. The second antibody is attached to an enzyme, and in a final step, a substrate containing the enzyme is added. The subsequent reaction produces a detectable signal, most commonly a color change of the substrate. The concept of ELISA methods is well known in the art, and various ways of performing ELISA are considered part of the methods for evaluating the antibodies of the present invention. In particular, the ability of the antibodies of the present invention to bind C1q can be determined by ELISA, comprising the following steps: (i) coating the antibody onto a 96-well plate, (ii) adding 3% serum, (iii) adding anti-human C1q antibody, (iv) developing the color of the plate, and (v) measuring OD. 405 Therefore, in one embodiment, the antibody comprises a modified Fc region such that the 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 the wild-type antibody, wherein C1q binding is determined by an 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 color of the plate, and (v) measuring OD. 405 nm.

[0749] As used in this article, the term "Fc receptor" or "FcR" refers to a protein present on the surface of certain cells. An FcR binds to the Fc region of an antibody. Several different types of FcRs exist, classified according to the type of antibody they recognize. For example, the Fcγ (gamma) receptor binds to IgG type antibodies.

[0750] The terms "Fcγ receptor," "Fc gamma receptor," or "FcγR" used in this article refer to a group of Fc receptors belonging to the immunoglobulin superfamily, and are the most important Fc receptors for inducing opsonization (encapsulation) of 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.

[0751] 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 the binding of various effector molecules to the Fc region. In the context of this invention, "Fc binding," "Fc receptor binding," "FcR binding," and "antibody Fc region binding to FcR" refer to the binding of the Fc region to an Fc receptor (FcR) or an effector molecule. The terms "FcγR binding" and "FcγRI binding" refer to binding to or via the Fc region to an Fc gamma receptor and Fc gamma receptor I, respectively. When a CD3 antibody binds to a T cell, the wild-type Fc region of the CD3 antibody binds to an FcR present on other cells (e.g., monocytes), leading to nonspecific Fc-mediated T cell activation. Such nonspecific Fc-mediated T cell activation may be undesirable. T cells can also be activated by targeted or target-specific T cell activation. Such targeted T-cell activation can be highly desirable for many indications, such as cancer treatment. The term "targeted T-cell activation" as used herein refers to the use of a bispecific antibody to guide T cells to specific cells, such as tumor cells, comprising 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 facilitated by using a bispecific antibody, wherein one binding region 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 occur, and therefore such unwanted nonspecific Fc-mediated T-cell activation via Fc-mediated crosslinking should be avoided and can be inactivated by preparing an Fc region that is inactive for such activity. Thus, the interaction between the inactive Fc region and the present Fc receptor is prevented.

[0752] The antibodies of this invention may contain modifications to the Fc region. When an antibody contains such modifications, it may become an inactive or non-activated antibody. As used herein, the terms "inactive," "non-active," or "non-activated" mean at least an Fc region that cannot bind to any Fcγ receptor, induces Fc-mediated cross-linking via FcR, induces FcR-mediated target antigen cross-linking via the Fc region, or cannot bind to the C1q Fc region. Inactivity of the Fc region of a humanized or chimeric CD3 antibody is suitably tested using a monospecific form of antibody; however, such identified inactive Fc regions may be used with bispecific or other humanized or chimeric multispecific CD3 antibodies.

[0753] Several variants can be constructed to prepare the Fc region of antibodies inactive to the interaction with the Fc gamma receptor and C1q, for use in the development of therapeutic antibodies. Examples of such variants are described in this paper.

[0754] Therefore, in one embodiment, the antibody comprises a modified Fc region such that, compared with a wild-type antibody, 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%, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0755] The term "reduction" when referring to T-cell proliferation means the ability of the antibodies of the present invention to reduce, minimize, or even completely inhibit T-cell proliferation compared to T-cell proliferation via binding to wild-type antibodies. The ability of the antibodies to reduce T-cell proliferation can be evaluated by PBMC-based functional assays. 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 from any species to demonstrate a reduction in T-cell proliferation, provided that the PBMCs of the species used are within the antibody's cross-reactivity profile, such as human, cynomolgus monkey, or rhesus monkey.

[0756] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay used to evaluate the functional characteristics of the antibodies of the present invention, such as the ability of the antibodies to affect T-cell proliferation or CD69 expression, wherein the only cells present are peripheral blood mononuclear cells. Therefore, in one embodiment, T-cell proliferation is measured by a method comprising the following steps: incubating PBMCs for three days at 37°C in a 5% (vol / vol) CO2 humidified incubator with an antibody in the range of 1-1000 ng / mL; adding a compound, such as BrdU, which is incorporated into the DNA of the proliferating cells; incubating for 5 hours, precipitating the cells, drying the cells, optionally storing the cells at 4°C, coating the cells onto an ELISA plate, incubating with anti-BrdU-peroxidase at room temperature for 90 minutes, developing the color with 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) for approximately 30 minutes, terminating the reaction with 100 μL of 2% oxalic acid, and measuring the absorbance at 405 nm in a suitable microplate reader.

[0757] The term “proliferation” used in this article refers to cell growth in the context of cell division.

[0758] The term "BrdU" used in this article 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 will be incorporated into the DNA of proliferating cells. After cell fixation, the incorporated BrdU can be detected by ELISA using anti-BrdU peroxidase. BrdU incorporation is therefore a measure of proliferation.

[0759] In one embodiment, the antibody comprises a modified Fc region such that, compared with a wild-type antibody, 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0760] Specifically, the term "reduction" when referring to the expression level of the T cell activation marker CD69 means a reduction in CD69 expression compared to the expression level of wild-type antibodies when T cells are bound to CD3 and interact with the Fc receptor. The ability of an antibody to reduce CD69 expression can be evaluated by a PBMC-based functional assay. Therefore, in one embodiment, CD69 expression is measured by a method comprising the following steps: incubating PBMCs at 37°C in a 5% (vol / vol) CO2 humidified incubator for 16–24 hours with an antibody ranging from 1–1000 ng / mL; washing the cells; staining the cells at 4°C with mouse anti-human CD28-PE and mouse anti-human CD69-APC antibodies; and determining CD69 expression on CD28-positive cells by flow cytometry.

[0761] As used in this article, the term "CD69" refers to cluster 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 CD69 expression. CD69 functions as a signaling receptor involved in cellular activation events, including proliferation, as a signaling receptor in lymphocytes, including natural killer cells and platelets, and as an inducer of specific genes.

[0762] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay used to evaluate the functional characteristics of the antibodies of the present invention, such as the ability of the antibodies 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 antibodies at 37°C in a 5% (vol / vol) CO2 humidified incubator for approximately 16-24 hours; (ii) washing the cells; (iii) staining the cells at 4°C with mouse anti-human CD28-PE and mouse anti-human CD69-APC antibodies; and (iv) when evaluating CD69 expression, determining CD69 expression on CD28-positive cells by flow cytometry.

[0763] Amino acids in the Fc region that play a major role in the interaction with C1q and Fc Gamma receptors can be modified. Examples of modifyable amino acid positions include positions L234, L235, and P331. Combinations of these, such as L234F / L235E / P331S, can cause a significant reduction in binding to human CD64, CD32A, CD16, and C1q.

[0764] Therefore, in one embodiment, the amino acids at at least one position corresponding to L234, L235, and P331 can be A, A, and S, respectively ([1],

[28] ). Furthermore, amino acid substitutions at L234F and L235E can produce an Fc region with abolished interactions with the Fc gamma receptor and C1q (

[29] -

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

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

[32] ). Therefore, in one embodiment, the amino acids at at least one position corresponding to D270, K322, P329 and P331 can be A, A, A and A, respectively.

[0765] An alternative method to minimize the interaction between the Fc region and the Fc gamma receptor and C1q is by removing the glycosylation site of the antibody. Mutation at position N297, for example, Q, A, and E, removes the glycosylation site that is critical for IgG-Fc gamma receptor interaction. Therefore, in one embodiment, the amino acid corresponding to the position N297 can be G, Q, A, or E (

[33] ). Another alternative method to minimize the interaction between the Fc region and the Fc gamma receptor can be obtained by the following mutations: P238A, A327Q, P329A, or E233P / L234V / L235A / G236del (

[31] ).

[0766] Alternatively, although human IgG2 and IgG4 subclasses are considered to be inherently deficient in their interactions with C1q and Fc gamma receptors, interactions with Fcγ 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 of 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 in the human IgG2 heavy chain corresponding to the L234 and G237 positions can be A and A, respectively. In one embodiment, the amino acid in the human IgG4 heavy chain corresponding to the L235 position can be E.

[0767] Other methods for further minimizing interactions with Fc gamma receptors and C1q in IgG2 antibodies include those described in

[36] and

[37] .

[0768] Regarding interactions with Fc gamma receptors and complement, the hinge region of an antibody may also be important (

[38] -

[39] ). Therefore, mutations or deletions in the hinge region can affect the effector function of the antibody.

[0769] As used in this article, "crosslinking" refers to an indirect bridging via the Fab arm (monovalent or bivalent) of an antibody that binds to the target antigen on a cell containing the Fc region of the antibody-binding antibody. Therefore, an antibody that binds to the target antigen on a cell containing the target antigen can crosslink with another cell expressing the FcR.

[0770] As used in this article, "non-specific killing" refers to the killing of cells by tumor-target antigen-independent activation via the cytotoxic function of T cells or other effector cells. Therefore, non-specific killing means that cells carrying tumor targets can be killed, for example, by cytotoxic T cells, rather than by antibodies binding to the tumor target through, for example, induction of CDC.

[0771] An inactive Fc region can be obtained by modifying one or more of at least five specific amino acid positions in the Fc region.

[0772] In one embodiment, the antibody comprises an Fc region containing first and second immunoglobulin heavy chains.

[0773] Therefore, in one embodiment, the antibody comprises first and second immunoglobulin heavy chains, wherein in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions L234, L235, D265, N297 and P331 of the human IgG1 heavy chain are not L, L, D, N and P, respectively.

[0774] In one embodiment, in both the first and second heavy chains, one or more amino acids at positions L234, L235, D265, N297, and P331 corresponding to the human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0775] In another embodiment, in at least one of the first and second heavy chains, one or more amino acids at positions L234, L235 and D265 of the human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0776] As used herein, the term “corresponding amino acid” refers to the amino acid position number of the human IgG1 heavy chain. Unless otherwise stated or contradicted by context, the amino acids in constant region sequences are numbered herein according to the Eu number 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 has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain, as determined by alignment with other amino acids or segments, typically using standard sequence alignment programs such as ALIGN, ClustalW, or similar programs at default settings. It is well known in the art how sequences or segments in sequences are aligned and thus positions in the sequence corresponding to the amino acid positions of the present invention are determined.

[0777] In the context of this invention, amino acids can be defined as described above.

[0778] The term "amino acid is not" or similar expressions, when referring to amino acids in the heavy chain, should be understood to mean that the amino acid is any other amino acid that is not the specific amino acid mentioned. For example, the amino acid at position L234 of the human IgG1 heavy chain is not L, meaning that the amino acid can be any other naturally occurring or non-naturally occurring amino acid other than L.

[0779] In one embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is not D.

[0780] In one embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is not D, and the amino acids at positions N297 and P331 of the 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 acid at position D265 of the human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0782] As used herein, the term "hydrophobic" for amino acid residues refers to amino acid residues selected from A, C, F, G, H, I, L, M, R, T, V, W, and Y. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acid corresponding to position D265 of the human IgG1 heavy chain is selected from the amino acids composed of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0783] As used herein, the term "polar" for amino acid residues refers to any amino acid residue selected from C, D, E, H, K, N, Q, R, S, and T. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human heavy chain is selected from C, E, H, K, N, Q, R, S, and T.

[0784] In another embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is an aliphatic uncharged, aromatic, or acidic amino acid.

[0785] As used herein, the term "aliphatic uncharged" for amino acid residues refers to any amino acid residue selected from A, G, I, L, and V. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, G, I, L, and V.

[0786] As used herein, the term "aromatic" for amino acid residues refers to any amino acid residue selected from F, T, and W. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from F, T, and W.

[0787] As used herein, the term "acidic" with respect to amino acid residues refers to any amino acid residue selected from D and E. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from D and E.

[0788] In a specific implementation, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, E, F, G, I, L, T, V, and W.

[0789] In one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is not D.

[0790] In one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is not D, and the amino acids at positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0791] In one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0792] As used herein, the term "hydrophobic" for amino acid residues refers to amino acid residues selected from A, C, F, G, H, I, L, M, R, T, V, W, and Y. Therefore, in one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from the amino acids composed of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0793] As used herein, the term "polar" for amino acid residues refers to any amino acid residue selected from C, D, E, H, K, N, Q, R, S, and T. Therefore, in one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human heavy chain is selected from 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 position D265 of the human IgG1 heavy chain is selected from amino acids composed of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0794] In one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human heavy chain is selected from C, E, H, K, N, Q, R, S, and T.

[0795] In another embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is an aliphatic uncharged, aromatic, or acidic amino acid.

[0796] As used herein, the term "aliphatic uncharged" for amino acid residues refers to any amino acid residue selected from A, G, I, L, and V. Therefore, in one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, G, I, L, and V.

[0797] As used herein, the term "aromatic" for amino acid residues refers to any amino acid residue selected from F, T, and W. Therefore, in one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from F, T, and W.

[0798] As used herein, the term "acidic" with respect to amino acid residues refers to any amino acid residue selected from D and E. Therefore, in one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from D and E.

[0799] In a specific implementation, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, E, F, G, I, L, T, V, and W.

[0800] In a further embodiment, in at least one of the first and second heavy chains, the amino acid at position N297 of the human IgG1 heavy chain is not N.

[0801] In one embodiment, in at least one of the first and second heavy chains, the amino acid at position N297 of the human IgG1 heavy chain is not N, and the amino acid at position P331 of the human IgG1 heavy chain is P.

[0802] In one embodiment, in both the first and second heavy chains, the amino acid at position N297 corresponding to the human IgG1 heavy chain is not N.

[0803] In one embodiment, in both the first and second heavy chains, the amino acid at position N297 of the human IgG1 heavy chain is not N, and the amino acid at position P331 of the human IgG1 heavy chain is P.

[0804] In a further embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 corresponding to the human IgG1 heavy chain are not L and L, respectively.

[0805] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0806] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are selected from A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, and V.

[0807] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0808] As used herein, the term "hydrophobic" for amino acid residues refers to amino acid residues selected from A, C, F, G, H, I, L, M, R, T, V, W, and Y. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from A, C, F, G, H, I, M, R, T, V, W, and Y.

[0809] As used herein, the term "polarity" for amino acid residues refers to any amino acid residue selected from C, D, E, H, K, N, Q, R, S, and T. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from amino acids composed of C, D, E, H, K, N, Q, R, S, and T.

[0810] In a specific implementation, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0811] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 corresponding to the human IgG1 heavy chain are not L and L, respectively.

[0812] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0813] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0814] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 corresponding to the human IgG1 heavy chain are each selected from A, C, F, G, H, I, M, R, T, V, W, and Y.

[0815] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 corresponding to the human IgG1 heavy chain are each selected from amino acids composed of C, D, E, H, K, N, Q, R, S, and T.

[0816] In a specific implementation, in both the first and second heavy chains, the amino acids at positions L234 and L235 corresponding to the human IgG1 heavy chain are each selected from A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0817] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are aliphatic uncharged, aromatic, or acidic amino acids.

[0818] As used herein, the term "aliphatic uncharged" for amino acid residues refers to any amino acid residue selected from A, G, I, L, and V. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from A, G, I, and V.

[0819] As used herein, the term "aromatic" for amino acid residues refers to any amino acid residue selected from F, T, and W. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from F, T, and W.

[0820] As used herein, the term "acidic" with respect to amino acid residues refers to any amino acid residue selected from D and E. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from D and E.

[0821] In a specific embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 are each selected from A, D, E, F, G, I, T, V, and W.

[0822] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are F and E, or A and A, respectively.

[0823] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are F and E, or A and A, respectively, and the amino acids at positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0824] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are F and E, or A and A, respectively.

[0825] In one embodiment, in both the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are F and E, or A and A, respectively, and the amino acids corresponding to positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0826] In a specific embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are F and E, respectively.

[0827] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are F and E, respectively.

[0828] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are A and A, respectively.

[0829] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 corresponding to the human IgG1 heavy chain are A and A, respectively.

[0830] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are not L, L, and D, respectively.

[0831] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0832] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are selected from 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 A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V and W.

[0833] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0834] As used herein, the term "hydrophobic" for amino acid residues refers to amino acid residues selected from A, C, F, G, H, I, L, M, R, T, V, W, and Y. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acid corresponding to position D265 of the human IgG1 heavy chain is selected from A, C, F, G, H, I, L, M, R, T, V, W, and Y, and the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from A, C, F, G, H, I, M, R, T, V, W, and Y.

[0835] As used herein, the term "polarity" for amino acid residues refers to any amino acid residue selected from C, D, E, H, K, N, Q, R, S, and T. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from C, D, E, H, K, N, Q, R, S, and T, and the amino acid corresponding to position D265 of the human heavy chain is selected from C, E, H, K, N, Q, R, S, and T.

[0836] In a specific embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid corresponding to position D265 of the human IgG1 heavy chain is selected from A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0837] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 of the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0838] In one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, C, F, G, H, I, L, M, R, T, V, W, and Y, and the amino acids at positions L234 and L235 of the human IgG1 heavy chain are each selected from A, C, F, G, H, I, M, R, T, V, W, and Y.

[0839] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are each selected from C, D, E, H, K, N, Q, R, S, and T, and the amino acid at position D265 of the human heavy chain is selected from C, E, H, K, N, Q, R, S, and T.

[0840] In a specific implementation, in both the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of the human IgG1 heavy chain are each selected from A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid corresponding to position D265 of the human IgG1 heavy chain is selected from A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0841] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are aliphatic uncharged, aromatic, or acidic amino acids.

[0842] As used herein, the term "aliphatic uncharged" for amino acid residues refers to any amino acid residue selected from A, G, I, L, and V. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, G, I, L, and V, and the amino acids at positions L234 and L235 of the human IgG1 heavy chain are each selected from A, G, I, and V.

[0843] As used herein, the term "aromatic" for amino acid residues refers to any amino acid residue selected from F, T, and W. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain are each selected from F, T, and W.

[0844] As used herein, the term "acidic" with respect to amino acid residues refers to any amino acid residue selected from D and E. Therefore, in one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain are each selected from D and E.

[0845] In a specific embodiment, in at least one of the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, E, F, G, I, L, T, V and W, and the amino acids at positions L234 and L235 are each selected from A, D, E, F, G, I, T, V and W.

[0846] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are not L, L, and D, respectively.

[0847] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0848] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 of the human IgG1 heavy chain are aliphatic uncharged, aromatic, or acidic amino acids.

[0849] In one embodiment, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, G, I, L, and V, and the amino acids at positions L234 and L235 of the human IgG1 heavy chain are each selected from A, G, I, and V.

[0850] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are each selected from D and E.

[0851] In a specific implementation, in both the first and second heavy chains, the amino acid at position D265 of the human IgG1 heavy chain is selected from A, E, F, G, I, L, T, V and W, and the amino acids at positions L234 and L235 are each selected from A, D, E, F, G, I, T, V and W.

[0852] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

[0853] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A, and the amino acids corresponding to positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0854] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

[0855] In one embodiment, in both the first and second heavy chains, the amino acids corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A, and the amino acids corresponding to positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0856] In a specific implementation, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are F, E, and A, respectively.

[0857] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are F, E, and A, respectively.

[0858] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are A, A, and A, respectively.

[0859] In one embodiment, in both the first and second heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are A, A, and A, respectively.

[0860] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions L234, L235, D265, N297 and P331 of the human IgG1 heavy chain are F, E, A, Q and S, respectively.

[0861] In one embodiment, in both the first and second heavy chains, the amino acids corresponding to positions L234, L235, D265, N297, and P331 of the human IgG1 heavy chain are F, E, A, Q, and S, respectively.

[0862] In one embodiment, the antibody according to the invention comprises the VH sequence shown in any of the sequences of SEQ ID NOs:107; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293; the VL sequence shown in SEQ ID NO:8; and amino acids F, E and A, respectively, at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain in at least one or two heavy chains. This provides an embodiment of an anti-CD3 antibody with reduced affinity for human CD3ε compared to a reference antibody comprising the VH and VL sequences shown in SEQ ID NO:4 and 8, wherein the antibody further comprises an inactive Fc region.

[0863] In one specific embodiment, the antibody according to the invention comprises the VH sequence shown in any of the sequences shown in SEQ ID NOs:107;59;245;299;285;55;185;179;237;177 and 293, the VL sequence shown in SEQ ID NO:10, and amino acids F, E and A, respectively, at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain in at least one or two heavy chains. This provides an embodiment of an anti-CD3 antibody with reduced affinity for human CD3ε compared to reference antibodies comprising the VH and VL sequences shown in SEQ ID NOs:4 and 8, wherein the antibody further comprises an inactive Fc region and a VL region allowing for enhanced production.

[0864] In another embodiment, the antibody according to the 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 two heavy chains, the amino acids corresponding to the L234, L235 and D265 positions in the human IgG1 heavy chain are F, E and A, respectively.

[0865] In one embodiment of the invention, the human IgG1 heavy chain has the IgG1m(f) sequence as shown in SEQ ID NO:407. In another embodiment, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1m(f) shown in SEQ ID NO:407 are F, E, and A, respectively.

[0866] In one embodiment of the invention, the human IgG1 heavy chain has an IgG1m(f) sequence as shown in SEQ ID NO:409.

[0867] In one aspect, the antibody according to the invention comprises the human IgLC2 / IgLC3 constant domain λ light chain of SEQ ID NO:408.

[0868] In one aspect, the antibody of the present invention may be modified in the light chain (LC) and / or heavy chain (HC) to increase expression levels and / or production yield. In one embodiment, the antibody of the present invention may be modified in the light chain (LC). Such modification is known in the art and can be performed according to the methods described, for example, in 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.

[0869] In one aspect, antibodies according to the invention can be modified in the VH and / or VL regions to modify antibody affinity, for example, by decreasing or increasing antibody affinity. This can be advantageous in some cases and lead to increased efficacy. Specifically, a low-affinity CD3 arm may influence the activity of circulating T cells at tumor sites, thereby leading to better engagement of tumor cells with T cells, see [link to relevant documentation]. See Webster et al., Molecular Immunology 44 (2007). Specifically, this can be used in a bispecific form, where the CD3 antibody serves as one of the binding arms. Modifications that lead to decreased antibody affinity are known in the art; see, for example, Webster et al., Int J Cancer Suppl. 1988; 3:13-6.

[0870] Therefore, in one embodiment, the antibody of the present invention comprises a light chain variable (VL) region having CDR1, CDR2 and CDR3 having sequences 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 CDR sequences selected from one of the following groups;

[0871] a) The CDR sequence shown in SEQ ID NO:54,2,3[T31M];

[0872] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0873] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0874] d) CDR1, CDR2 and shown in SEQ ID NO:1,2,176[H101G]

[0875] CDR3 sequence;

[0876] e) CDR1, CDR2 and shown in SEQ ID NO:1,2,184[H101N]

[0877] CDR3 sequence;

[0878] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0879] g) CDR1, CDR2 and shown in SEQ ID NO:1,2,236[S110A]

[0880] CDR3 sequence;

[0881] h) CDR1, CDR2 and shown in SEQ ID NO:1,2,244[S110G]

[0882] CDR3 sequence;

[0883] i) CDR1, CDR2 and shown in SEQ ID NO:1,2,284[Y114M]

[0884] CDR3 sequence;

[0885] j) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,292[Y114R]; and

[0886] k) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,298[Y114V].

[0887] On the other hand, the present invention provides an antibody that binds to human CD3, comprising a binding region having a light chain variable (VL) region and a heavy chain variable (VH) region having the sequence shown in SEQ ID NO 10, wherein CDR1, CDR2, and CDR3 of the VH region have sequences selected from one of the following groups:

[0888] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3[T31M];

[0889] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0890] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0891] d) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,176[H101G];

[0892] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184[H101N];

[0893] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0894] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236[S110A];

[0895] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244[S110G];

[0896] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284[Y114M];

[0897] j) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,292[Y114R]; and

[0898] k) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,298[Y114V].

[0899] This provides an embodiment comprising a T41K mutation in the VL region as shown in SEQ ID NO:10, thereby allowing for increased production of the antibody.

[0900] In one aspect, the present invention relates to multispecific antibodies that at least comprise a first binding region of an antibody according to any aspect or embodiment described herein, and one or more binding regions binding to one or more different targets to the first binding region. Such multispecific antibodies may be bispecific antibodies.

[0901] Therefore, in one aspect, the present invention relates to a bispecific antibody comprising a first binding region of an antibody according to any aspect or embodiment described herein, and a second binding region that binds to a different target to the first binding region.

[0902] The term "multispecific antibody" refers to an antibody that is specific to at least two, for example, at least three, typically non-overlapping epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cells or different cells or cell types.

[0903] The term "bispecific antibody" refers to an antibody that is specific to at least two distinct, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, these targets may be on the same cells or different cells or cell types.

[0904] In one embodiment, the bispecific antibody comprises a first and a second heavy chain.

[0905] Embodiments involving modifications to the Fc region and embodiments involving specific amino acid substitutions are contemplated as part of any bispecific antibody of the present 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 describing the provision of 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 describing the provision of an inactive Fc region). Therefore, 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.

[0906] Examples of bispecific antibody molecules that can be used in this invention include (i) a single antibody having two arms containing different antigen-binding regions, (ii) a single-chain antibody specific for two different epitopes, for example via two scFvs tandemly linked by an additional peptide linker; and (iii) a dual variable domain antibody (DVD-Ig). TM (iv) chemically linked bispecific (Fab')2 fragments; (v) each of the light and heavy chains contains two variable domains linked by short peptide bonds. It is a fusion of two single-chain biantibodies, producing a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) a flexible antibody, which is a combination of scFv and a biantibody, producing a multivalent molecule; (vii) so-called "dock and latch" molecules. According to the "dimerization and docking domain" in protein kinase A, when applied to Fab, it can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) the so-called Scorpion molecule, which contains, for example, two scFvs fused to the two ends of a human Fab arm; and (ix) a biantibody.

[0907] In one embodiment, the bispecific antibody of the present invention is a biantibody, a cross-body, or a bispecific antibody obtained through controlled Fab arm exchange, for example... (e.g., those described in

[41] ), as described in this invention.

[0908] Examples of different types of bispecific antibodies include, but are not limited to: (i) IgG-like molecules having complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual-targeting molecules wherein each side of the molecule contains a Fab fragment or a portion of a Fab fragment of at least two different antibodies; (iii) IgG fusion molecules wherein a full-length IgG antibody is fused with an additional Fab fragment or a portion of a Fab fragment; (iv) Fc fusion molecules wherein a single-chain Fv molecule or a stable biantibody is fused with a heavy chain constant domain, an Fc region, or a portion thereof; (v) Fab fusion molecules wherein different Fab fragments are fused together with a heavy chain constant domain, an Fc region, or a portion thereof; and (vi) ScFv-based and biantibody-based heavy chain antibodies (e.g., domain antibodies, ), where different single-chain Fv molecules or different biantibodies or different heavy-chain antibodies (e.g., domain antibodies, They may fuse with each other, or with another protein or carrier molecule that is fused to the heavy chain constant domain, Fc-region, or a portion thereof.

[0909] Examples of IgG-like molecules with complementary CH3 domains include, but are not limited to, those with complementary CH3 domains. (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 2012Nov 1), DIG and PIG bodies (Pharmabcine,WO2010134666,WO2014081202), chain exchange modified structural 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 antibody (Roche,WO2009080254) and Molecules (Genmab A / S,

[41] ).

[0910] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, dual-targeting (DT)-Ig (GSK / Domantis, WO2009058383), dual-antibody (Genentech, Bostrom et al. 2009. Science 323, 1610–1614), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star,

[54] ), and Zybodies. TM (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr; 5(2): 208-18), using the shared light chain method (Crucell / Merus,

[55] ), κλBodies (NovImmune, WO2012023053) and (CovX / Pfizer, Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17, 501–506).

[0911] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig.TM (Abbott,

[56] ), dual-domain bispecific antibody (Unilever; Sanofi Aventis,

[57] ), IgG-like bispecific antibody (ImClone / EliLilly, Lewis et al. Nat Biotechnol. 2014 Feb; 32(2):191-8), Ts2Ab (MedImmune / AZ, Dimasi et al. J Mol Biol. 2009 Oct 30; 393(3):672-92) and BsAb (Zymogenetics, WO2010111625), HERCULES (Biogen Idec,

[58] ), scFv fusion (Novartis), scFv fusion (Changzhou AdamBiotech Inc,

[59] ) and TvAb (Roche,

[59] ,

[60] ).

[0912] 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), and dual affinity redirecting technology (Fc-DART). TM ) (MacroGenics,

[62] ,

[63] ) and dual (ScFv)2-Fab (National Research Center for Antibody Medicine–China).

[0913] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), dual-action or Bis-Fab (Genentech). (DNL)(ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0914] Examples of ScFv-, biantibody-based antibodies and domain-based antibodies include, but are not limited to, bispecific T-cell adaptors. (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Redirection Technology (DART)) TM (MacroGenics), single-chain biantibody (Academic, Lawrence FEBS Lett. 1998 Apr 3; 425(3):479-84), TCR-like antibody (AIT, Receptor Logics), human serum albumin ScFv fusion (Merrimack, WO2010059315) and COMBODY molecule (Epigen Biotech, Zhu et al. Immunol CellBiol. 2010 Aug; 88(6):667-75), dual-targeting (Ablynx, Hmila et al., FASEBJ. 2010), a dual-targeting antibody targeting only the heavy chain domain.

[0915] It is also anticipated that any monospecific antibody that meets the assay conditions described herein can form the basis for bispecific antibodies. That is, a bispecific antibody in which one of the binding regions binds CD3 can be derived from any monospecific CD3 antibody tested in a functional assay and meeting the requirements described herein. Such bispecific antibodies can be provided by the method described in

[41] , which is incorporated herein by reference.

[0916] In one aspect, the bispecific antibody of the present invention comprises a first Fc region containing a first CH3 region and a second Fc region containing a second CH3 region, wherein the sequences of the first and second CH3 regions are different, and such that the heterodimeric interaction between the first and second CH3 regions is stronger than each homodimeric interaction between the first and second CH3 regions. Further details regarding these interactions and how they are achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0917] Therefore, 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 at least one amino acid in the first heavy chain corresponding to positions selected from T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain is substituted, and at least one amino acid in the second heavy chain corresponding to positions selected from T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain is substituted, wherein the first and second heavy chains are not substituted at the same positions. In this case, the term "substitution" means that an amino acid at a specific amino acid position is replaced by another naturally occurring or non-naturally occurring amino acid. Therefore, an amino acid "substituted" at a position corresponding to a position in the human IgG1 heavy chain means that the amino acid at that specific position is different from the amino acid naturally occurring in the IgG1 heavy chain.

[0918] In one embodiment, the amino acid at position K409 of the human IgG1 heavy chain in the first heavy chain is not K, L, or M, and optionally the amino acid at position F405 of the human IgG1 heavy chain is F, and at least one amino acid at position T366, L368, K370, D399, F405, and Y407 of the human IgG1 heavy chain in the second heavy chain is substituted.

[0919] In one embodiment, the amino acid at position K409 of the human IgG1 heavy chain in the first heavy chain is not K, L, or M, and the amino acid at position F405 of the human IgG1 heavy chain in the second heavy chain is not F, and optionally the amino acid at position K409 of the human IgG1 heavy chain is K.

[0920] In one embodiment, the amino acid at position F405 of the human IgG1 heavy chain in the first heavy chain is not F, R, or G, and the amino acid at position T366, L368, K370, D399, Y407, and K409 of the human IgG1 heavy chain in the second heavy chain is replaced.

[0921] In one embodiment, the amino acid at position K409 of the human IgG1 heavy chain in the first heavy chain is not K, L, or M, and the amino acid at position F405 of the human IgG1 heavy chain is not F.

[0922] In a further embodiment, the amino acid at position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at position K409 of the human IgG1 heavy chain in the second heavy chain is R, or vice versa.

[0923] Therefore, in one embodiment, the amino acid at position K409 of the human IgG1 heavy chain in the first heavy chain is R, and the amino acid at position F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0924] In a further embodiment, the humanized or chimeric CD3 antibody of the present invention comprises, in at least one of the first and second heavy chains, one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E, and D265A; and an amino acid at the position corresponding to F405 that is not F. In one embodiment, the humanized or chimeric CD3 antibody of the present invention comprises, in at least one of the first and second heavy chains, one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E, and D265A; and an additional substitution at the K409 position, such as K409R. Specifically, in one embodiment, the humanized or chimeric CD3 antibody of the present invention comprises, in both the first and second heavy chains, one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E, and D265A; and a substitution at the F405 position, such as F405L. In one embodiment, the humanized or chimeric CD3 antibody of the present invention comprises, in both the first and second heavy chains, one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E, and D265A; and an additional substitution at the K409 position, such as K409R. Such an antibody can be used to generate bispecific antibodies.

[0925] Therefore, in a further embodiment, the amino acids 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; the amino acid corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L; and the amino acid corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0926] In one embodiment, the amino acids corresponding to positions L234, L235, D265, N297, and P331 of the 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 corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L; and the amino acid corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0927] In an alternative embodiment, the amino acids 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; the amino acid corresponding to position K409 of the human IgG1 heavy chain in the first heavy chain is R; and the amino acid corresponding to position F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0928] In one embodiment, the amino acids corresponding to the positions L234, L235, D265, N297, and P331 of the 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 corresponding to the position K409 of the human IgG1 heavy chain in the first heavy chain is R; and the amino acid corresponding to the position F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0929] In another embodiment, the amino acids corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E, and A, respectively; the amino acid corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L; and the amino acid corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0930] In one embodiment, the amino acids corresponding to the positions L234, L235, D265, N297, and P331 of the human IgG1 heavy chain in both the first and second heavy chains are F, E, A, N, and P, respectively; the amino acid corresponding to the position F405 of the human IgG1 heavy chain in the first heavy chain is L; and the amino acid corresponding to the position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0931] In an alternative embodiment, the amino acids corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E, and A, respectively; the amino acid corresponding to position K409 of the human IgG1 heavy chain in the first heavy chain is R; and the amino acid corresponding to position F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0932] In one embodiment, the amino acids corresponding to the positions L234, L235, D265, N297, and P331 of the human IgG1 heavy chain in both the first and second heavy chains are F, E, A, N, and P, respectively; the amino acid corresponding to the position K409 of the human IgG1 heavy chain in the first heavy chain is R; and the amino acid corresponding to the position F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0933] As described herein, T cells are recruited to specific target cells, such as cancer or tumor cells, providing a method for killing the target cells. T cell-mediated killing can be achieved with a bispecific antibody that targets CD3 in a first binding region and another target in a second binding region. Thus, in one embodiment, the first binding region is as described herein for any embodiment of a humanized or chimeric CD3 antibody, 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, one half of the bispecific antibody is a humanized or chimeric antibody that binds CD3 according to the invention, and the other half may be a humanized, chimeric, completely non-human, or completely human antibody that binds to a second target. Therefore, in one embodiment, the antibody comprises first and second heavy chains, and first and second light chains, wherein the first heavy chain and the first light chain are humanized or chimeric and are connected by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are fully human and are connected by a disulfide bridge to form a second binding region, wherein the first binding region, according to any aspect or embodiment described herein, and the second binding region bind different targets. In one embodiment, the antibody comprises first and second heavy chains, and first and second light chains, wherein the first heavy chain and the first light chain are humanized or chimeric and are connected by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are humanized or chimeric and are connected by a disulfide bridge to form a second binding region, wherein the first binding region, according to any aspect or embodiment described herein, and the second binding region bind different CD3 epitopes from the first binding region.

[0934] The term “disulfide bridge” used in this article refers to a covalent bond between two cysteine ​​residues, and the interaction can also be referred to as the Cys-Cys interaction.

[0935] As used herein, the term "target" refers to the molecule to which the binding region of the antibody of the present invention binds. When used in the context of antibody binding, the term includes any antigen targeted by the resulting antibody.

[0936] In one specific implementation, the first heavy chain and the first light chain are humanized or chimeric and are connected by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are fully human and are connected by a disulfide bridge to form a second binding region, wherein the first binding region binds to different targets according to any aspect or implementation thereof; and wherein the amino acids at 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.

[0937] In one specific implementation, the first heavy chain and the first light chain are humanized or chimeric and connected by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region binds a different CD3 epitope according to any aspect or implementation described herein; and wherein the amino acids at 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.

[0938] In one specific implementation, the first heavy chain and the first light chain are humanized or chimeric and connected by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are fully human and connected 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 implementation described herein; and wherein the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E, and A, respectively.

[0939] In one specific implementation, the first heavy chain and the first light chain are humanized or chimeric and connected by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region binds a different CD3 epitope according to any aspect or implementation described herein; and wherein the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E, and A, respectively.

[0940] In another aspect, the present invention relates to a method for reducing the binding affinity of an antibody to human CD3 compared to a reference antibody containing a heavy chain variable (VH) region, wherein the VH region comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO:1, 2, and 3, the method comprising introducing a mutation in one of the three CDR sequences of the reference antibody, the mutation being selected from mutations at one of the following positions: T31M, T31P, N57, H101, S110, and Y114, wherein the positions are numbered according to the reference sequence of SEQ ID NO:4.

[0941] The amino acids in the VH region are numbered according to the amino acid sequence in SEQ ID NO:4. The numbering follows a direct numerical numbering scheme from the first amino acid to 125 in the direction from the N-terminus to the C-terminus. Figure 2 The numerical designation corresponding to the position of SEQ ID NO:4 is shown. Furthermore, the CDR area has been annotated according to the IMGT definition.

[0942] In one embodiment of the invention, the method includes introducing a T31M or T31P mutation. Position T31 is defined according to SEQ ID NO:4.

[0943] In one embodiment of the invention, the method includes introducing a mutation at position N57. Position N57 is defined in SEQ ID NO:4. In one embodiment, the mutation is N57E.

[0944] In one embodiment of the invention, the method includes introducing a mutation at position H101. Position H101 is defined in SEQ ID NO:4. In one embodiment, the mutation is H101G or H101N.

[0945] In one embodiment of the invention, the method includes introducing a mutation at position Y114. Position Y114 is defined in SEQ ID NO:4. In one embodiment, the mutation is Y114, Y114R, or Y114V.

[0946] In one embodiment of the invention, the method includes introducing a mutation in a mutation in a VH CDR3 region corresponding to a location selected from H101, S110, and Y114.

[0947] In one embodiment of the invention, the method includes introducing a mutation into the VH CDR3 region, selected from H101G, H101N, S110A, S110G, Y114M, Y114R, and Y114V.

[0948] In one embodiment of the invention, the method includes introducing a mutation, wherein the binding affinity of the antibody to the human CD3ε peptide having SEQ ID NO:402 corresponds to 1.6 x 10⁻⁶. -8 M to 9.9x10 -8 M or 1.0x10 -7 Up to 9.9x10 -7 M of K D Values, such as those determined by biological layer interferometry.

[0949] In one embodiment of the invention, the method includes introducing a mutation, wherein the antibody has a binding affinity for the human CD3ε peptide having SEQ ID NO:402 corresponding to 1.4 x 10⁻⁶. -8 M to 1.0x10 -8 M or 9.9x10 -9 Up to 1x10 -9 K D Values, such as those determined by biological layer interferometry.

[0950] 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 x 10⁻⁶. -8 M to 9.9x10 -8 M or 1.0x10 -7 Up to 9.9x10 -7 M of K D Values, such as those determined by biological layer interferometry.

[0951] On the other hand, the present invention relates to a method for increasing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody containing a heavy chain variable region (VH) region, wherein the VH region contains CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, 2, 3, and the method includes introducing a mutation in the VH CDR3 corresponding to position G105, wherein the position is numbered according to the reference sequence of SEQ ID NO:4.

[0952] In one embodiment, the method includes introducing a mutation at position G105. Position G105 is defined in SEQ ID NO:4. In one embodiment, the mutation is G105P.

[0953] In one embodiment of the invention, the method includes 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 CDR of the VH region of the reference antibody shown in SEQ ID NO: 1, 2, 3.

[0954] In one embodiment of the invention, a method for increasing or decreasing binding affinity includes a binding region comprising a heavy chain variable region (VH), wherein the VH region comprises a CDR1, CDR2, and CDR3 sequence selected from the following:

[0955] a) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:54,2,3[T31M];

[0956] b) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:58,2,3[T31P];

[0957] c) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,106,3[N57E];

[0958] d) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,176[H101G];

[0959] e) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,184[H101N];

[0960] f) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,220[G105P];

[0961] g) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,236[S110A];

[0962] h) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,244[S110G];

[0963] i) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,284[Y114M];

[0964] j) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1,2,292[Y114R]; and

[0965] k) The CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:1,2,298[Y114V].

[0966] In another embodiment of the invention, the method includes introducing a mutation in the CDR2 region of the VH region corresponding to N57E. In another embodiment of the invention, the method includes introducing a mutation in the CDR3 region of the VH region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V. On the other hand, the invention relates to methods for reducing or increasing the binding affinity of an antibody to CD3, wherein the antibody comprises a binding region containing a heavy chain variable (VH) region, wherein the VH region contains a mutation in one of the three CDR sequences of a reference antibody shown in CDR1 SEQ ID: 1, CDR2 SEQ ID: 2, and CDR3 SEQ ID: 3, wherein the antibody contains a mutation at 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.

[0967] In one embodiment of the invention, the method includes introducing a mutation in the VH region CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the invention, the method includes introducing a mutation in the VH region CDR2 region corresponding to N57E. In yet another embodiment of the invention, the method includes introducing a mutation in the VH region CDR3 region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V.

[0968] On the other hand, the present invention relates to a method for reducing the binding affinity of an antibody that binds to CD3 compared to a reference antibody containing a heavy chain variable (VH) region, wherein the VH region comprises CDR1, CDR2, and CDR3 having the CDR sequences shown in SEQ ID NO:1, 2, and 3, the method comprising introducing a mutation in one of the CDR1, CDR2, or CDR3 sequences of the VH region shown in SEQ ID NO:1, 2, or 3.

[0969] In one embodiment of the invention, the method includes introducing a mutation in one of 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.

[0970] In one embodiment of the invention, the method includes 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 NO1. When the mutation is represented by X, the resulting CDR1 sequence can present as GFTFNXYA. In one embodiment, the three CDR sequences of the VH region can have the following sequences: CDR1 GFTFNXYA, CDR2 IRSKYNNYAT, and CDR3 VRHGNFGNSYVSWFAY In one implementation, the mutation at position T31 in CDR1 of the VH region is either a T31M or a T31P mutation.

[0971] In one embodiment of the invention, the method includes 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 can present as IRSKYNXYAT. In one embodiment, the three CDR sequences of the VH region can have the following sequences: CDR1 GFTFNTYA, CDR2 IRSKYNXYAT, and CDR3 VRHGNFGNSYVSWFAY In one implementation, the mutation at position N57 in CDR2 of the VH region is the N57E mutation.

[0972] In one embodiment of the invention, the method includes introducing a mutation into the CDR3 sequence in 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 can present as follows: VRXGNFGNSYVSWFAY In one implementation, the three CDR sequences in the VH region may have the following sequences: CDR1GFTFNTYA, CDR2IRSKYNNYAT, and CDR3. VRXGNFGNSYVSWFAY In one implementation, the mutation at position H101 in the VH region CDR3 is either an H101G or H101N mutation.

[0973] In one embodiment of the invention, the method includes introducing a mutation into the CDR3 sequence in the VH region 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 can present as follows: VRHGNFGNSYVXWFAY In one implementation, the three CDR sequences in the VH region may have the following sequences: CDR1GFTFNTYA, CDR2IRSKYNNYAT, and CDR3. VRHGNFGNSYVXWFAY In one implementation, the mutation at position H101 in CDR3 of the VH region is an S110A or S110G mutation.

[0974] In one embodiment of the invention, the method includes introducing a mutation into the CDR3 sequence in the VH region 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 can present as follows: VRHGNFGNSYVSWFAX In one implementation, the three CDR sequences in the VH region may have the following sequences: CDR1GFTFNTYA, CDR2IRSKYNNYAT, and CDR3. VRHGNFGNSYVSWFAX In one implementation, the mutation at position Y114 in CDR3 of the VH region is a Y114M, Y114R, or Y114V mutation.

[0975] In one embodiment of the invention, the method includes introducing up to three mutations, up to two mutations, or up to one 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.

[0976] In one embodiment of the invention, the method includes 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 an antibody, wherein the mutations preferably do not alter the binding of the antibody to CD3 compared to the same antibody without the mutation.

[0977] In one embodiment of the invention, the method includes introducing a mutation selected from T31M or T31P into the VH region CDR1 sequence. In another embodiment of the invention, the method includes introducing a mutation into the N57E VH region CDR2 sequence. In yet another embodiment of the invention, the method includes introducing a mutation into the VH region CDR3 sequence selected from H101G, H101N, S110A, S110G, Y114M, Y114R, and Y114V.

[0978] On the other hand, the present invention relates to a method for increasing the binding affinity of an antibody that binds to CD3 compared to a reference antibody containing a heavy chain variable (VH) region, wherein the VH region comprises a CDR sequence having CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1, 2, and 3, the method comprising introducing a mutation in one of the CDR1, CDR2, or CDR3 sequences of the VH region shown in SEQ ID NO:1, 2, or 3.

[0979] In one embodiment of the invention, the method includes introducing a mutation into the CDR3 sequence in the VH region corresponding to position G105, wherein the CDR3 sequence is as shown in SEQ ID NO 3. When the mutation is represented by X, the resulting CDR3 sequence can present as follows: VRHGNFXNSYVSWFAY In one implementation, the three CDR sequences in the VH region may have the following sequences: CDR1GFTFNTYA, CDR2IRSKYNNYAT, and CDR3. VRXGNFGNSYVSWFAY In one implementation, the mutation at position G105 in CDR3 of the VH region is the G105P mutation.

[0980] Nucleic acid constructs, expression vectors, and host cells

[0981] In one aspect, the present invention relates to nucleic acid constructs encoding one or more sequences shown in Table 1. Therefore, the present invention relates to nucleic acid constructs encoding any one of the sequences shown in SEQ ID NO: 107; 221; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293.

[0982] 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 present invention, an expression vector comprising the nucleic acid construct of the present invention, a host cell comprising the expression vector, and a method for producing the antibody by culturing the host cell under suitable conditions for producing and optionally recovering the antibody. Humanized CD3 antibodies are also referred to as "huCD3".

[0983] 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.

[0984] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of a heavy chain and a light chain CDR sequence, wherein the VH CDR sequence is selected from: SEQ ID NO. 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 in the CDR sequences.;

[0985] 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 CDR sequences of the VL region CDR1, CDR2, CDR3 region comprise the CDR sequences shown in SEQ ID NO: 6, GTN, 7 and the CDR sequences of the VH region CDR1, CDR2, CDR3 region are selected from: CDR1, CDR2, CDR3 shown in SEQ ID NOs.: 54, 2, 3; CDR1, CDR2, CDR3 shown in SEQ ID NO: 58, 2, 3; CDR1, CDR2, CDR3 shown in SEQ ID NO: 1, 106, 3; CDR1, CDR2, CDR3 shown in SEQ ID NO: 1, 2, 176; CDR1, CDR2, CDR3 shown in SEQ ID NO: 1, 2, 184; CDR1, CDR2, CDR3 shown in SEQ ID NO: 1, 2, 220; and CDR1, CDR2, CDR3 shown in SEQ ID NO: 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.

[0986] In specific embodiments, the expression vector comprises a nucleic acid sequence encoding one or more variants of the above-described amino acid sequences, said 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 modification, such as deletion or insertion, preferably substitution, such as conserved or non-conserved substitution, or having at least 80% identity with any of said sequences, such as at least 85%, 90%, or 95%, such as 96%, 97%, 98%, or 99% identity with any of said amino acid sequences. The invention also relates to nucleic acid sequences that differ from the above-described nucleic acid sequences but encode the same amino acid sequence as the antibodies of the invention due to differences in the genetic code. For example, the nucleic acid sequences may differ 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 those skilled in the art.

[0987] In a further embodiment, the expression vector further comprises a nucleic acid sequence encoding a constant region of the light chain, heavy chain, or both the light and heavy chains of an antibody (e.g., a human antibody).

[0988] The expression vectors described above can be used for recombinant generation of the antibodies of this invention.

[0989] In the context of this invention, the expression vector can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (containing a nucleic acid sequence of a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, humanized or chimeric CD3 antibody-encoding nucleic acid is contained in a naked DNA or RNA vector, including, for example, linear expression elements (as described in, for example,

[64] ), dense nucleic acid vectors (as described in, for example,

[65] and / or

[66] ), plasmid vectors such as pBR322, pUC 19 / 18, or pUC 118 / 119, "midge" minimum size nucleic acid vectors (as described in, for example,

[67] ), or as a precipitated nucleic acid vector construct, such as CaPO4. - -Constructions of the precipitate (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] ).

[0990] In one embodiment, the vector is suitable for expressing humanized or chimeric CD3 antibodies in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (

[74] ), pET vectors (Novagen, MadisonWI), etc.

[0991] 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 α-factors, alcohol oxidases, and PGH (reviewed in

[75] and

[76] ).

[0992] 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) into the cytoplasm or cell culture medium. Such sequences are known in the art and include secretion leader sequences or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER-retaining signals, mitochondrial transport sequences, chloroplast transport sequences), membrane localization / anchoring sequences (e.g., termination transfer sequences, GPI anchor sequences), etc., which are well-known in the art.

[0993] In the expression vectors of the present invention, the humanized or chimeric CD3 antibody-encoding nucleic acid may contain or be associated with any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer, as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origin of replication of plasmid products in *E. coli*, antibiotic resistance genes as selective markers, and / or suitable cloning sites (e.g., polymer adapters). The nucleic acid constructs and / or vectors may also contain inducible promoters, such as CMV IE, as opposed to constitutive promoters (those skilled in the art will appreciate that these terms are, in effect, descriptors of gene expression levels under certain conditions).

[0994] In one implementation, the humanized or chimeric CD3 antibody-encoding expression vector is located in a host cell or host animal, and / or delivered to the host cell or host animal via a viral vector.

[0995] Such expression vectors can be used to recombinantly generate humanized or chimeric CD3 antibodies.

[0996] In one aspect, the present invention provides a host cell comprising the expression vector of the present invention.

[0997] In one aspect, any humanized or chimeric CD3 antibody described in any aspect or embodiment herein is provided using a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell that produces the antibody. Therefore, 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 that includes a sequence encoding the expression of the humanized or chimeric CD3 antibody described herein. In another embodiment, the host cell comprises a non-integrated nucleic acid sequence, such as a plasmid, granule, phage particle, or linear expression element, that includes a sequence encoding the expression of the humanized or chimeric CD3 antibody described herein.

[0998] As used herein, the term "recombinant host cell" (or simply "host cell") refers to the cell into which the expression vector, nucleic acid construct, or sequence is introduced. It should be understood that this term refers not only to the specific test cell but also to the progeny of said cells. Because certain modifications may occur during passage due to mutations or environmental influences, said progeny may indeed differ from the parent cells, 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, NSO cells, and lymphocytes; and prokaryotic cells, such as Escherichia coli; and other eukaryotic hosts, such as plant cells and fungi.

[0999] In a further aspect, the present invention relates to a method for generating the humanized or chimeric CD3 antibody of the present invention, the method comprising the following steps:

[1000] a) Culturing the host cells of the present invention as described above, and

[1001] b) Recover and / or purify the antibodies of the present invention from the culture medium.

[1002] In a further aspect, the nucleotide sequence encoding the humanized or chimeric CD3 antibody further encodes a second portion, such as a therapeutic peptide. Exemplary therapeutic peptides are further described 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:

[1003] a) Culturing host cells, said host cells containing an expression vector comprising the said nucleotide sequence, and

[1004] b) Recover and / or purify humanized or chimeric CD3 antibody fusion proteins from the culture medium.

[1005] Composition

[1006] In one aspect, the present invention provides compositions comprising antibodies or bispecific antibodies comprising any of the aspects and embodiments described herein.

[1007] In one aspect, the present invention provides a pharmaceutical composition comprising an antibody or bispecific antibody as defined in any of the aspects and embodiments described herein and a pharmaceutically acceptable carrier.

[1008] Pharmaceutical compositions may be formulated using conventional techniques, such as those described in

[77] , with pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients.

[1009] Pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, should be suitable for the humanized or chimeric antibodies of the present invention and the chosen route of administration. The suitability of the carriers and other components of the pharmaceutical composition is determined based on the absence of any significant negative impact on the biological properties required for antigen binding to 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.)).

[1010] The pharmaceutical compositions of the present invention may further include diluents, fillers, salts, buffers, detergents (e.g., nonionic 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 the pharmaceutical composition.

[1011] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and route of administration and is non-toxic to the patient. The selected dose level will depend on various pharmacokinetic factors well known in the medical field, including the activity of the specific composition of the present invention or its amide, the route of administration, the frequency of administration, the excretion rate of the specific compound used, the duration of treatment; other drugs, compounds, and / or materials used in combination with the specific composition used; and factors such as the age, sex, weight, condition, general health, and previous medical history of the patient being treated.

[1012] The pharmaceutical composition may be administered via any suitable route and method. 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 those skilled in the art.

[1013] In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.

[1014] The phrases “parenteral administration” and “via gastrointestinal administration” as used in this article refer to administration methods other than enteral and local administration, usually by injection, and including epidermal, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injections and infusions.

[1015] In one embodiment, the pharmaceutical composition is administered via intravenous or subcutaneous injection or infusion.

[1016] In a preferred embodiment, the pharmaceutical composition is administered subcutaneously.

[1017] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delay agents, which are physiologically compatible with the humanized or chimeric antibodies of the present invention.

[1018] Examples of suitable aqueous and non-aqueous carriers for use 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; carboxymethyl cellulose colloidal solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffer solutions. Other carriers are well known in the pharmaceutical industry.

[1019] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions of the present invention is contemplated unless any conventional media or reagent is incompatible with the active compound. When referring to "active compound," the humanized or chimeric antibodies of the present invention are also contemplated.

[1020] Appropriate flowability can be achieved, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersants, and by using surfactants.

[1021] The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable antioxidants, 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 chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[1022] The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyols, such as mannitol, sorbitol, glycerol, or sodium chloride.

[1023] The pharmaceutical compositions of the present invention may further comprise one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, or buffers, which may improve 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 controlled-release formulations, 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, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with waxes, 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] ).

[1024] In one embodiment, the humanized or chimeric antibody of the present invention may 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 provisional preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art. Unless any conventional media or reagent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated. Other active or therapeutic compounds may also be incorporated into the composition.

[1025] Injectable pharmaceutical compositions must generally be sterile and stable under preparation and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be an aqueous or non-aqueous solvent or dispersion medium, including, for example, water, ethanol, polyols (e.g., 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 flowability can be maintained, for example, by using a coating material such as lecithin, or, in the case of a dispersant, by maintaining the desired particle size and by using a surfactant. In many cases, it is preferred to include an isotonic agent in the composition, such as sugars, polyols such as glycerol, mannitol, sorbitol, or sodium chloride. Extended absorption of the injectable composition can be achieved by including a delay-absorption agent in the composition, such as monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound with, as needed, one or a combination of the ingredients listed above, into a suitable solvent, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile solvent containing a basic dispersion matrix and other desired components, such as those listed above. In the case of sterile powders used to prepare sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient from its previously sterile filtered solution plus any other desired components.

[1026] Sterile injectable solutions can be prepared by incorporating the required amount of active compound with one or a combination of the ingredients listed above, as needed, into a suitable solvent, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile solvent containing a base dispersion medium and any other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient from its previously sterile filtered solution plus any other desired ingredients.

[1027] Therapeutic applications

[1028] In another aspect, the present invention relates to humanized or chimeric antibody or pharmaceutical compositions of the present invention as defined in any aspect or embodiment described herein, which are used as pharmaceuticals.

[1029] In another aspect, the present invention relates to humanized or chimeric antibody or pharmaceutical compositions of the present invention as defined in any aspect or embodiment described herein, for the treatment of diseases.

[1030] In one embodiment of the present invention, a bispecific antibody, composition, or pharmaceutical composition is used to treat a disease.

[1031] In one embodiment of the present invention, a bispecific antibody, composition, or pharmaceutical composition is used to treat a disease, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

[1032] The humanized or chimeric antibody or pharmaceutical compositions of the present invention can be used to treat any cancer in which an effector mechanism of cytotoxic T cells is required. For example, the humanized or chimeric antibody can be administered to cells, for example, in vitro or in vitro cultured, or to a human subject, for example, in vivo, to treat or prevent conditions such as cancer, inflammatory, or autoimmune conditions. The term "subject" as used herein generally refers to a person who responds to the humanized or chimeric antibody or pharmaceutical composition. A subject may, for example, include a patient with a condition that can be corrected or improved by modulating target function or by directly or indirectly causing cell killing.

[1033] On the other hand, the present invention provides a method for treating or preventing conditions such as cancer, wherein the recruitment of T cells will contribute to the treatment or prevention, the method comprising administering a therapeutically effective amount of the humanized or chimeric antibody or pharmaceutical composition of the present invention to a subject in need. The method generally comprises administering to the subject an amount of humanized or chimeric antibody effective in treating or preventing said condition.

[1034] In one particular aspect, the present invention relates to methods of treating cancer, comprising administering the humanized or chimeric antibody or pharmaceutical composition of the invention as defined in any aspect and embodiment described herein to a subject in need.

[1035] In another aspect, the present invention relates to the use or method defined in any aspect or embodiment described herein, wherein the humanized or chimeric antibody is a bispecific antibody that specifically binds to both CD3 and a cancer-specific target or a target 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 (e.g., synovial sarcoma), painless or invasive forms of B-cell lymphoma, chronic lymphocytic leukemia, or acute lymphoblastic leukemia.

[1036] The effective dose and dosing regimen of humanized or chimeric antibodies depend on the disease or condition to be treated and can be determined by those skilled in the art.

[1037] Physicians with ordinary skills in the art can readily determine and prescribe the required effective amount of the pharmaceutical composition. For example, a physician may start with a dose of the humanized or chimeric antibody used in the pharmaceutical composition below the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Generally, a suitable dose of the composition of the present invention is the amount of the lowest dose of humanized or chimeric antibody that effectively produces a therapeutic effect according to a specific dosing regimen. Such an effective dose generally depends on the factors described above.

[1038] 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 predicting efficacy in human tumors. Alternatively, this property of the composition can be evaluated by in vitro assays known to those skilled in the art to examine the ability of humanized or chimeric antibodies to inhibit cell growth or induce cytotoxicity. A therapeutically effective amount of the therapeutic compound, i.e., the therapeutically humanized or chimeric antibody or pharmaceutical composition of the present invention, can reduce tumor size or otherwise improve symptoms in a subject. Those skilled 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 specific composition or chosen route of administration.

[1039] Exemplary, non-limiting ranges for the therapeutically effective amount of the humanized or chimeric antibody of the present invention are about 0.001-30 mg / kg, for example about 0.001-20 mg / kg, for example about 0.001-10 mg / kg, for example about 0.001-5 mg / kg, for example about 0.001-2 mg / kg, for example about 0.001-1 mg / kg, for example 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.

[1040] Administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and may be administered, for example, near the target site.

[1041] The dosage regimens used in the above treatment methods and uses are adjusted to provide the best possible response (e.g., therapeutic response). For example, a single bolus may be given, several separate doses may be given over time, or the dose may be reduced or increased proportionally as indicated by the urgency of the treatment situation.

[1042] In one implementation, the efficacy of the treatment is monitored during treatment, for example at predetermined time points.

[1043] If desired, the effective daily dose of the pharmaceutical composition may be administered as sub-dose given individually at appropriate time intervals throughout the day, two, three, four, five, six or more times, optionally in unit dose form. In another embodiment, the humanized or chimeric antibody or pharmaceutical composition may be administered via slow, continuous infusion over a prolonged period, such as more than 24 hours, to minimize unwanted side effects.

[1044] Although it is possible to administer the humanized or chimeric antibodies of the present invention alone, it is preferred to administer them as part of the pharmaceutical composition described above.

[1045] Effective doses of the humanized or chimeric antibodies of the present invention may also be administered on a dosing schedule of once a week, once every two weeks, or once every three weeks. The dosing schedule may be limited, for example, to 8 weeks, 12 weeks, or until clinical progress is confirmed. Alternatively, effective doses of the humanized or chimeric antibodies of the present invention may be administered every one, two, or three weeks.

[1046] In one implementation, the humanized or chimeric antibody can be administered via infusion at a dose of mg / m³. 2The calculated weekly dose is administered. Such a dose can be, for example, based on the mg / kg dose provided above, according to the following formula: dose (mg / kg) x 70: 1.8. Such administration can be repeated, for example, 1-8 times, or, for example, 3-5 times. Administration can be carried out by continuous infusion over a period of 2-24 hours, for example, 2-12 hours. In one embodiment, the humanized or chimeric antibody can be administered by slow continuous infusion over a longer period of time, for example, more than 24 hours, to reduce toxic side effects.

[1047] In one implementation, when administered once weekly, the humanized or chimeric antibody can be given as a fixed-dose once-weekly dose up to eight times, for example, four to six times. Such a regimen can be repeated once or more as needed, for example, after 6 or 12 months. Such a fixed dose can be based, for example, on the mg / kg dose provided above, with a body weight estimated at 70 kg. The dose can be determined or adjusted by measuring the amount of the humanized or chimeric antibody of the present invention in the blood after administration, for example, by obtaining a biological sample and using an anti-individual genotype antibody targeting the binding region of the humanized or chimeric antibody of the present invention.

[1048] In one implementation, humanized or chimeric antibodies can be administered via maintenance therapy, for example, once a week for a period of 6 months or more.

[1049] 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 alleviated.

[1050] For ease of administration and uniform dosage, parenteral compositions may be formulated in unit dosage form. As used herein, unit dosage form refers to a physically discrete unit suitable as a single dose to a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect and a desired pharmaceutical carrier. The specifications of the unit dosage form of the present invention are indicated by and directly dependent on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of formulating such active compounds for the treatment of individuals with varying sensitivities.

[1051] 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. This can be particularly useful in patients where tumors are difficult to locate due to other biological factors.

[1052] Diagnostic applications

[1053] Using compositions comprising the humanized or chimeric antibodies described herein, the humanized or chimeric antibodies of the present invention can also be used for diagnostic purposes. Therefore, 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 diseases, and for monitoring the progress of therapeutic treatment, monitoring disease progression, evaluating post-treatment status, monitoring disease recurrence, assessing the risk of disease occurrence, etc.

[1054] In one aspect, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, comprising administering a humanized or chimeric antibody of the present invention, a composition of the present invention, or a pharmaceutical composition of the present invention to a subject, optionally wherein the humanized or chimeric antibody is labeled with a detectable reagent.

[1055] In one aspect, the humanized or chimeric antibodies of the present invention are used in vitro, for example, to diagnose diseases or diseases involving pathogenesis by detecting the level of a target in a sample obtained from a patient or in cells expressing the target on their cell surface. Cells expressing a specific target and bound by the humanized or chimeric antibody indicate a disease. This can be achieved, for example, by contacting the test sample, optionally together with a control sample, with the humanized or chimeric antibody of the present invention under conditions that allow the antibody to bind to the target. Complex formation can then be detected (e.g., using an ELISA). When the control sample is used in conjunction with the test sample, the levels of the humanized or chimeric antibody or antibody-target complex are analyzed in both samples, and a statistically significant higher level of the humanized or chimeric antibody or antibody-target complex in the test sample indicates a higher level of the target in the test sample compared to the control sample.

[1056] Examples of routine immunoassays in which the humanized or chimeric antibodies of the present invention can be used include, but are not limited to, ELISA, RIA, FACS analysis, plasma resonance analysis, chromatographic analysis, tissue immunohistochemistry, Western blotting and / or immunoprecipitation.

[1057] Therefore, in one embodiment, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, comprising administering an antibody, bispecific antibody, composition, or pharmaceutical composition of any aspect or embodiment described herein to a subject, optionally wherein the antibody is labeled with a detectable marker.

[1058] In one embodiment, the present invention relates to a method for detecting the presence of a target or cells expressing a target in a sample, comprising:

[1059] - To contact the sample with the 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

[1060] - Analyze whether a complex is formed. Typically, the sample is a biological sample.

[1061] In one implementation, the sample is a tissue sample known or suspected to contain a specific target and / or cells expressing the target. For example, in situ detection of target expression can be achieved by removing a histological sample from a patient and providing the humanized or chimeric antibody of the present invention to such a sample. The humanized or chimeric antibody can be provided by applying or coating the sample with the humanized or chimeric antibody, which is then detected using a suitable tool. It may then be possible not only to detect the presence of the target or cells expressing the target, but also to detect the distribution of the target or cells expressing the target in the examined tissue (e.g., in the case of evaluating the spread of cancer cells). Using the present invention, those skilled in the art will readily understand that any of a variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[1062] In the above-described assay, the humanized or chimeric antibody may be labeled with a detectable substance to allow the bound antibody to be detected. Alternatively, the bound (first)-specific humanized or chimeric antibody may be detected by labeling with a detectable substance and binding an antibody to the first-specific humanized or chimeric antibody. Furthermore, in the above-described assay, diagnostic compositions comprising antibodies or bispecific antibodies of any aspect or embodiment described herein may be used. Therefore, in one aspect, the present invention relates to diagnostic compositions comprising antibodies or bispecific antibodies of any aspect or embodiment described herein.

[1063] Target levels in samples can also be evaluated using a competitive immunoassay with target standards labeled with detectable substances and unlabeled target-specific humanized or chimeric antibodies. In this type of assay, a biological sample, labeled target standards, and target-specific humanized or chimeric antibodies are combined, and the amount of labeled target standards binding 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 labeled target standards bound to the target-specific humanized or chimeric antibody.

[1064] Suitable labeling for target-specific humanized or chimeric antibodies, secondary antibodies, and / or target standards used in in vitro diagnostic techniques includes, but is not limited to, various enzymes, cofactors, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine 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.

[1065] In one aspect, the target-specific humanized or chimeric antibodies of the present invention are used for in vivo imaging of tissues expressing the target, such as tumors. For in vivo methods, antibody fragments such as (Fab')2, Fab, and Fab' fragments are particularly advantageous due to their rapid distribution kinetics.

[1066] In vivo imaging can be performed using any suitable technique. For example, using... 99 Tc, 131 I, 111 Target-specific humanized or chimeric antibodies (e.g., antibodies or fragments) labeled with In or other gamma-ray emitting isotopes can be used to image the accumulation or distribution of the target-expressing antibody in tissues such as tumors using a gamma scintillation camera (e.g., the ElscintApex 409ECT device), typically employing a low-energy high-resolution collimator or a low-energy universal collimator. Alternatively, using... 89 Zr、 76 Br, 18 F or other positron-emitting radionuclides can be used in tumors with positron emission tomography (PET) to image the distribution of target-specific humanized or chimeric antibodies or antibody fragments. Images obtained using such techniques can be used to evaluate the biodistribution of the target in a patient, mammal, or tissue, for example, when the target is used as a biomarker of the presence of cancer / tumor cells. Variations of this technique may include the use of magnetic resonance imaging (MRI) to improve imaging via gamma camera techniques. Conventional immunoscintillation methods and principles are described, for example, in

[79] ,

[80] , and

[81] . Furthermore, such images can also, or alternatively, serve as the basis for surgical techniques to remove tumors. In addition, such in vivo imaging techniques can allow for the 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 conventional analytical techniques. All of these methods are characteristic of the present invention.

[1067] The in vivo imaging and other diagnostic methods provided by this invention can be used specifically to detect micrometastases in human patients (e.g., patients who have not been previously diagnosed with cancer or patients in the recovery / remission phase of cancer).

[1068] 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 with a radiopaque reagent to facilitate 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 are analyzed. Through this technique and any other diagnostic method provided herein, the present invention provides methods for screening the presence of disease-related cells in human patients or biological samples obtained from human patients and / or for evaluating the distribution of target-specific humanized or chimeric antibodies prior to target-specific ADC therapy.

[1069] For diagnostic imaging, radioisotopes can bind directly to target-specific humanized or chimeric antibodies or indirectly through the use of intermediate functional groups. Useful intermediate functional groups include chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (see, for example,

[82] ).

[1070] In addition to radioisotopes and radiopaque reagents, diagnostic methods may be performed using target-specific antibodies conjugated with dyes (e.g., biotin-streptoantibiotin complexes), contrast agents, fluorescent compounds, or molecules and enhancers for magnetic resonance imaging (MRI) (e.g., paramagnetic ions) (see, for example,

[83] , which describes MRI techniques and the preparation of antibodies conjugated with MRI enhancers). Such diagnostic / detection reagents may be selected from reagents and fluorescent compounds used for MRI. To load target-specific humanized or chimeric antibodies with radioactive metals or paramagnetic ions, it may be necessary to react them with a reagent having a long tail to which a multiple chelating group is attached to bind the ion. Such a tail may be a polymer such as polylysine, polysaccharide, or another derivatized or derived chain with a side-attached group, to which the chelating group may be attached, for example, porphyrin, polyamine, crown ether, dithiocarbazone, polyoxime, etc., known for this purpose. The chelating agent may be conjugated to the target-specific humanized or chimeric antibody using standard chemistry.

[1071] Therefore, the present invention provides diagnostic target-specific humanized or chimeric antibodies, wherein the target-specific humanized or chimeric antibodies are conjugated to a contrast agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound contrast enhancement) or a radionuclide (which may be, for example, γ-, β-, α-, Auger electron- or positron-emitting isotopes).

[1072] In one aspect, the present invention relates to diagnostic compositions comprising the antibodies or bispecific antibodies of the present invention.

[1073] In a further aspect, the present invention relates to a kit for detecting the presence of target antigens or cells expressing targets in a sample, comprising:

[1074] - Target-specific humanized or chimeric antibodies of the present invention; and

[1075] - Instructions for use of the kit.

[1076] Therefore, in one aspect, the present invention provides a kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising the following steps:

[1077] a) Contact the sample with the antibody or bispecific antibody of the present invention under conditions that allow for the formation of a complex between the antibody or bispecific antibody and CD3; and

[1078] b) Analyze whether a complex is formed.

[1079] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing a target-specific humanized or chimeric antibody, and one or more reagents for detecting the binding of the target-specific humanized or chimeric antibody to a target. The reagents may include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include a second or third antibody or reagents 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 comprising, in a suitable container, one or more target-specific humanized or chimeric antibodies of the present invention in labeled or unlabeled form, reagents for incubation in an indirect assay, and a substrate or derivatizing reagent (depending on the nature of the label) for detection in such an assay. Control reagents and instructions for use may also be included.

[1080] Diagnostic kits are also available for use with target-specific humanized or chimeric antibodies, such as labeled target-specific antibodies, to detect the presence of a target in tissue samples or a host. In such diagnostic kits, as well as in kits for therapeutic use described elsewhere herein, the target-specific humanized or chimeric antibody is typically provided in lyophilized form in a container, either alone or in combination with another antibody specific to the target cell or peptide. Typically, a pharmaceutically acceptable carrier (e.g., an inert diluent) and / or its components, such as Tris, phosphate or carbonate buffers, stabilizers, preservatives, biocides, inert proteins such as serum albumin (typically in separate containers for mixing), and additional reagents (also typically in separate containers) may also be included. Some kits also include a second antibody capable of binding to the target-specific humanized or chimeric antibody, typically present in a separate container. The second antibody is typically conjugated with a tag 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 in this article, target-specific humanized or chimeric antibodies can be used to define subsets of cancer / tumor cells and characterize such cells and associated tumor tissues.

[1081] Anti-individual genotype antibodies

[1082] In a further aspect, the present invention relates to anti-individual genotype antibodies, which are combined with the humanized or chimeric antibodies of the present invention as described herein.

[1083] In one embodiment, the present invention relates to an anti-individual genotype antibody incorporating an antibody or bispecific antibody according to any one of the claims of the present invention.

[1084] Anti-genotype (Id) antibodies are antibodies that recognize unique determinants typically associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing animals of the same species and genotype as the source of the CD3 monoclonal antibody with a monoclonal antibody to which anti-Id has been prepared. The immunized animals typically recognize and respond to the individual genotype determinants of the immunizing antibody by producing antibodies against these individual genotype determinants (anti-Id antibodies). Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of this invention.

[1085] Anti-Id antibodies can also be used as "immunogens" to induce an immune response in another animal, producing so-called anti-anti-Id antibodies. Anti-anti-Id antibodies may be epitopes identical to the original monoclonal antibody that induces the anti-Id antibody. Therefore, by using antibodies targeting individual genotypic determinants of monoclonal antibodies, it is possible to identify other clones expressing antibodies with the same specificity. Anti-Id antibodies can be altered (thus producing anti-Id antibody variants) and / or derived using any suitable technique, such as those described elsewhere herein with respect to CD3-specific antibodies of the present invention. For example, monoclonal anti-Id antibodies can be conjugated to a vector such as keyhole cirrhosis hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice typically contains anti-anti-Id antibodies with binding properties similar to (if not identical to) the original / parental CD3 antibodies.

[1086] sequence

[1087] Table 1

[1088]

[1089]

[1090]

[1091]

[1092]

[1093]

[1094]

[1095]

[1096]

[1097]

[1098]

[1099]

[1100]

[1101]

[1102]

[1103]

[1104]

[1105]

[1106]

[1107]

[1108]

[1109]

[1110]

[1111]

[1112]

[1113]

[1114]

[1115]

[1116]

[1117]

[1118]

[1119]

[1120]

[1121]

[1122]

[1123]

[1124]

[1125]

[1126]

[1127]

[1128]

[1129]

[1130] The CDR section is annotated according to the IMGT definition. Example

[1131] Example 1 - Generation of humanized CD3 antibodies and non-activated antibody variants

[1132] Humanization of CD3 antibody

[1133] Humanization of a mouse CD3 antibody (US 8,236,308, described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK) using its modified form of germline humanization (CDR-transplantation) technology (EP 0629240). Using this technology, one distinct VH chain (SEQ ID NO:4) and two distinct VL chains (SEQ ID NO:8, 10) were designed. By combining these one VH with the two VL chains, two distinct antibodies were generated. The humanized variant is described herein as huCD3. Therefore, the humanized variant of the present invention comprising VH and VL is described, for example, IgG1-huCD3-H1L1, meaning that the particular variant has an IgG1 isotype, is humanized CD3, and comprises the amino acid sequence referred to as "H1" and defined according to SEQ ID NO:4, and the amino acid sequence referred to as "L1" and defined according to SEQ ID NO:8. Therefore, H1 refers to the variable heavy chain region VH1, L1 refers to the variable light chain region VL1, and so on.

[1134] Specifically, the variants IgG1-huCD3-H1L1 (humanized CD3 containing 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 containing the VH1 sequence shown in SEQ ID NO:4 and the VL sequence shown in SEQ ID NO:10).

[1135] b12 antibody

[1136] In some embodiments, antibody b12, an HIV-1 gp120 specific antibody (Barbas, CF. J MolBiol. 1993 Apr 5; 230(3): 812-23.), is used as a negative control and is referred to as “IgG1-b12”.

[1137] Express

[1138] Antibodies expressed as IgG1,κ or IgG1,λ, with or without the non-activating mutations described below and mutations in the CH3 domain, are capable of producing bispecific antibodies via the methods described below. Essentially as described by the manufacturer, a mixture of plasmid DNA encoding both the heavy and light chains of the antibody is transiently transfected into Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US).

[1139] Antibody purification

[1140] The culture supernatant was filtered through a 0.2 μm empty-end filter onto a 5 mL LAbSelect SuRe column (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluent 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 eluent 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 aseptically filtered through a 0.2 μm empty-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.

[1141] Example 2: Generation of mutant libraries

[1142] Point mutations were generated by random mutagenesis using the Quick Change Mutagenesis Kit (Stratagene, according to manufacturer's instructions) and expression plasmids HC (p33HGTE-huCD3-H1) and LC (p33L-huCD3-L1-T41K) as templates. The HC plasmids encoded the monovalent UniBody-TE form as described in WO2011110642. Each selected position was randomized using primers containing the NNS codons at the selected position (N = G, A, T or C and S = G or C). The mutant library was transformed into OneShotDH5α (Invitrogen) according to the manufacturer's instructions.

[1143] Colony selection and LEE PCR

[1144] For each mutation site, 96 clones were picked and added to 50 μL of LEE (linear expression element) PCR buffer (5 μL 10x AccuPrime PCR buffer, 44.6 μL water (B. Braun), 0.1 μL CMVP f (MAR5) and 0.1 μL TkpAr (MAR1) primers (100 μM stock solution), and 0.2 μL Accuprime Taq (Invitrogen) to amplify the expression cassette (promoter to polyA) from the expression plasmid. LEE PCR was performed by incubating the mixture at 2' 94°C, [30" 94°C, 30" 55°C, 5' 68°C] 35x, 10' 72°C, and stored at 4°C until further use.

[1145] Each library (12) of 96 colonies was sequenced using Sanger sequencing (Beckman Coulter Genomics, UK).

[1146] Table 2: Primer sequences for LEE PCR

[1147]

[1148] Example 3: Expression of mutant library and IgG quantification

[1149] For each mutant (12 × 96 in total), 1.11 μL of HC and 1.11 μL of LC LEE PCR product were diluted in 2.78 μL of water. Individual wells in a 96-well plate were transfected using 5 μL of DNA dilution buffer.

[1150] Add 0.4 μL of ExpiFectamine to each well. 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 of DNA dilution buffer 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 of Expi293F. TM In the cell. In all programs, the oscillator is equipped with Expi293F. TM Cells were plated to maintain cell suspension. After transfection, cells were incubated at 37°C / 8% CO2 for 5 days.

[1151] Five days after transfection, the supernatant was collected. The antibody concentration in the supernatant was measured using OctetRED (ForteBio, US) via biolayer interferometry.

[1152] Example 4: Generation of CD3 / TCR-LC13 screening library

[1153] Freestyle 293-F cells (Invitrogen, US) were co-transfected with expression constructs encoding human α and β chains (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, the cells were frozen until further use.

[1154] Example 5: Screening for affinity mutants

[1155] Homogenization assay (dose-response)

[1156] Based on sequence data, mutants are selected where the sequence traces show a high PHRED score, indicating the absence of multiple mutations. For each mutation, multiple redundant clones are selected when available.

[1157] The binding of recombinant UniBody molecules to cell culture supernatants was determined using a homogeneous antigen-specific binding assay employing Fluorometric Micro Volume Assay Technology (FMAT; Applied Biosystems, Foster City, CA, USA). In the assay, the binding of antibody or monovalent antibody molecules in the designed samples 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 (a negative control not expressing human TCR) was analyzed in dose-response mode. Normalized IgG levels in the samples prior to dose-response binding were measured using an Octet instrument (Fortebio, Menlo Park, USA).

[1158] Diluted serial samples were added to cells to allow binding to CD3. Binding of the monovalent antibody molecules was then 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. Samples were scanned using an Applied Biosystems 8200 Cell Detection System (8200CDS), and total fluorescence within the sample concentration range was used as readout. A sample was considered positive when the count was above 50, and the count x fluorescence (total fluorescence) was at least three times that of the negative control.

[1159] Heatmap

[1160] From the homogeneous dose-response screening, binding curves were fitted using a 4-parameter sigmoid model. From the fitted curves, the maximum binding for each mutant was determined. For each mutant, the average maximum binding was calculated and plotted as follows: Figure 1 The ratio between the average maximum value and wt is shown.

[1161] Comparison

[1162] The selected HC mutants generated in these libraries were compared and... Figure 2 Described in the text. The CDR area has been approved by IMGT. Define comments. The sequence numbering follows the direct numbering scheme for comments.

[1163] Example 6: Generation of bispecific antibodies via 2-MEA-induced Fab arm exchange

[1164] The bispecific antibody according to the present invention can be produced by using the methods disclosed in WO2011131746 and WO2013060867 (Genmab).

[1165] For example, a mutation at position F405L can be introduced into one parental antibody of the IgG1 isotype, and a mutation at position K409R can be introduced into another parental antibody of the IgG1 isotype.

[1166] The two parental antibodies, each at a final concentration of 0.5 mg / mL (equimolar concentration), were 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 was stopped by removing the reducing agent 2-MEA using a centrifuge column (Microcon centrifuge filter, 30k, Millipore), according to the manufacturer's instructions.

[1167] Bispecific antibodies can be filtered through a 0.2 μm dead-end filter, and their final concentration can be determined by measuring the absorbance (A280) at 280 nm.

[1168] Example 7: Combining data

[1169] For all the following binding assays, a selected group of heavy chain variants of huCD3-H1L1 were tested in different forms:

[1170] Table 3: Selected affinity variants of huCD3-H1L1 in the monovalent antibody-TE form

[1171]

[1172]

[1173] Octet binding affinity assay of CD3 affinity mutant in monovalent antibody-TE form

[1174] Affinities for the selected VH variants (Table 3) were determined using biolayer interferometry on a ForteBio OctetHTX. CD3 affinity mutants in monovalent antibody-TE form (2 μg / mL) were loaded for 600 s to an anti-human Fc capture (AHC) biosensor (ForteBio, Portsmouth, UK; catalog number 18-5060), aiming for a response of 0.4 nm. The antibody in UniBody-TE form was used to specifically measure the monovalent interaction affinity between the CD3 affinity mutant and the CD3ε27-GSKα ligand. Binding (1000 s) and dissociation (1000 s) of CD3ε27-GSKα (100 and 1000 nM) were measured after baseline (150 s). The CD3ε27-GSKα protein consists of a human CD3ε peptide (aa1-27) fused to the N-terminus of κLC (SEQ ID NO:402). For calculation, the theoretical molecular weight of CD3ε27-GSKα, 27.1 kDa, based on the amino acid sequence, was used. The experiment was conducted while shaking at 1000 rpm and 30 °C.

[1175] Data were analyzed using ForteBio data analysis software v8.1, employing a 1:1 model and a global perfect fit, with a binding time of 1000 s and a dissociation time of 200 s. The data trajectory was corrected by subtracting a reference curve (a CD3 affinity mutant without CD3ε27-GSKα), aligning the Y-axis with the baseline for the last 5 s, and applying inter-step correction and Savitzky-Golay filtering.

[1176] Table 4: Equilibrium dissociation constants (KD) of the selected variants

[1177]

[1178]

[1179] nd = Undetermined

[1180] T cell binding of the affinity variant of humanized CD3 (UniTE-huCD3-H1L1-LT41K) in flow cytometry (FACS)

[1181] T cell binding of purified VH affinity variants of humanized CD3 (IgG1-huCD3-H1L1) antibody was measured using fluorescence-activated cell sorting on a FACSCanto 752 (BD Biosciences). T cells were isolated from erythrocyte sedimentation rate (ESR) chromatographic analysis of anticoagulated human donor blood samples and resuspended at 1.8 × 10⁶ cells / mL in PBS / 0.1% BSA / 0.02% azide. 50 μL of T cell suspension and 50 μL of antibody dilution were combined in 96-well plates on ice, incubated at 4 °C for 30 min, and washed twice with PBS / 0.1% BSA / 0.02% azide. Next, 50 μL of goat anti-human IgG F(ab')2 (109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) conjugated with secondary antibody R-phycoerythrin (PE) in PBS / 0.1% BSA / 0.02% azide (diluted 1 / 200) was added for staining. The mixture was incubated at 4°C for 30 min, followed by washing twice with PBS / 0.1% BSA / 0.02% azide. Cells were resuspended in 120 μL of PBS / 0.1% BSA / 0.02% azide, and the geometric mean fluorescence intensity of PE was measured. Binding curves were analyzed using nonlinear regression (S-shaped dose-response with variable slope) with GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA), and the apparent affinity (KD) was derived from the half-maximal binding concentration. Figure 4 The binding curves of the affinity variant of humanized CD3 (UniTE-huCD3-H1L1-LT41K) are shown. Figure 5 Binding curves of the low-affinity variant of humanized CD3 (UniTE-huCD3-H1L1-LT41K) are shown.

[1182] Table 5: Summary of binding data between monovalent antibody and CD3 affinity mutant in TE form

[1183]

[1184]

[1185] For all of the following binding assays, preferred heavy chain variants of the selected groups were tested (see Table 5).

[1186] Octet binding affinity determination of the IgG1-huCD3-H1L1-FEAL affinity mutant: The affinity of the selected CD3 affinity variant of the IgG1-huCD3-H1L1-FEAL form was determined using biolayer interferometry on a ForteBio Octet HTX (ForteBio, UK) (Table 6). The anti-human Fc capture biosensor (CAT: 18-5060, ForteBio, UK) was loaded with hIgG (1 μg / mL) for 600 s. After baseline (200 s), association (1000 s) and dissociation (2000 s) of CD3E27-GSKa were determined using a CD3E27-GSKa concentration range of 27.11 μg / mL–0.04 μg / mL (1000 nM–1.4 nM) and a three-fold dilution step (sample diluent, CAT: 18-5028, ForteBio, UK). For calculation, the theoretical molecular weight of CD3E27-GSKα, 27.11 kDa, based on the amino acid sequence, was used. Experiments were conducted with shaking at 1000 rpm and 30°C. Each antibody was tested in at least two independent experiments (Table 6).

[1187] Data were analyzed using a 1:1 model with a global perfect fit, 1000 s binding time, and 100 s dissociation time, using ForteBio data analysis software v8.1. Data trajectories were corrected by subtracting a reference curve (antibody without CD3E27-GSKα), aligning the Y-axis to the baseline for the last 10 s, and applying inter-step correction and Savitzky-Golay filtering. Data traces with responses <0.05 nm were excluded from the analysis.

[1188] Table 6

[1189]

[1190] T-cell binding affinity assays for the IgG1-huCD3-H1L1-FEAL affinity mutant were performed using RosetteSep human T-cell enrichment mixture (Cat: 15021C.1, Stemcell Technologies, France) to isolate T cells from donor erythrocyte sedimentation rate (ESR) amber (Sanquin, Amsterdam, The Netherlands) according to the manufacturer's instructions. Briefly, 50 μL of the T-cell isolation mixture was added to 1 mL of ESR amber and incubated at room temperature for 20 min. Next, the ESR amber was diluted with PBS (Cat: 3623140, B. Braun, Germany) (1:3, v / v) and gently transferred to 50 mL Falcon tubes (Cat: 227261, Greinerbio-one, The Netherlands) containing 15 mL of lymphocyte separation medium (Cat: 17-829E, Lonza, Switzerland). Without immobilization, centrifuge the tubes at 1200xg for 20 minutes at room temperature. Collect T cells from the density medium and wash twice with PBS.

[1191] Resuspend 2 × 10⁶ T cells / mL in FACS buffer and transfer 50 μL to a 96-well round-bottom plate (CAT: 650101, Greinerbio-one, The Netherlands). Add 50 μL of a 5-fold diluted antibody solution at 5 μg / mL and incubate at 4°C for 30 min. Centrifuge the 96-well plate at 300 x g for 5 min at 4°C and discard the supernatant. Wash the cells twice on ice with ice-cold FACS buffer, and add 1:200 diluted secondary antibody (anti-IgG Fcγ-PE(fab)'2, CAT: 109-116-098, Jackson Immuno Research, UK) to 100 μL / well and incubate for 30 min, washing twice with FACS buffer. Measure fluorescence intensity on a FACS Canto and calculate the geometric mean using FlowJo V10 software. Figures were generated using GraphPad (V6.04). See also Figure 5 .

[1192] Example 9: In vitro cytotoxicity screening of CD3 affinity mutants

[1193] Cytotoxicity of CD3 affinity mutants to solid tumor cell lines (Almar blue assay)

[1194] T cells from donor erythrocyte sedimentation rate (ESR) amber layer (Sanquin, Amsterdam, The Netherlands) were isolated using RosetteSep human T cell enrichment mixture (Cat: 15021C.1, Stemcell Technologies, France) according to the manufacturer's instructions. NCI-N87 (25,000 cells / well) was then used. Figure 6A SKOV3 (16,000 cells / well) Figure 6B ) and MDA-MB-231 (16,000 cells / well) Figure 6C Cells were seeded into flat-bottomed 96-well plates (cat: 655180, Greiner-bio-one, The Netherlands) and allowed to adhere for 3-5 hours at 37°C. T cells were added to 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 10% alpha blue (cat: DAL1100, Life Technologies, The Netherlands) solution prepared in RPMI-1640 medium containing 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 multi-label plate reader (PerkinElmer, US). Cells treated with staphylococcalin (cat: S6942, Sigma-Aldrich, US) were set as 100% kill, and untreated cells were set as 0% kill. Viable cells were calculated by subtracting staphylococcal-treated cells from all groups, and the percentage was plotted against the untreated group. The graph was created using GraphPad (V6.04). See Figure 6.

[1195] Cytotoxicity of CD3 affinity mutants to blood cell lines (chromium release assay)

[1196] 5 x 10⁶ Daudi cells / mL were incubated for 1 h in complete medium containing 100 μCi chromium under shaking conditions at 37°C. The cells were then washed twice in PBS and resuspended in 5 mL of complete cell culture medium (RPMI 1640 containing 10% donor bovine serum and iron). 5,000 Daudi cells were seeded into 96-well round-bottom plates. T cells from donor erythrocyte sedimentation rate (ESR) amber layer (purchased from Sanquin, Amsterdam, The Netherlands) were isolated according to the manufacturer's instructions using the RosetteSep human T cell enrichment mixture (Cat: 15021C.1, Stemcell Technologies, France). T cells were added to the Daudi cells at a (tumor cells:T cells) ratio of 1:10, followed by a two-fold diluted antibody solution. The plates were incubated at 37°C for 24 h. After 24 h, the plates were centrifuged at 300 x g for 3 min, the supernatant was collected, and radioactivity was measured. See also Figure 7 .

[1197] Table 7

[1198]

[1199] Example 10: Tumor efficacy of CD3xHER2 bispecific antibody in NOD-SCID mouse co-transplantation model (human PBMC + NCI-N87 cells)

[1200] The in vivo antitumor efficacy of several CD3xHER2 bispecific antibodies was evaluated in a subcutaneous NCI-N87 co-graft model (Figure 8). The CD3 arm of the bispecific antibody used humanized WT CD3 (huCD3-FEAL) and four different CD3 affinity variants (N57E, H101K, S110A, Y114M). The HER2-targeting arm was identical in all cases (Herceptin FEAR).

[1201] BisG1-huCD3-FEALx1014-Hersetin-FEAR

[1202] BisG1-huCD3-N57E-FEALx1014-Hersetin-FEAR

[1203] BisG1-huCD3-H101K-FEALx1014-Hersetin-FEAR

[1204] BisG1-huCD3-S110A-FEALx1014-Hersetin-FEAR

[1205] BisG1-huCD3-Y114M-FEALx1014-Hersetin-FEAR

[1206] In this model, HLA-A-matched unstimulated human 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).

[1207] Mice were sorted into treatment groups (n=4 per treatment group). On day 0, each female NOD-SCID mouse (NOD.CB-17-Prkdc) was subcutaneously (sc) inoculated with a mixture of HLA-A-matched hPBMCs (5x10E6, Sanquin) and NCI-N87 (5x10E6) cells in 200 μL PBS / 0.1% BSA. scid / J), 6-11 weeks old (Charles-River) right ventral side. Following direct injection into tumor cells, five different CD3xHER2 antibodies were administered intravenously (150 μL) at two different concentrations (0.5 and 0.05 mg / kg) for all bispecific antibodies. Tumor volume was measured at least twice a week. Tumor volume (mm) was measured using calipers (PLEXX). 3 The calculation is: 0.52 × (length) × (width) 2 .

[1208] NCI-N87 cells (ATCC#CRL-5822, gastric cancer 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, and 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 growth phase, with counts excluded by trypan blue exclusion.

[1209] For each study, hPBMCs were isolated from the erythrocyte sedimentation rate (ESR) serosanquin layer 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 filter, and resuspended to a concentration of 50 x 10⁶ cells / mL in PBS / 0.1% BSA.

[1210] 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 on day 44. Statistical analysis of tumor volume on day 44 (the last day when all groups remained intact) (Mann-Whitney) showed that at a dose of 0.05 mg / kg, BisG1-huCD3-FEALx1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR, and BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR showed significant tumor growth inhibition (p<0.05) compared to the control (PBMC), while BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR and BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR did not. At a dose of 0.5 mg / kg, BisG1-huCD3-FEALx1014-Herceptin-FEAR and all CD3-arm affinity variants showed significant tumor growth inhibition (p<0.05) compared with the PBS (PBMC) control group, except for BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR.

[1211] 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 only significantly reduced NCI-N87 tumor volume (p<0.05) at a dose of 0.5 mg / kg. At both tested doses, BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR did not affect NCI-N87 tumor growth.

[1212] Reference List

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[12] WO 2012 / 162067

[1225]

[13] WO 2008 / 119567

[1226]

[14] Fundamental Immunology Ch.7, Paul, W., ed., 2nd ed. Raven Press, NY (1989)

[1227]

[15] Lefranc MP, et al., 2003, Dev Comp Immunol. Jan; 27(1):55-77

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[16] Brochet, X. et al., 2008, Nucl. Acids Res.36, W503-508

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[17] Giudicelli,V.,Brochet,X,Lefranc,M.-P.,2011,Cold Spring HarbProtoc.Jun 1;2011(6)

[1230]

[18] Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch.15

[1231]

[19] WO92 / 22653

[1232]

[20] EP 0629240

[1233]

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Claims

1. A humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region, the binding region comprising a heavy chain variable (VH) region, wherein the VH region contains a mutation in one of 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, the mutation being located at one of the following positions: T31M, T31P, N57, H101, G105, S110 and Y114, wherein the position is numbered according to the reference sequence of SEQ ID NO:

4.

2. The antibody of claim 1, wherein the binding affinity of the antibody to human CD3 is reduced or increased 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.

3. The antibody of any one of claims 1 to 2, wherein the antibody comprises a T31M or T31P mutation.

4. The antibody of any one of claims 1 to 2, wherein the antibody comprises a mutation at position N57.

5. The antibody of any one of claims 1, 2 and 4, wherein the antibody comprises an N57E mutation.

6. The antibody of any one of claims 1 to 2, wherein the antibody comprises a mutation at position H101.

7. The antibody of any one of claims 1, 2 and 6, wherein the antibody comprises an H101G or H101N mutation.

8. The antibody of any one of claims 1 to 2, wherein the antibody comprises a mutation at position G105.

9. The antibody of any one of claims 1, 2 and 8, wherein the antibody comprises a G105P mutation.

10. The antibody of any one of claims 1 to 2, wherein the antibody comprises a mutation at position Y114.

Images