Humanized or chimeric CD3 antibodies

Abstract

To provide humanized or chimeric antibodies binding CD3 and further provide bispecific antibodies, compositions, pharmaceutical compositions, use of the antibodies in the treatment of a disease, and methods of treatment.SOLUTION: Disclosed are humanized or chimeric CD3 antibodies with an optimized affinity to CD3. Disclosed are humanized or chimeric CD3 antibodies which are optimized compared to a reference antibody such as an antibody specified by a specific sequence. In another aspect, the present invention relates to antibodies or bispecific antibodies, compositions, or pharmaceutical compositions of the present invention for use as a medicament.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present 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 disease. [Background technology]

[0002] background Cluster of Differentiation 3 (CD3) has been known for a long time and has therefore been of interest in many areas. In particular, antibodies raised against CD3 or against the T cell receptor complex of which CD3 is a component are known. In vitro characterization of recombinant chimeric CD3 isotype variants as well as a number of humanized OKT3 effector function mutant antibodies have been described [1].

[0003] CD3 antibodies (e.g., muromonab-CD3) have been widely used in the treatment of acute allograft rejection. Moreover, treatment with the anti-CD3 monoclonal antibody hOKT3 gamma 1 (Ala-Ala) results in improved C-peptide responses and clinical parameters for at least 2 years after the onset of type 1 diabetes in the absence of ongoing immunosuppressive drugs [2].

[0004] A promising approach to improve targeted antibody therapy relies on the specific delivery of cytotoxic cells to antigen-expressing cancer cells. This concept of using T cells to efficiently kill tumor cells has been described previously [3]. However, early clinical studies were rather disappointing, mainly due to the low efficacy, severe adverse effects (cytokine storm) and immunogenicity of bispecific antibodies [4]. Advances in the design and application of bispecific antibodies have partially overcome the initial obstacle of cytokine storm, improving clinical efficacy and eliminating dose-limiting toxicity [5].

[0005] For example, certain bispecific antibodies that target antigens on tumor cells with one arm and, for example, CD3 on T cells with the other arm and contain an active Fc fragment that provides Fc receptor binding, have been shown to induce tumor cell death. Upon binding, complexes can form between T cells, tumor cells, and effector cells that bind to the antibody Fc region, leading to tumor cell death [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heavy chain heterodimer and has been successful in treating cancer-associated ascites after intraperitoneal application [6]. However, the mouse / rat hybrid is immunogenic [7] and cannot be applied for long-term treatment in humans. High frequency treatment-related adverse events attributable to catumaxomab included cytokine release-related symptoms (i.e. fever, nausea, vomiting, chills, tachycardia, and hypotension) [8]-[9], which are related to the strong polyclonal T cell activation by catumaxomab (through its active Fc fragment). Another antibody, ertumaxomab (HER2 × CD3), which induces cytotoxicity in HER2-expressing cell lines, is in phase II clinical development for metastatic breast cancer

[10] -

[11] .

[0006] The potency of CD3 bispecific antibodies and other CD3 bispecific antibody-based formats depends on several properties of the bispecific antibody, such as the affinity of the CD3 arm and / or the affinity of the second arm for the target, as well as the target copy number on the target cell. Some CD3 bispecific antibodies show high potency when CD3 affinity is low (EpCam×CD3 - Bortoletto 2002 PMID 12385030, MT103 / blinatumomab vs. TandAb - Molhoj 2007 PMID 17083975), while other CD3 bispecifics show high potency with high CD3 affinity (Reusch 2015, Mabs, PMID 25875246). In some cases, for example, high CD3 affinity is required when arming ex vivo expanded activated T cells from a patient with a bispecific antibody composed of an anti-CD3 targeting arm and a second arm against a selected tumor-associated antigen. In the latter case, CD3 affinity should be high to retain the interaction with the expanded T cells when the product is infused back into the patient to mediate tumor cell cytolysis (Reusch 2006 Clin Cancer Res PMID 16397041). However, high affinity antibodies against CD3, in contrast to low affinity ligands, are less effective at TCR triggering at low copy numbers because they present an approximately 1:1 stoichiometry and linear dose-response curve, indicating a single cycle rather than a continuous triggering mode of T cell response (Viola 1996 Science, PMID 8658175). In other words, the low affinity of the CD3 arm may allow T cells to flexibly migrate from one target and / or target cell to another (Hoffman 2005, PMID: 15688411).

[0007] Low CD3 affinity can potentially prevent biased localization of bispecific antibodies to T cells (due to first encounter in circulation), thus improving biodistribution and minimizing interference with normal T cell immune responses. Depending on the target and target copy number of the second arm, the indication, and / or the route of administration, the desired CD3 affinity can be customized to enhance the maximum efficacy of the product. A panel of CD3 variants covering a range of CD3 affinities may be necessary to meet these specific tailoring needs per antibody product.

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

[12] -

[13] , but such cross-reactive antibodies require further improvement. Summary of the Invention

[0009] It is an object of the present invention to provide a humanized or chimeric CD3 antibody with an optimized affinity for CD3. It is therefore an object of the present invention to provide a humanized or chimeric CD3 antibody that is optimized compared to a reference antibody, such as the antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. Such an antibody may therefore have a reduced or enhanced affinity for CD3 compared to the reference antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. It is a further object of the present invention to provide an antibody that has a lower binding affinity for CD3 than the antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. The inventors have found that antibodies having a binding affinity for the CD3 peptide shown in SEQ ID NO:402 that is reduced compared to a reference antibody having a VH region sequence shown in SEQ ID NO:4 maintain the same or similar cytotoxic activity in vitro and in vivo compared to the reference antibody. Another object of the present invention is to provide a CD3 antibody having a binding affinity for CD3 that is reduced compared to a reference antibody defined by the VH sequence of SEQ ID NO:4 and the VL sequence of SEQ ID NO:8, but retains the same cytolytic activity as the reference antibody. It is yet another object of the present invention to provide an antibody having a higher binding affinity for CD3 than the antibody defined by the VH sequence of SEQ ID NO:4 and the VL sequence of SEQ ID NO:8.

[0010] The present invention provides, in one aspect, a humanized or chimeric antibody that binds to a human CD3 antibody, the antibody comprising a binding region comprising a heavy chain variable (VH) region, the VH region comprising a mutation in one of three CDR sequences of a reference antibody having VH CDR sequences shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3, the mutation being at one of the positions selected from the group consisting of T31M, T31P, N57, H101, G105, S110, and Y114, the positions being 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 125th amino acid in the N-terminal to C-terminal direction. The numerical numbering of the positions corresponding to SEQ ID NO: 4 is shown in FIG. 2. Additionally, the VH CDR regions were annotated according to the IMGT definition.

[0011] In one embodiment of the invention, the antibody has a binding affinity to human CD3 that is reduced or enhanced 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.

[0012] In some embodiments of the invention, an antibody having a binding affinity for a CD3 peptide (e.g., a human CD3 molecule such as SEQ ID NO: 402) that is reduced compared to a reference antibody may maintain the same cytolytic activity against target cells as the reference antibody.

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

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

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

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

[0017] In one embodiment, the present invention provides a humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 regions having CDR sequences selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 12, 2, 3; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 14, 2, 3; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 16, 2, 3; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 18, 2, 3; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 20, 2, 3; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 22, 2, 3; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 24, 2, 3; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 26, 2, 3; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 28, 2, 3; j) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 30, 2, 3; k) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 32, 2, 3; l) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 34, 2, 3; m) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 36, 2, 3; n) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 38, 2, 3; o) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 40, 2, 3; p) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 42, 2, 3; q) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 44, 2, 3; r) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 46, 2, 3; s) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 48, 2, 3; t) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 50, 2, 3; u) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 52, 2, 3; v) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 54, 2, 3; w) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 56, 2, 3; x) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3; y) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 60, 2, 3; z) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 62, 2, 3; aa) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 64, 2, 3; bb) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 66, 2, 3; cc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 68, 2, 3; dd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 70, 2, 3; ee) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 72, 2, 3; ff) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 74, 2, 3; gg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 76, 2, 3; hh) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 78, 2, 3; ii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 80, 2, 3; jj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 82, 2, 3; kk) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 84, 2, 3; ll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 86, 2, 3; mm) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 88, 2, 3; nn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 90, 2, 3; oo) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 92, 2, 3; pp) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 2, 3; qq) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 96, 2, 3; rr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 98, 2, 3; ss) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 100, 3; tt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 102, 3; uu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 104, 3; vv) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3; ww) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 108, 3; xx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 110, 3; yy) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 112, 3; zz) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 114, 3; aaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 116, 3; bbb) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 118, 3; ccc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 120, 3; ddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 122, 3; eee) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 124, 3; fff) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 126, 3; ggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 128, 3; hhh) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 130, 3; iii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 132, 3; jjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 134, 3; kkk) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 136, 3; lll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 138, 3; mmm) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 140, 3; nnn) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 142, 3; ooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 144, 3; ppp) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 146, 3; qqq) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 148, 3; rrr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 150, 3; sss) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 152, 3; ttt) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 154, 3; uuu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 156, 3; vvv) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 158, 3; www) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 160, 3; xxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 162, 3; yyy) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 164, 3; zzz) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 166, 3; aaaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 168, 3; bbbb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 170; cccc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 172; dddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 174; eeee) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176; ffff) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 178; gggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 180; hhhh) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 182; iiii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184; jjjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 186; kkkk) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 188; lll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 190; mmmm) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 192; nnnn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 194; oooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 196; pppp) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 198; qqqq) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 200; rrrr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 202; ssss) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 204; tttt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 206; uuuu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 208; vvvv) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 210; wwww) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 212; xxxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 214; yyyy) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 216; zzzz) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 218; aaaaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 220; bbbbb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 222; ccccc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 224; ddddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 226; eeeee) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 228; fffff) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 230; ggggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 232; hhhhh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 234; iii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236; jjjjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 238; kkkkk) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 240; lllll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 242; mmmmm) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244; nnnnn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 246; ooooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 248; ppppp) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 250; qqqqq) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 252; rrrrr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 254; sssss) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 256; tttt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 258; uuuuu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 260; vvvvv) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 262; wwwww) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 264; xxxxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 266; yyyyy) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 268; zzzzz) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 270; aaaaaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 272; bbbbbb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 274; cccccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 276; dddddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 278; eeeeee) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 280; ffffff) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 282; gggggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284; hhhhhh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 286; iiiiii) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 288; jjjjjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 290; kkkkkk) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292; llllll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 294; mmmmmm) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 296; nnnnnn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 298, and oooooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 300.

[0018] That is, the inventors of the present invention found in a first aspect of the present invention that a humanized or chimeric antibody of said sequence has a binding affinity for the CD3 peptide of SEQ ID NO: 402 that is optimized compared to a reference antibody such as the antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. The reference antibody defined by the VL sequences of SEQ ID NO: 4 and SEQ ID NO: 8 has a binding affinity of 1.5×10 -8 M for the CD3 peptide of SEQ ID NO: 402. In some embodiments of the invention, the antibody has a binding affinity of, for example, 1.6×10, as determined by biolayer interferometry, as described in Table 6 in Example 7. -8 M~9.9×10 -8 Binding affinity of M, or e.g. 1.0 x 10 -7 ~9.9×10 -7 M binding affinity, 1.5 x 10 -8 M. In some embodiments of the invention, the antibody has a binding affinity for the CD3 peptide of SEQ ID NO: 402 that is lower than, for example, 1.4×10 -8 ~1.0×10 -8 M, e.g. 9.9 x 10 -9 ~1×10 -9 M, or for example 9.9 x 10 -9 ~1×10 -9 M, 1.5 x 10 -8 The binding affinity for the CD3 peptide of SEQ ID NO: 402 is higher than K D Equivalent to the value.

[0019] In one aspect of the invention, the invention relates to a humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 regions having CDR sequences selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V], and l) CDR1, CDR2, and CDR3 sequences having at least 90% or at least 95% amino acid sequence identity across the three CDR sequences to any one of the three CDR sequences set forth in a) to k), provided that the CDR1, CDR2, and CDR3 sequences do not have the sequences set forth in SEQ ID NOs: 1, 2, and 3.

[0020] In another aspect, the present invention relates to a humanized or chimeric antibody, wherein the binding region comprises a light chain variable (VL) region, the VL region comprising CDR1, CDR2, and CDR3 regions having the CDRs shown in SEQ ID NO: 6, GTN, 7.

[0021] In yet another aspect, the present invention relates to a method for reducing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 3, comprising the step of introducing a mutation in one of the three CDR sequences of the reference antibody selected from a mutation in one of the positions selected from the group of T31M, T31P, N57, H101, S110 and Y114, wherein the positions are numbered according to the reference sequence of SEQ ID NO: 4.

[0022] In one embodiment of the present invention, the method comprises introducing a mutation in the VH region CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the present invention, the method comprises introducing a mutation in the VH region CDR2 region corresponding to N57E. In yet another embodiment of the present invention, the method comprises introducing a mutation in the VH region CDR3 region selected from H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V.

[0023] In one embodiment, the CD3 is human CD3ε.

[0024] In another aspect, the present invention relates to a bispecific antibody comprising a first binding region of an antibody of the present invention and a second binding region that binds to a target different from the first antigen-binding region.

[0025] In another aspect, the present invention relates to a nucleic acid construct encoding one or more amino acid sequences of the present invention.

[0026] In another aspect, the present invention relates to an expression vector comprising (i) a nucleic acid sequence encoding the heavy chain sequence of a humanized or chimeric antibody of the present invention, (ii) a nucleic acid sequence encoding the light chain sequence of a humanized or chimeric antibody of the present invention, or (iii) both (i) and (ii).

[0027] In another aspect, the present invention relates to a host cell comprising the expression vector of the present invention.

[0028] In another aspect, the invention relates to a composition comprising the antibody or bispecific antibody of the invention.

[0029] In another aspect, the invention relates to a pharmaceutical composition comprising the antibody or bispecific antibody of the invention and a pharma- ceutically acceptable carrier.

[0030] In another aspect, the present invention relates to the antibody or bispecific antibody, composition, or pharmaceutical composition of the present invention for use as a medicament.

[0031] In another aspect, the invention relates to an antibody or bispecific antibody, a composition, or a pharmaceutical composition of the invention for use in the treatment of a disease.

[0032] In another aspect, the present invention relates to a method of treating a disease comprising the step of administering to a subject in need thereof an antibody or bispecific antibody, composition, or pharmaceutical composition of the invention.

[0033] In another aspect, the invention relates to a method of subcutaneous or topical administration of an antibody or bispecific antibody.

[0034] In one aspect, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, the method comprising the step of administering to a subject a humanized or chimeric antibody of the invention, a composition of the invention, or a pharmaceutical composition of the invention, optionally labeled with a detectable agent.

[0035] In another aspect, the invention relates to a method for producing an antibody or bispecific antibody of the invention, the method comprising the steps of a) culturing a host cell of the invention, and b) purifying said antibody from the culture medium.

[0036] In another aspect, the invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to any one of the embodiments disclosed herein.

[0037] In one embodiment, the diagnostic composition is a companion diagnostic used to screen and select patients who will benefit from treatment with the bispecific antibody.

[0038] In another aspect, the present invention relates to a method for detecting the presence of a CD3 antigen or a CD3-expressing cell in a sample, comprising the steps of a) contacting said sample with an antibody or bispecific antibody of the invention under conditions allowing the formation of a complex between said antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

[0039] In another aspect, the present invention relates to a kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample comprising i) an antibody or bispecific antibody of the invention and ii) instructions for using the kit.

[0040] In another aspect, the present invention relates to an anti-idiotypic antibody or an anti-idiotypic antibody pair that binds to an antibody of the present invention. [The present invention 1001] A humanized or chimeric antibody that binds to human CD3, the antibody comprising a binding region comprising a heavy chain variable (VH) region, the VH region comprising a mutation in one of three CDR sequences of a reference antibody having CDR sequences as shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3, the mutation being at one of the positions selected from the group consisting of T31M, T31P, N57, H101, G105, S110, and Y114, the positions being numbered according to the reference sequence of SEQ ID NO: 4. [The present invention 1002] The antibody of the present invention, wherein the binding affinity of the antibody to human CD3 is reduced or enhanced 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. [The present invention 1003] The antibody of any one of claims 1001 to 1002, comprising a T31M or T31P mutation. [The present invention 1004] The antibody of any of claims 1001 to 1002, comprising a mutation at position N57. [The present invention 1005] Any of the antibodies 1001, 1002 and 1004 of the present invention, comprising an N57E mutation. [The present invention 1006] The antibody of any of claims 1001 to 1002, comprising a mutation at position H101. [The present invention 1007] The antibody of any of claims 1001, 1002, and 1006, comprising an H101G or H101N mutation. [The present invention 1008] The antibody of any of claims 1001 to 1002, comprising a mutation at position G105. [The present invention 1009] Any of the antibodies 1001, 1002, and 1008 of the present invention, comprising a G105P mutation. [The present invention 1010] The antibody of any of claims 1001 to 1002, comprising a mutation at position Y114. [The present invention 1011] The antibody of any of claims 1001, 1002, and 1010, comprising a Y114M, Y114R, or Y114V mutation. [The present invention 1012] The VH region is a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and l) CDR1, CDR2, and CDR3 sequences having at least 90% or at least 95% amino acid sequence identity across the three CDR sequences to any one of the three CDR sequences shown in a) to k), except that the CDR1, CDR2, and CDR3 sequences do not have the sequences shown in SEQ ID NOs: 1, 2, and 3. The antibody of any of claims 1001 to 1002, comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of: [The present invention 1013] The VH region is a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V], and l) CDR1, CDR2 and CDR3 sequences as defined in a) to k), which have up to 5 further mutations or substitutions, up to 4 further mutations or substitutions, up to 3 further mutations or substitutions, up to 2 further mutations or substitutions or up to 1 further mutation or substitution across the three CDR sequences, which mutations or substitutions preferably do not alter the binding affinity to human CD3. The antibody of any of claims 1001 to 1002, comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of: [The present invention 1014] The antibody of the present invention, wherein said further mutations or substitutions are conservative, physical, or functional amino acids. [The present invention 1015] The VH region is a) the VH sequence shown in SEQ ID NO: 55 [T31M]; b) the VH sequence shown in SEQ ID NO: 59 [T31P]; c) the VH sequence shown in SEQ ID NO: 107 [N57E]; d) the VH sequence shown in SEQ ID NO: 177 [H101G]; e) the VH sequence shown in SEQ ID NO: 185 [H101N]; f) the VH sequence shown in SEQ ID NO: 221 [G105P]; g) the VH sequence shown in SEQ ID NO: 237 [S110A]; h) the VH sequence shown in SEQ ID NO: 245 [S110G]; i) the VH sequence shown in SEQ ID NO: 285 [Y114M]; j) the VH sequence shown in SEQ ID NO: 293 [Y114R]; k) the VH sequence shown in SEQ ID NO: 299 [Y114V], and l) a VH sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to any one of the sequences set forth in a) to k). The antibody of any of claims 1001 to 1002, having a VH sequence selected from the group consisting of: [The present invention 1016] The VH region is a) the VH sequence shown in SEQ ID NO: 55 [T31M]; b) the VH sequence shown in SEQ ID NO: 59 [T31P]; c) the VH sequence shown in SEQ ID NO: 107 [N57E]; d) the VH sequence shown in SEQ ID NO: 177 [H101G]; e) the VH sequence shown in SEQ ID NO: 185 [H101N]; f) the VH sequence shown in SEQ ID NO: 221 [G105P]; g) the VH sequence shown in SEQ ID NO: 237 [S110A]; h) the VH sequence shown in SEQ ID NO: 245 [S110G]; i) the VH sequence shown in SEQ ID NO: 285 [Y114M]; j) the VH sequence shown in SEQ ID NO: 293 [Y114R], and k) VH sequence shown in SEQ ID NO: 299 [Y114V] The humanized or chimeric antibody of any of claims 1001 to 1002, having a VH sequence selected from the group consisting of: [The present invention 1017] Any of the antibodies of the present invention, wherein the binding region comprises a light chain variable (VL) region, the VL region comprising CDR1, CDR2, and CDR3 regions having the CDR sequences shown in SEQ ID NO: 6, GTN, and SEQ ID NO: 7. [The present invention 1018] The binding domain comprises a light chain variable (VL) domain, the VL domain comprising: a) the VL sequence shown in SEQ ID NO: 8; b) the VL sequence shown in SEQ ID NO: 10, or c) a VL sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to any one of the sequences shown in a) to b). Any of the antibodies of the present invention, selected from the group consisting of: [The present invention 1019] The VL region comprises CDR1, CDR2, and CDR3 regions having the sequences shown in SEQ ID NO: 6, GTN, and SEQ ID NO: 7, and the VH region comprises a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; and k) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V] The antibody of the present invention, comprising CDR1, CDR2, and CDR3 regions having CDR sequences selected from the group consisting of: [The present invention 1020] Any of the antibodies of the present invention, wherein the human CD3 is human CD3ε defined in SEQ ID NO:399. [The present invention 1021] The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. D Value 1.6×10 -8 M~9.9×10 -8 M or 1.0×10 -7 ~9.9×10 -7 Any of the antibodies of the present invention, which corresponds to M. [The present invention 1022] The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. DValue 1.4×10 -8 ~1.0×10 -8 M or 9.9×10 -9 ~1×10 -9 Any of the antibodies of the present invention, which corresponds to M. [The present invention 1023] Any of the antibodies of the present invention which are humanized antibodies. [The present invention 1024] Any of the antibodies of the present invention which are full length antibodies. [The present invention 1025] Any of the antibodies of the present invention, which is an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [The present invention 1026] Any of the antibodies of the present invention which are monovalent, bivalent, or multivalent. [The present invention 1027] Any of the antibodies of the invention comprising an Fc region comprising a first and a second immunoglobulin heavy chain. [The present invention 1028] Any of the antibodies of the invention, wherein the antibody comprises a first and a second immunoglobulin heavy chain, and in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively. [The present invention 1029] The antibody of the invention, wherein in at least one of said first and second immunoglobulin heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D. [The present invention 1030] The antibody of the invention, wherein in at least one of said first and second heavy chains the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N. [The present invention 1031] The antibody of the invention, wherein in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively. [The present invention 1032] The antibody of any of 1028 and 1031, wherein in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E; or A and A, respectively. [The present invention 1033] The antibody of the invention 1032, wherein in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively. [The present invention 1034] The antibody of the invention 1032, wherein in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively. [The present invention 1035] The antibody of the invention, wherein in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively. [The present invention 1036] The antibody of the invention, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively. [The present invention 1037] The antibody of any of claims 1035 to 1036, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively. [The present invention 1038] The antibody of any of claims 1035 to 1036, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are A, A, and A, respectively. [The present invention 1039] The antibody of the present invention, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, Q, and S, respectively. [The present invention 1040] A bispecific antibody comprising a first binding region of any of the antibodies of the present inventions 1001 to 1039 and a second binding region that binds to a target different from the first antigen-binding region. [The present invention 1041] A bispecific antibody of the present invention comprising a first and a second heavy chain. [The present invention 1042] The bispecific antibody of any of the present inventions 1040 to 1041, wherein each of the first and second heavy chains comprises at least a hinge region, a CH2 region and a CH3 region, wherein in the first heavy chain, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted, and in the second heavy chain, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain is substituted, and the first and second heavy chains are not substituted at the same positions. [The present invention 1043] The bispecific antibody of the invention 1042, wherein the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in said first heavy chain and the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R in said second heavy chain, or vice versa. [The present invention 1044] The bispecific antibody of any of claims 1040 to 1043, wherein said first binding region is according to any of claims 1001 to 1027, and said second binding region binds to a target different from said first binding region. [The present invention 1045] A method for reducing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, 2, 3, the method comprising introducing a mutation selected from a mutation at one of the positions selected from the group of T31M, T31P, N57, H101, S110, and Y114 in one of the three CDR sequences of the reference antibody, the positions being numbered according to the reference sequence of SEQ ID NO: 4. [The present invention 1046] The method of claim 1045, wherein the mutation is a T31M or T31P mutation. [The present invention 1047] The method of claim 1045, comprising the step of introducing a mutation corresponding to position N57 in said VH CDR2 region. [The present invention 1048] The method of claim 1047, wherein said mutation at position N57 is an N57E mutation. [The present invention 1049] The method of claim 1045, comprising the step of introducing mutations corresponding to positions selected from the group of H101, S110, and Y114 in said VH CDR3 region. [The present invention 1050] 1049. The method of claim 1049, wherein said mutation in said VH CDR3 region is selected from the group consisting of H101G, H101N, S110A, S110G, Y114M, Y114R, and Y114V. [The present invention 1051] 1. A method for enhancing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 3, comprising introducing a mutation corresponding to position G105 in the VH CDR3, said positions being numbered according to the reference sequence of SEQ ID NO: 4. [The present invention 1052] 1052. The method of claim 1051, wherein said mutation at position G105 is a G105P mutation. [The present invention 1053] The method of any one of claims 1045 to 1052, comprising the step of introducing up to five additional mutations, up to four additional mutations, up to three additional mutations, up to two additional mutations, or up to one additional mutation into the CDR of the VH region of the reference antibody shown in SEQ ID NO: 1, 2, or 3. [The present invention 1054] The antibody having enhanced or decreased binding affinity comprises a binding region comprising a heavy chain variable (VH) region, the VH region comprising: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; and k) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V] The method of any of claims 1045 to 1053, comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of: [The present invention 1055] The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. D Value 1.6×10 -8 M~9.9×10 -8 M or 1.0×10 -7 ~9.9×10 -7 Any of the methods of 1045 to 1054 of the present invention, which corresponds to M. [The present invention 1056] The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. D Value 1.4×10 -8 ~1.0×10 -8 M or 9.9×10 -9 ~1×10 -9 Any of the methods of 1045 to 1054 of the present invention, which corresponds to M. [The present invention 1057] The method of any of claims 1045 to 1057, wherein the human CD3ε is expressed on a T cell. [The present invention 1058] The method of any of claims 1045 to 1057, wherein said human CD3ε is in isolated form, for example an isolated human CD3ε peptide. [The present invention 1059] A nucleic acid construct encoding one or more amino acid sequences of any one of 1001 to 1044 of the present invention. [The present invention 1060] (i) a nucleic acid sequence encoding the heavy chain sequence of any one of humanized or chimeric antibodies 1001 to 1016 of the present invention; (ii) a nucleic acid sequence encoding the light chain sequence of any one of humanized or chimeric antibodies 1017 to 1018 of the present invention; or (iii) Both (i) and (ii) An expression vector comprising: [The present invention 1061] A host cell comprising an expression vector of the present invention. [The present invention 1062] A host cell of the invention 1061 which is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell. [The present invention 1063] A composition comprising any one of the antibodies of the present invention Nos. 1001 to 1027 or the bispecific antibodies of the present invention Nos. 1028 to 1044. [The present invention 1064] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1027 or any one of the bispecific antibodies of the present inventions 1028 to 1044, and a pharma- ceutically acceptable carrier. [The present invention 1065] The antibody of any one of the present inventions 1001 to 1044, the composition of the present invention 1063, or the pharmaceutical composition of the present invention 1064 for use as a medicine. [The present invention 1066] The antibody of any of 1001 to 1044, the composition of 1063, or the pharmaceutical composition of 1064 for use as a medicine in the treatment of cancer, an infectious disease, or an autoimmune disease. [The present invention 1067] The antibody of any of the present inventions 1001 to 1027, the bispecific antibody of the present inventions 1028 to 1044, the composition of the present invention 1063, or the pharmaceutical composition of the present invention 1064 for use in treating a disease. [The present invention 1068] Use of the antibody of any of the inventions 1001 to 1027 or the bispecific antibody of the inventions 1028 to 1044 for the preparation of a medicament for the treatment of a disease. [The present invention 1069] The use of the present invention 1068 for the treatment of a disease which is cancer, an infectious disease, or an autoimmune disease. [The present invention 1070] A method for treating a disease, comprising the step of administering to a subject in need thereof an antibody of any of the present inventions 1001 to 1027, a bispecific antibody of the present inventions 1028 to 1044, a composition of the present invention 1063, or a pharmaceutical composition of the present invention 1064. [The present invention 1071] The method of claim 1070, wherein the disease is cancer, an infectious disease, or an autoimmune disease. [The present invention 1072] A method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, comprising the step of administering to a subject an antibody of any one of the present inventions 1001 to 1027, a composition of the present invention 1063, or a pharmaceutical composition of the present invention 1064, wherein the antibody is optionally labeled with a detectable agent. [The present invention 1073] A method for producing any one of the antibodies 1001 to 1027 of the present invention, comprising: a) culturing a host cell according to any one of claims 1061 to 1062 of the present invention; and b) purifying the antibody from the culture medium The method comprising: [The present invention 1074] A diagnostic composition comprising any one of the antibodies of the present invention Nos. 1001 to 1027 or any one of the bispecific antibodies of the present invention Nos. 1028 to 1044. [The present invention 1075] 1. A method for detecting the presence of CD3 antigen or CD3 expressing cells in a sample, comprising: a) contacting the sample with any one of the antibodies of the present inventions 1001 to 1027 or any one of the bispecific antibodies of the present inventions 1028 to 1044 under conditions allowing the formation of a complex between the antibody or the bispecific antibody and CD3; and b) analyzing whether a complex is formed The method comprising: [The present invention 1076] A kit for detecting the presence of CD3 antigen or CD3 expressing cells in a sample, comprising: i) any one of the antibodies 1001 to 1027 of the present invention or any one of the bispecific antibodies 1028 to 1044 of the present invention; and ii) Instructions for use of the kit. [The present invention 1077] An anti-idiotype antibody which binds to any one of the antibodies of the present invention 1001 to 1027 or any one of the bispecific antibodies of the present invention 1028 to 1044. [Brief description of the drawings]

[0041] [Figure 1] Heat plot of binding ratios of mutant vs. wt UniTE-huCD3-H1L1-T41K molecules. Ratios above 1 indicate stronger binding than wt, while ratios below 1 indicate weaker binding than wt. Binding was determined on CD3 / TCR-LC13 transfected Freestyle 293-F cells. [Diagram 2] Alignment of selected CD3 affinity variants in 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. [Diagram 3] T cell binding curves of selected VH affinity variants of the humanized CD3 (UniTE-huCD3-H1L1-T41K) antibody as determined by flow cytometry. The affinity variants shown cover a wide range of T cell binding capacities between wild type and undetectable responses. [Figure 4] T cell binding curves of selected VH affinity variants of humanized CD3 (UniTE-huCD3-H1L1-T41K) antibody determined by flow cytometry showing very low, undetectable T cell binding. [Diagram 5] T cell binding curves of selected VH affinity variants of humanized CD3 (BisG1-huCD3-H1L1-X-FEAL / 1014-Herceptin-FEAR) antibody determined by flow cytometry. The affinity variants shown cover a wide range of T cell binding capacities between wild type and undetectable responses. [Figure 6A]Cytotoxicity of CD3 affinity variants against solid tumor cell lines measured by Alamar Blue assay. (A) NCI-N87 cells, effector cell (T cell):tumor cell (NCI-N87 cell) ratio = 3:1, 48 h incubation, n = 2 donors. The affinity variants tested shown cover a wide range of cytotoxicity between wild-type response and no observed cytotoxicity for all tumor cell lines tested. [Figure 6B] Cytotoxicity of CD3 affinity variants against solid tumor cell lines measured by Alamar Blue assay. (B) SKOV3 cells, T cell:SKOV3 cell ratio=4:1, 48 h incubation, n=2 donors. The affinity variants tested shown cover a wide range of cytotoxicity between wild-type response and no observed cytotoxicity for all tumor cell lines tested. [Figure 6C] Cytotoxicity of CD3 affinity variants against solid tumor cell lines measured by Alamar Blue assay. (C) MDA-MB-231 cells, T cell:MDA-MB-231 cell ratio=8:1, 48 h incubation, n=2 donors. The affinity variants tested shown cover a wide range of cytotoxicity between wild-type response and no observed cytotoxicity for all tumor cell lines tested. [Figure 7] Cytotoxicity of CD3 affinity variants against blood (Daudi) cell lines measured by chromium release assay. T cell:Daudi cell ratio=10:1, 24 hour incubation, 1 donor. The affinity variants tested shown cover a wide range of cytotoxicity between wild type response and no observed cytotoxicity for the tumor cell lines tested. [Figure 8A]Cytotoxicity of CD3xHER2 bispecific antibodies in a NCI-N87, human PBMC co-implant model in NOD-SCID mice. NOD-SCID mice were co-inoculated with CD3 affinity antibodies at a dose level of 0.05 mg / kg and NCI-N87 tumor cells using HLA-A matched unstimulated human PBMC as a source of human T cells. Humanized WT CD3 (huCD3) and four different CD3 affinity mutants (N57E, H101K, S110A, Y114M) were tested. (A) Mean tumor volume (n=4 per group) followed over time after treatment with 0.05 mg / kg antibody. [Figure 8B] Cytotoxicity of CD3xHER2 bispecific antibodies in a NCI-N87, human PBMC co-implant model in NOD-SCID mice. NOD-SCID mice were co-inoculated with CD3 affinity antibodies at a dose level of 0.5 mg / kg and NCI-N87 tumor cells using HLA-A matched unstimulated human PBMC as a source of human T cells. Humanized WT CD3 (huCD3) and four different CD3 affinity mutants (N57E, H101K, S110A, Y114M) were tested. (B) Mean tumor volume followed over time after treatment with 0.5 mg / kg antibody (n=4 per group). [Figure 8C] Cytotoxicity of CD3xHER2 bispecific antibodies in a NCI-N87, human PBMC co-implant model in NOD-SCID mice. NOD-SCID mice were co-inoculated with CD3 affinity antibodies at a dose level of 0.05 mg / kg and NCI-N87 tumor cells using HLA-A matched unstimulated human PBMC as a source of human T cells. Humanized WT CD3 (huCD3) and four different CD3 affinity mutants (N57E, H101K, S110A, Y114M) were tested. (C) Mean tumor volume at day 44 after treatment with 0.05 mg / kg antibody on day 0 (n=4 per group). Statistics were performed on the data in C. [Figure 8D]Cytotoxicity of CD3xHER2 bispecific antibodies in a NCI-N87, human PBMC co-implant model in NOD-SCID mice. NOD-SCID mice were co-inoculated with CD3 affinity antibodies at a dose level of 0.5 mg / kg and NCI-N87 tumor cells using HLA-A matched unstimulated human PBMC as a source of human T cells. Humanized WT CD3 (huCD3) and four different CD3 affinity mutants (N57E, H101K, S110A, Y114M) were tested. (D) Mean tumor volume at day 44 after treatment with 0.5 mg / kg antibody on day 0 (n=4 per group). Statistics were performed on the data in D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3 and has an optimized affinity for CD3. Thus, it is an object of the present invention to provide a humanized or chimeric CD3 antibody that is optimized compared to a reference antibody, such as the antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. It is a further object of the present invention to provide an antibody that has an optimized in vivo efficacy compared to a reference antibody, such as the antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. It is a further object of the present invention to provide an antibody that has a lower binding affinity for CD3 than the antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8. It is yet another object of the present invention to provide an antibody that has a higher binding affinity for CD3 than the antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8.

[0043] In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, the antibody comprising a binding region comprising a heavy chain variable (VH) region, the VH region comprising a mutation in one of the three CDR sequences of a reference antibody having CDR sequences shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3, the mutation being at one of the positions selected from the group consisting of T31M, T31P, N57, H101, G105, S110, and Y114, the positions being 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 125th amino acid in the N-terminal to C-terminal direction. The numerical numbering of the positions corresponding to SEQ ID NO: 4 is shown in FIG. 2. Additionally, the CDR regions were annotated according to the IMGT definition.

[0044] In one embodiment of the invention, the antibody has a binding affinity to human CD3 that is reduced or enhanced 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.

[0045] In some embodiments of the invention, an antibody having a binding affinity for a CD3 peptide (e.g., a CD3 molecule such as SEQ ID NO: 402) that is reduced compared to a reference antibody may maintain the same cytolytic activity against target cells as the reference antibody.

[0046] In one embodiment of the invention, the antibody comprises a T31M or T31P mutation, wherein position T31 is according to SEQ ID NO:4.

[0047] In one embodiment of the invention the antibody comprises a mutation at position N57. Position N57 is according to SEQ ID NO: 4. In one embodiment the mutation is N57E.

[0048] In one embodiment of the invention the antibody comprises a mutation at position H101. Position H101 is according to SEQ ID NO: 4. In one embodiment the mutation is H101G or H101N.

[0049] In one embodiment of the invention the antibody comprises a mutation at position G105. Position G105 is according to SEQ ID NO: 4. In one embodiment the mutation is G105P.

[0050] In one embodiment of the invention the antibody comprises a mutation at position Y114. Position Y114 is according to SEQ ID NO: 4. In one embodiment the mutation is Y114M, Y114R or Y114V.

[0051] The reference antibodies defined by the VL sequences of SEQ ID NO: 4 and SEQ ID NO: 8 were synthesized at 1.5×10 -8 K of M D The antibody has a binding affinity for the CD3 peptide of SEQ ID NO:402 corresponding to a value of 0.01 to 0.01.

[0052] In some embodiments of the invention, the antibody has a titer of, for example, 1.6×10 -8 M~9.9×10 -8 Binding affinity of M, or e.g. 1.0 x 10 -7 ~9.9×10 -7 M binding affinity, 1.5 x 10 -8 M. In some embodiments of the invention, the antibody has a binding affinity for the CD3 peptide of SEQ ID NO: 402 that is lower than, for example, 1.4×10 -8 ~1.0×10 -8 M, e.g. 9.9 x 10 -9 ~1.0×10 -9 M, 1.5 x 10 -8 M has a binding affinity for the CD3 peptide of SEQ ID NO: 402 that is higher than M.

[0053] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 having CDR sequences selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 12, 2, 3; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 14, 2, 3; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 16, 2, 3; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 18, 2, 3; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 20, 2, 3; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 22, 2, 3; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 24, 2, 3; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 26, 2, 3; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 28, 2, 3; j) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 30, 2, 3; k) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 32, 2, 3; l) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 34, 2, 3; m) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 36, 2, 3; n) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 38, 2, 3; o) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 40, 2, 3; p) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 42, 2, 3; q) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 44, 2, 3; r) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 46, 2, 3; s) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 48, 2, 3; t) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 50, 2, 3; u) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 52, 2, 3; v) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 54, 2, 3; w) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 56, 2, 3; x) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3; y) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 60, 2, 3; z) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 62, 2, 3; aa) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 64, 2, 3; bb) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 66, 2, 3; cc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 68, 2, 3; dd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 70, 2, 3; ee) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 72, 2, 3; ff) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 74, 2, 3; gg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 76, 2, 3; hh) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 78, 2, 3; ii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 80, 2, 3; jj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 82, 2, 3; kk) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 84, 2, 3; ll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 86, 2, 3; mm) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 88, 2, 3; nn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 90, 2, 3; oo) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 92, 2, 3; pp) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 94, 2, 3; qq) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 96, 2, 3; rr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 98, 2, 3; ss) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 100, 3; tt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 102, 3; uu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 104, 3; vv) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3; ww) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 108, 3; xx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 110, 3; yy) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 112, 3; zz) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 114, 3; aaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 116, 3; bbb) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 118, 3; ccc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 120, 3; ddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 122, 3; eee) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 124, 3; fff) the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 126, 3; ggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 128, 3; hhh) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 130, 3; iii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 132, 3; jjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 134, 3; kkk) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 136, 3; lll) CDR sequences shown in SEQ ID NO: 1, 138, 3; mmm) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 140, 3; nnn) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 142, 3; ooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 144, 3; ppp) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 146, 3; qqq) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 148, 3; rrr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 150, 3; sss) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 152, 3; ttt) CDR sequences shown in SEQ ID NOs: 1, 154, 3; uuu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 156, 3; vvv) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 158, 3; www) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 160, 3; xxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 162, 3; yyy) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 164, 3; zzz) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 166, 3; aaaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 168, 3; bbbb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 170; cccc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 172; dddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 174; eeee) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176; ffff) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 178; gggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 180; hhhh) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 182; iiii) CDR sequences shown in SEQ ID NO: 1, 2, 184; jjjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 186; kkkk) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 188; lll) CDR sequences shown in SEQ ID NO: 1, 2, 190; mmmm) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 192; nnnn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 194; oooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 196; pppp) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 198; qqqq) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 200; rrrr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 202; ssss) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 204; tttt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 206; uuuu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 208; vvvv) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 210; wwww) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 212; xxxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 214; yyyy) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 216; zzzz) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 218; aaaaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 220; bbbbb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 222; ccccc) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 224; ddddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 226; eeeee) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 228; fffff) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 230; ggggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 232; hhhhh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 234; iii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236; jjjjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 238; kkkkk) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 240; lllll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 242; mmmmm) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244; nnnnn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 246; ooooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 248; ppppp) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 250; qqqqq) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 252; rrrrr) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 254; sssss) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 256; tttt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 258; uuuuu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 260; vvvvv) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 262; wwwww) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 264; xxxxx) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 266; yyyyy) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 268; zzzzz) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 270; aaaaaa) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 272; bbbbbb) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 274; cccccc) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 276; dddddd) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 278; eeeeee) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 280; ffffff) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 282; gggggg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284; hhhhhh) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 286; iiiiii) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 288; jjjjjj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 290; kkkkkk) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292; llllll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 294; mmmmmm) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 296; nnnnnn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 298, and oooooo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 300.

[0054] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 regions having CDR sequences selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V], and l) CDR1, CDR2, and CDR3 sequences having at least 90% or at least 95% amino acid sequence identity across the three CDR sequences to any one of the three CDR sequences set forth in a) to k), provided that the CDR1, CDR2, and CDR3 sequences do not have the sequences set forth in SEQ ID NOs: 1, 2, and 3.

[0055] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 regions having CDR sequences selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and a. and l) CDR1, CDR2 and CDR3 sequences as defined in a) to k), which have up to 5 further mutations or substitutions, up to 4 further mutations or substitutions, up to 3 further mutations or substitutions, up to 2 further mutations or substitutions or up to 1 further mutation or substitution across the three CDR sequences, which mutations or substitutions preferably do not alter the binding affinity to human CD3.

[0056] In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising a heavy chain variable (VH) region, said VH region comprising one of the VH sequences from the group consisting of: a) the VH sequence shown in SEQ ID NO: 13; b) the VH sequence shown in SEQ ID NO: 15; c) the VH sequence shown in SEQ ID NO: 17; d) the VH sequence shown in SEQ ID NO: 19; e) the VH sequence shown in SEQ ID NO: 21; f) the VH sequence shown in SEQ ID NO: 23; g) the VH sequence shown in SEQ ID NO: 25; h) the VH sequence shown in SEQ ID NO: 27; i) the VH sequence shown in SEQ ID NO: 29; j) the VH sequence shown in SEQ ID NO: 31; k) the VH sequence shown in SEQ ID NO: 33; l) the VH sequence shown in SEQ ID NO: 35; m) the VH sequence shown in SEQ ID NO: 37; n) the VH sequence shown in SEQ ID NO: 39; o) the VH sequence shown in SEQ ID NO: 41; p) the VH sequence shown in SEQ ID NO: 43; q) the VH sequence shown in SEQ ID NO: 45; r) the VH sequence shown in SEQ ID NO: 47; s) the VH sequence shown in SEQ ID NO: 49; t) the VH sequence shown in SEQ ID NO: 51; u) the VH sequence shown in SEQ ID NO: 53; v) the VH sequence shown in SEQ ID NO: 55; w) the VH sequence shown in SEQ ID NO: 57; x) the VH sequence shown in SEQ ID NO: 59; y) the VH sequence shown in SEQ ID NO: 61; z) the VH sequence shown in SEQ ID NO: 63; aa) the VH sequence shown in SEQ ID NO: 65; bb) the VH sequence shown in SEQ ID NO: 67; cc) the VH sequence shown in SEQ ID NO: 69; dd) the VH sequence shown in SEQ ID NO: 71; ee) the VH sequence shown in SEQ ID NO: 73; ff) the VH sequence shown in SEQ ID NO: 75; gg) the VH sequence shown in SEQ ID NO: 77; hh) the VH sequence shown in SEQ ID NO: 79; ii) the VH sequence shown in SEQ ID NO: 81; jj) the VH sequence shown in SEQ ID NO: 83; kk) the VH sequence shown in SEQ ID NO: 85; ll) the VH sequence shown in SEQ ID NO: 87; mm) the VH sequence shown in SEQ ID NO: 89; nn) the VH sequence shown in SEQ ID NO: 91; oo) the VH sequence shown in SEQ ID NO: 93; pp) the VH sequence shown in SEQ ID NO: 95; qq) the VH sequence shown in SEQ ID NO: 97; rr) the VH sequence shown in SEQ ID NO: 99; ss) the VH sequence shown in SEQ ID NO: 101; tt) the VH sequence shown in SEQ ID NO: 103; uu) the VH sequence shown in SEQ ID NO: 105; vv) the VH sequence shown in SEQ ID NO: 107; ww) the VH sequence shown in SEQ ID NO: 109; xx) the VH sequence shown in SEQ ID NO: 111; yy) the VH sequence shown in SEQ ID NO: 113; zz) the VH sequence shown in SEQ ID NO: 115; aaa) the VH sequence shown in SEQ ID NO: 117; bbb) the VH sequence shown in SEQ ID NO: 119; ccc) the VH sequence shown in SEQ ID NO: 121; ddd) the VH sequence shown in SEQ ID NO: 123; eee) the VH sequence shown in SEQ ID NO: 125; fff) the VH sequence shown in SEQ ID NO: 127; ggg) the VH sequence shown in SEQ ID NO: 129; hhh) the VH sequence shown in SEQ ID NO: 131; iii) the VH sequence depicted in SEQ ID NO: 133; jjj) the VH sequence shown in SEQ ID NO: 135; kkk) the VH sequence shown in SEQ ID NO: 137; lll) the VH sequence shown in SEQ ID NO: 139; mmm) the VH sequence shown in SEQ ID NO: 141; nnn) the VH sequence shown in SEQ ID NO: 143; ooo) the VH sequence shown in SEQ ID NO: 145; ppp) the VH sequence shown in SEQ ID NO: 147; qqq) the VH sequence shown in SEQ ID NO: 149; rrr) the VH sequence shown in SEQ ID NO: 151; sss) the VH sequence shown in SEQ ID NO: 153; ttt) the VH sequence shown in SEQ ID NO: 155; uuu) the VH sequence shown in SEQ ID NO: 157; vvv) the VH sequence shown in SEQ ID NO: 159; www) VH sequence shown in SEQ ID NO: 161; xxx) the VH sequence shown in SEQ ID NO: 163; yyy) the VH sequence shown in SEQ ID NO: 165; zzz) the VH sequence shown in SEQ ID NO: 167; aaaa) the VH sequence shown in SEQ ID NO: 169; bbbb) the VH sequence shown in SEQ ID NO: 171; cccc) the VH sequence shown in SEQ ID NO: 173; dddd) the VH sequence shown in SEQ ID NO: 175; eeee) the VH sequence shown in SEQ ID NO: 177; ffff) the VH sequence shown in SEQ ID NO: 179; gggg) the VH sequence shown in SEQ ID NO: 181; hhhh) the VH sequence shown in SEQ ID NO: 183; iiii) the VH sequence shown in SEQ ID NO: 185; jjjj) the VH sequence shown in SEQ ID NO: 187; kkkk) the VH sequence shown in SEQ ID NO: 189; lll) the VH sequence shown in SEQ ID NO: 191; mmmm) the VH sequence shown in SEQ ID NO: 193; nnnn) the VH sequence shown in SEQ ID NO: 195; oooo) the VH sequence shown in SEQ ID NO: 197; pppp) the VH sequence shown in SEQ ID NO: 199; qqqq) the VH sequence shown in SEQ ID NO: 201; rrrr) the VH sequence shown in SEQ ID NO: 203; ssss) the VH sequence shown in SEQ ID NO: 205; tttt) the VH sequence shown in SEQ ID NO: 207; uuuu) the VH sequence shown in SEQ ID NO: 209; vvvv) the VH sequence shown in SEQ ID NO: 211; wwww) the VH sequence shown in SEQ ID NO: 213; xxxx) the VH sequence shown in SEQ ID NO: 215; yyyy) the VH sequence shown in SEQ ID NO: 217; zzzz) the VH sequence shown in SEQ ID NO: 219; aaaaa) the VH sequence shown in SEQ ID NO: 221; bbbbb) the VH sequence shown in SEQ ID NO: 223; ccccc) the VH sequence shown in SEQ ID NO: 225; ddddd) the VH sequence shown in SEQ ID NO: 227; eeeee) the VH sequence shown in SEQ ID NO: 229; fffff) the VH sequence shown in SEQ ID NO: 221; ggggg) the VH sequence shown in SEQ ID NO: 223; hhhhh) the VH sequence shown in SEQ ID NO: 225; iiiiii) the VH sequence depicted in SEQ ID NO: 227; jjjjj) the VH sequence shown in SEQ ID NO: 229; kkkkk) the VH sequence shown in SEQ ID NO: 231; lllll) the VH sequence shown in SEQ ID NO: 233; mmmmm) the VH sequence shown in SEQ ID NO: 235; nnnnn) the VH sequence shown in SEQ ID NO: 237; ooooo) the VH sequence shown in SEQ ID NO: 239; ppppp) the VH sequence shown in SEQ ID NO: 241; qqqqq) the VH sequence shown in SEQ ID NO: 243; rrrrr) the VH sequence shown in SEQ ID NO: 245; sssss) the VH sequence shown in SEQ ID NO: 247; tttt) the VH sequence shown in SEQ ID NO: 249; uuuuu) the VH sequence shown in SEQ ID NO: 251; vvvvv) the VH sequence shown in SEQ ID NO: 253; www) the VH sequence shown in SEQ ID NO: 255; xxxxx) the VH sequence shown in SEQ ID NO: 257; yyyyy) the VH sequence shown in SEQ ID NO: 259; zzzzz) the VH sequence shown in SEQ ID NO: 261; aaaaaa) the VH sequence shown in SEQ ID NO: 263; bbbbbb) the VH sequence shown in SEQ ID NO: 265; cccccc) the VH sequence shown in SEQ ID NO: 267; dddddd) the VH sequence shown in SEQ ID NO: 269; eeeeee) the VH sequence shown in SEQ ID NO: 271; ffffff) the VH sequence shown in SEQ ID NO: 273; gggggg) the VH sequence shown in SEQ ID NO: 275; hhhhhh) the VH sequence shown in SEQ ID NO: 277; iiiiii) the VH sequence shown in SEQ ID NO: 279; jjjjjj) the VH sequence shown in SEQ ID NO: 281; kkkkkk) the VH sequence shown in SEQ ID NO: 283; llllll) the VH sequence shown in SEQ ID NO: 285; mmmmmm) the VH sequence shown in SEQ ID NO: 287; nnnnnn) the VH sequence shown in SEQ ID NO: 289; oooooo) the VH sequence shown in SEQ ID NO: 291; pppppp) the VH sequence shown in SEQ ID NO: 293; qqqqqq) the VH sequence shown in SEQ ID NO: 295; rrrrrr) the VH sequence shown in SEQ ID NO: 297; ssssss) the VH sequence shown in SEQ ID NO: 299, and tttttt) VH sequence shown in SEQ ID NO:301.

[0057] In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising a heavy chain variable (VH) region, said VH region comprising one of the VH sequences selected from the group consisting of: a) the VH sequence shown in SEQ ID NO: 55 [T31M]; b) the VH sequence shown in SEQ ID NO: 59 [T31P]; c) the VH sequence shown in SEQ ID NO: 107 [N57E]; d) the VH sequence shown in SEQ ID NO: 177 [H101G]; e) the VH sequence shown in SEQ ID NO: 185 [H101N]; f) the VH sequence shown in SEQ ID NO: 221 [G105P]; g) the VH sequence shown in SEQ ID NO: 237 [S110A]; h) the VH sequence shown in SEQ ID NO: 245 [S110G]; i) the VH sequence shown in SEQ ID NO: 285 [Y114M]; j) the VH sequence shown in SEQ ID NO: 293 [Y114R], and k) The VH sequence shown in SEQ ID NO: 299 [Y114V].

[0058] In one embodiment of the invention, the humanized or chimeric antibody comprises a binding region comprising a light chain variable (VL) region, said VL region comprising CDR1, CDR2, and CDR3 with CDR sequences selected from the group consisting of: a) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 7; 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) 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) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 310, GTN, 7; g) 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) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 318, GTN, 7; k) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 320, GTN, 7; l) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 322, GTN, 7; m) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 324, GTN, 7; n) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 326, GTN, 7; o) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 328, GTN, 7; p) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 330, GTN, 7; q) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 332; r) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 334; s) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 336; t) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 338; u) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 340; v) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 342; w) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 344; x) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 346; y) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 348; z) 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) 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; gg) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 364; hh) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 366; ii) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 368; jj) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 370; kk) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 372; ll) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 374; mm) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 376; nn) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 378; oo) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 380; pp) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 382; qq) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 384; rr) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 386; ss) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 388; tt) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 390; uu) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 392, and vv) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 6, GTN, 394.

[0059] In another embodiment of the invention, the humanized or chimeric antibody comprises a binding region comprising a light chain variable (VL) region, said VL region comprising one of the VL sequences selected from the group consisting of: a) the VL sequence shown in SEQ ID NO: 8; and b) The VL sequence shown in SEQ ID NO: 10.

[0060] The term "antibody" as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, capable of specifically binding to an antigen under typical physiological conditions with a half-life of a significant period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other functionally defined period of time (e.g., a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a time sufficient for the antibody to mobilize effector activity). The binding region that interacts with the antigen (sometimes referred to herein as a binding domain, both terms having the same meaning) includes the variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant region of an antibody (Ab) may mediate the binding of the immunoglobulin to host tissues or host factors, such as various cells of the immune system (e.g., effector cells and T cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. As indicated above, the term antibody as used herein includes fragments of antibodies that retain the ability to specifically interact (e.g., bind) to an antigen, unless otherwise stated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, V L , V H , C L and C H (ii) a monovalent fragment consisting of one domain, or a monovalent antibody as described in WO2007059782 (Genmab A / S); 2 (iii) V fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; H Domain and C H(iv) an Fd fragment consisting essentially of one domain; and (iv) a V L Domains and V H Furthermore, the Fv fragment essentially consists of two domains, V L and V H are encoded by separate genes, but using recombinant methods, they can be L Area and V H They may be joined by synthetic linkers that allow the domains to pair into a single protein chain forming a monovalent molecule (known as single chain antibodies or single chain Fvs (scFvs); see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by the context. Although such fragments are generally included within the meaning of antibodies, they are, collectively and each individually, unique features of the present invention and exhibit distinct biological properties and usefulness. These and other useful antibody fragments in the context of the present invention are discussed further herein. Unless otherwise specified, it should be understood that the term antibody also encompasses polyclonal, monoclonal (mAb), chimeric and humanized antibodies, as well as antibody fragments which retain the ability to specifically bind to antigen (antigen-binding fragments) obtained by any known method, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. The antibodies produced can be of any isotype.

[0061] The term "immunoglobulin heavy chain", "heavy immunoglobulin chain" or "heavy chain" as used herein refers to one of the immunoglobulin chains. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains CH1, CH2, and CH3. The heavy chain constant region may further include a hinge region. The term "immunoglobulin" as used herein refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four of which may be interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g.,

[14] ). In the structure of immunoglobulins (e.g., IgG), the two heavy chains are connected to each other by disulfide bonds in the so-called "hinge region". Like the heavy chains, each light chain typically consists of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically consists of one domain, CL. The VH and VL regions can also be further subdivided into hypervariable regions (i.e., hypervariable regions that may be hypervariable in sequence and / or in the form of structurally defined loops), also called complementarity determining regions (CDRs), interspersed with highly conserved regions called framework regions (FRs). VH and VL each typically consist of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

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

[16] -

[17] .

[0062] As used herein, the term "isotype" refers to the immunoglobulin (sub)class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f) [SEQ ID NO:407]) encoded by the heavy chain constant region genes. Thus, in one embodiment, the antibody comprises an immunoglobulin heavy chain of the IgG1 class or any allotype thereof. Furthermore, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain.

[0063] The term "chimeric antibody" as used herein refers to an antibody whose variable region originates from a non-human species (e.g., from a rodent) and whose constant region originates from a different species, such as human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term used for various types of antibody modification and is a process well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques as described in

[18] . Chimeric antibodies can therefore be genetically engineered recombinant antibodies. Some chimeric antibodies are genetically or enzymatically engineered antibodies. As the production of chimeric antibodies is known to those skilled in the art, the production of chimeric antibodies of the present invention can also be performed by methods other than those described herein. Therapeutic chimeric monoclonal antibodies are developed to reduce antibody immunogenicity. They typically contain non-human (e.g., murine) variable regions specific for the antigen of interest and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" as used in connection with chimeric antibodies refers to the region that includes the CDRs and framework regions of both the heavy and light chains of an immunoglobulin.

[0064] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to have a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen-binding site onto a homologous human acceptor framework region (FR) (see

[19] -

[20] ). To fully reconstitute the binding affinity and specificity of the parent antibody, it may be necessary to substitute framework residues from the parent antibody (i.e., the non-human antibody) into the human framework region (backmutation). Structural homology modeling can help to identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily backmutations, may be applied to obtain a humanized antibody with favorable characteristics such as affinity or biochemical properties.

[0065] A humanized or chimeric antibody according to any aspect or embodiment of the present invention may be referred to as a "humanized or chimeric CD3 antibody," "a humanized or chimeric antibody of the invention," "a CD3 antibody," or "a CD3 antibody of the invention," all of which have the same meaning and purpose, except where the context is inconsistent.

[0066] Since the amino acid sequence of an antibody of non-human origin is different from that of human origin, the non-human antibody is potentially immunogenic when administered to a human patient. However, despite the antibody's non-human origin, its CDR segments are responsible for the antibody's ability to bind to its target antigen, and humanization aims to maintain the specificity and binding affinity of the antibody. Thus, humanization of a non-human therapeutic antibody is performed to minimize its immunogenicity in humans, while at the same time, the humanized antibody maintains the specificity and binding affinity of the antibody of non-human origin.

[0067] As used herein, the term "binding domain" refers to a region of an antibody that has the ability to bind to some molecule, such as a polypeptide present on a cell, bacterium or virion.

[0068] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target, which binding to the antigen or target is typically determined by, for example, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument, with the antigen as the ligand and the antibody as the analyte, at a rate of about 10 -6 M or less, e.g. 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K below M D and is at least 10-fold, e.g. at least 100-fold, e.g. at least 1,000-fold, e.g. at least 10,000-fold, e.g. at least 100,000-fold, e.g. at least 100,000-fold lower than the affinity for binding to a non-specific antigen (e.g., BSA, casein) that is neither the predetermined nor a closely related antigen. D The affinity of an antibody is determined by its K D Since it depends on the antibody's K D If K is very low (i.e., the antibody is highly specific), the affinity for the antigen can be 10,000-fold lower than the affinity for a non-specific antigen. D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0069] As used herein, the term "human CD3" refers to the human cluster of differentiation 3 protein, which is part of the T cell coreceptor protein complex and is composed of four distinct chains. As CD3 is also found in other species, the term "CD3" as used herein is not limited to human CD3, unless the context is otherwise contradictory. In mammals, this complex contains the CD3γ (gamma) chain (human CD3γ chain Swissprot P09693 or cynomolgus CD3γ Swissprot Q95LI7), the CD3δ (delta) chain (human CD3δ Swissprot P04234 or cynomolgus CD3δ Swissprot Q95LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766, or cynomolgus CD3ε Swissprot Q95LI5, or rhesus CD3ε Swissprot G7NCB9) and the CD3ζ (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus CD3ζ Swissprot Q09TK0). These chains associate with a molecule known as the T cell receptor (TCR) to generate an activation signal in T lymphocytes. The TCR and CD3 molecules together make up the TCR complex.

[0070] It is known to those skilled in the art that the amino acid sequences referred to as Swissprot numbers include signal peptides that are removed after translation of the protein. Thus, proteins such as CD3 present on the cell surface do not include signal peptides. In particular, the amino acid sequences listed in Table 1 do not contain such signal peptides. Proteins such as those listed in Table 1 can be referred to as "mature proteins". Thus, SEQ ID NO:398 represents the amino acid sequence of mature human CD3δ (delta), SEQ ID NO:399 represents the amino acid sequence of mature human CD3ε (epsilon), SEQ ID NO:403 represents the amino acid sequence of mature cynomolgus CD3ε, and SEQ ID NO:404 represents the amino acid sequence of mature rhesus CD3ε. Thus, the term "mature" as used herein refers to a protein that does not include any signal or leader sequence.

[0071] It is well known that the homology, length and cleavage site location of signal peptide sequences vary considerably among proteins. Signal peptides can be determined in different ways, for example, SEQ ID NO:399 of the present invention was determined according to the SignalP application (available at http: / / www.cbs.dtu.dk / services / SignalP / ).

[0072] In certain embodiments, the humanized or chimeric antibodies of the invention bind to the epsilon chain of CD3, such as the epsilon chain of human CD3 (SEQ ID NO:399). In yet another particular embodiment, the humanized or chimeric antibodies bind to an epitope within amino acids 1-27 of the N-terminal portion of human CD3ε (epsilon) (SEQ ID NO:402). In certain such embodiments, the antibodies may further cross-react with other non-human primate species, such as cynomolgus monkeys (cynomolgus CD3 epsilon SEQ ID NO:403) and / or rhesus monkeys (rhesus CD3 epsilon SEQ ID NO:404).

[0073] The antibodies of the invention, which comprise the CDR sequences defined herein and further comprise framework regions, may differ in sequences outside the CDR sequences, yet still retain full binding ability compared to the original antibody.The invention therefore also relates to antibodies which comprise amino acid sequences of variable regions which have a certain sequence identity with any of the sequences described herein.

[0074] The term "sequence identity" as used in the context of the present invention refers to the percentage identity between two sequences as a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap (i.e., % homology = 100 x number of identical positions / total number of positions). The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller

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

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

[23] (e.g., as used in Vector NTI Advance® software version 11.5 (Invitrogen Inc.)).

[0075] Thus, in one embodiment of the invention, the antibody comprises a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 regions having three CDR sequences selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and l) CDR1, CDR2, and CDR3 sequences having at least 90% or at least 95% amino acid sequence identity across the three CDR sequences to any one of the three CDR sequences set forth in a) to k), provided that the CDR1, CDR2, and CDR3 sequences do not have the sequences set forth in SEQ ID NOs: 1, 2, and 3.

[0076] As shown in sequence table 1 of this document, the VH region consists of a sequence of 125 amino acids. Thus, a second VH sequence of 125 amino acids, the 124 amino acid position of which is identical to one of the first VH sequences listed above, has 99.2% sequence identity with the first VH sequence. A second sequence of 125 amino acids, the 120 amino acid position of which is identical to one of the first VH sequences listed above, has 96% sequence identity with the first VH sequence. A second sequence of 125 amino acids, the 115 amino acid position of which is identical to one of the first VH sequences listed above, has 92% sequence identity with the first VH sequence.

[0077] In certain embodiments thereof, the VH region has at least 96% amino acid sequence identity to at least one of the VH sequences identified in said group.

[0078] In one embodiment of the invention, the mutations are located in the framework regions of the VH region. Thus, in some embodiments, the three CDR sequences of the VH region are 100% identical to the antibodies of the invention, but amino acid variations may occur in the framework regions of the VH region. Such amino acid variations in the framework regions preferably do not alter the binding affinity of the antibody to CD3 compared to the antibody when the CDRs are contained in the reference frame of SEQ ID NO: 407.

[0079] Mutations in the VH sequence that result in variations in sequence identity may preferably be conservative, physical, or functional amino acids. Substitution of an amino acid with a similar amino acid may increase the likelihood of retaining the functionality of the parent antibody.

[0080] In one embodiment of the invention, the antibody is a humanized antibody.

[0081] In one embodiment of the invention, the antibody is a full-length antibody.

[0082] The humanized antibodies of the present invention can be generated by comparing the heavy and light chain variable region amino acid sequences to a database of human germline variable region sequences to identify heavy and light chain human sequences with adequate homology to be used as human variable framework regions. A series of humanized heavy and light chain variable regions can be designed, for example, by grafting mouse CDRs into the framework regions (identified as described above) and, if necessary, backmutating (mutating one or more of the human amino acid residues in the framework regions back to the non-human amino acid at that particular position) residues identified as potentially critical for restoring antibody binding efficiency to their particular mouse sequences. The variant sequences with the lowest occurrence of potential T-cell epitopes, as determined by application of in silico techniques such as iTope™ and TCED™ (

[24] ,

[25] , and

[26] ), can then be selected.

[0083] Furthermore, the humanized antibodies of the present invention can also be "deimmunized". Deimmunization may be desirable since the presence of human T cell epitopes within a protein sequence, such as the humanized antibodies of the present invention, may increase the immunogenicity risk profile due to their potential to activate helper T cells. Such activation of helper T cells can be avoided by deimmunization. Deimmunization can be performed by introducing mutations into the amino acid sequence of the humanized antibody to remove T cell epitopes without significantly reducing the binding affinity of the antibody.

[0084] Thus, in one aspect of the invention, a humanized antibody can be produced by a method comprising the steps of: (i) comparing the entire non-human heavy and / or light chain variable sequence with a database of human germline sequences; (ii) obtaining a humanized sequence by selecting the human germline sequence having the highest homology to the non-human sequence; (iii) optimizing the humanized sequence by back mutation, if necessary; and (iv) expressing the sequence in a suitable expression system.

[0085] Thus, the full-length antibody of the present invention can be produced by a method comprising the steps of: (i) comparing the non-human heavy and light chain variable sequences with a database of human germline sequences; (ii) selecting the human germline sequences with the highest homology to the non-human sequences; (iii) obtaining humanized sequences by grafting the non-human CDRs into the selected human germline; (iv) optimizing the humanized sequences by back mutation, if necessary; (v) identifying the constant heavy and light chain sequences; and (vi) expressing the complete heavy and light chain sequences in a suitable expression system. Thus, the full-length antibody of the present invention can be produced as described in Example 1. Producing full-length antibodies starting from either CDR sequences or the entire variable region sequences is known to those skilled in the art. Thus, the method of making the full-length antibody of the present invention will be known to those skilled in the art.

[0086] As used herein, the term "complete heavy chain sequence" refers to a sequence consisting of a heavy chain variable sequence and a constant heavy chain sequence.

[0087] As used herein, the term "complete light chain sequence" refers to a sequence consisting of the light chain variable sequence and the constant light chain sequence.

[0088] The back mutations can be introduced by standard DNA mutagenesis techniques. Standard techniques for such DNA mutagenesis are described in

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

[0089] Thus, in one embodiment the antibody is a humanized antibody.

[0090] Chimeric antibodies can be made by replacing all of the constant region sequences of a non-human (e.g., mouse) antibody with constant region sequences of human origin. Thus, the chimeric antibody maintains the complete non-human variable region sequences. Thus, the chimeric antibodies of the present invention can be produced by a method that includes expressing the non-human heavy chain variable (SEQ ID NO:405), the non-human light chain variable sequence (SEQ ID NO:406), the human constant heavy chain sequence and the human constant light chain sequence in a suitable expression system, thereby producing a full-length chimeric antibody. Alternative methods can also be used. Such methods of producing chimeric antibodies are known to those skilled in the art, so that the skilled artisan will know how to produce the chimeric antibodies of the present invention. Thus, to produce the chimeric antibodies of the present invention, the mutations of the present invention will be introduced into the non-human (e.g., mouse) VH or VL sequence.

[0091] Thus, in one embodiment the antibody is a chimeric antibody.

[0092] In one embodiment, the antibody is a full-length antibody. As used herein, the term "full-length antibody" refers to an antibody (e.g., a parent antibody or a variant antibody) that contains all of the heavy and light chain constant and variable domains that normally correspond to those found in a wild-type antibody of that isotype.

[0093] In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0094] As used herein, the term "Fc region" refers to a region that includes, from the N-terminus to the C-terminus, at least the hinge region, the CH2 region, and the CH3 region. The Fc region may further include a CH1 region on the N-terminal side of the hinge region.

[0095] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to the Eu numbering system as set forth in Kabat.

[0096] Unless otherwise stated or contradicted by context, the amino acids of constant region sequences are numbered according to the Eu-index of numbering (described in

[27] ), and are referred to herein as "Eu numbering as described in Kabat," "Eu numbering of Kabat," or "in the Eu numbering system."

[0097] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 in the Eu numbering system. However, the CH1 region may be of any of the other subtypes described herein.

[0098] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 in the Eu numbering system. However, the CH2 region may be of any of the other subtypes described herein.

[0099] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 in the Eu numbering system. However, the CH3 region may be of any of the other subtypes described herein.

[0100] In one embodiment, the immunoglobulin heavy chain isotype is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain can be of any allotype within each immunoglobulin class, such as IgG1m(f) (SEQ ID NO:407). Thus, in one particular embodiment, the immunoglobulin heavy chain isotype is IgG1 or any allotype thereof, such as IgG1m(f) (SEQ ID NO:407).

[0101] When targeting the antigen CD3, which is part of the T cell receptor (TCR), a T cell-specific mechanism of cell killing is desirable. Other effector functions, such as complement activation, may not be necessary, so reducing effector functions is desirable. C1q binding is the first step in the complement cascade and therefore serves as an indicator of the complement-dependent cytotoxicity (CDC) ability of the antibody. If C1q binding to the antibody can be avoided, activation of the complement cascade can also be avoided.

[0102] Thus, in one embodiment, the antibody comprises an Fc region modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.9%, or 100% compared to a wild-type antibody, where C1q binding is determined by ELISA. In a preferred embodiment, the antibody comprises an Fc region modified such that binding of C1q to the antibody is reduced by at least 99%-100% compared to a wild-type antibody, where C1q binding is determined by ELISA.

[0103] The term "modified" as used herein refers to an amino acid sequence of an Fc region that is not identical to the amino acid sequence of a wild-type Fc region. That is, amino acid residues at specific positions of the wild-type Fc region are substituted, deleted, or inserted, for example to modify the binding site of C1q, the binding site of other effector molecules, or binding to Fc receptors (FcRs). Such modifications of the amino acid sequence can be prepared by substituting one or more amino acids with conservative amino acids, or by substituting one or more amino acids with alternative amino acids that are physically and / or functionally similar to the amino acids present in the wild-type. Substitutions can also be prepared by substituting non-conservative amino acids.

[0104] In the context of the present invention, amino acids can be described as conservative or non-conservative amino acids and can therefore be classified accordingly. Amino acid residues can also be classified into classes defined by alternative physical and functional properties. Thus, amino acid classes can be reflected in one or both of the following tables:

[0105] Conserved classes of amino acid residues TIFF2025038049000002.tif40128Alternative physical and functional classifications of amino acid residues TIFF2025038049000003.tif73143

[0106] In the context of the present invention, substitutions in an antibody, such as a humanized or chimeric antibody, include: Original amino acid - position - substituted amino acid It is shown in the form:

[0107] With reference to well-known amino acid nomenclature, three-letter or one-letter symbols are used to indicate any amino acid residue, including the symbols Xaa and X. Thus, the designation "L234F" or "Leu234Phe" means that the antibody contains a substitution of leucine with phenylalanine at amino acid position 234.

[0108] Substitution of an amino acid at a given position for any other amino acid is Original amino acid-position, i.e., for example, "L234" It is mentioned that:

[0109] For modifications in which the original and / or replacement amino acids may include two or more amino acids, but not all amino acids, those two or more amino acids can be separated by "," or " / ". For example, replacement of leucine with phenylalanine, arginine, lysine or tryptophan at position 234 is "Leu234Phe,Arg,Lys,Trp" or "Leu234Phe / Arg / Lys / Trp" or "L234F,R,K,W" or "L234F / R / K / W" or "L234→F,R,K or W".

[0110] With respect to the present invention, such designations may be used interchangeably and have the same meaning and purpose.

[0111] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 naturally occurring amino acids or with other amino acids, e.g., unnatural amino acids. For example, substitution of amino acid L at position 234 includes each of the following substitutions: 234A, 234C, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P, and 234Y. Note that this is equivalent to designating 234X, where X designates any amino acid other than the original amino acid. These substitutions can also be designated as L234A, L234C, etc., or L234A,C, etc., or L234A / C / , etc. The same is equally true for any and all positions mentioned herein, and any one of such substitutions is specifically included herein.

[0112] The antibodies of the invention may also include deletions of amino acid residues. Such deletions are designated "del" and include, for example, designations such as L234del. Thus, in such embodiments, the leucine at position 234 has been deleted from the amino acid sequence.

[0113] The terms "amino acid" and "amino acid residue" may be used interchangeably herein.

[0114] In one embodiment of the invention, the antibody comprises a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 regions having three CDR sequences selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V], and l) CDR1, CDR2 and CDR3 sequences as defined in a) to k), which have up to 5 further mutations or substitutions, up to 4 further mutations or substitutions, up to 3 further mutations or substitutions, up to 2 further mutations or substitutions or up to 1 further mutation or substitution across the three CDR sequences, which mutations or substitutions preferably do not alter the binding affinity to human CD3.

[0115] In one embodiment of the invention, the further mutations or substitutions are conservative, physically or functional amino acids.

[0116] In some embodiments, the binding to CD3 can be binding to full length CD3, such as CD3 present on T cells. In other embodiments, the binding to CD3 can be binding to a CD3 peptide, for example, as shown in SEQ ID NO: 402. Binding to the CD3 peptide, and whether any further mutations can alter binding to CD3, can be determined by biolayer interferometry as disclosed in Example 7.

[0117] In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0118] As used herein, the term "C1q binding" refers to the binding of C1q to an antibody when the antibody is bound to its antigen. As used herein, the term "binding to its antigen" refers to the binding of an antibody to its antigen both in vivo and in vitro.

[0119] As used herein, the term "reduced" when referring to C1q binding refers to the ability of the antibodies of the invention to reduce, minimize, or completely inhibit C1q binding to the antibodies of the invention when compared to C1q binding to a wild-type antibody.

[0120] As used herein, the term "reduced" or "reducing" or any variant thereof, when used in relation to the binding affinity of an antibody that binds to human CD3, refers to a lower binding affinity when compared to a reference binding affinity. In this context, the reference binding affinity may be the binding affinity of a reference antibody defined by the VH sequence of SEQ ID NO: 4 and the VL sequence of SEQ ID NO: 8, when bound to a CD3 peptide such as SEQ ID NO: 402 and determined by biolayer interferometry as described in Example 7.

[0121] As used herein, the term "binding affinity" refers to the binding of an antibody to a predetermined antigen or target, typically at a K D As used herein, the term "K" refers to binding that occurs with an affinity equivalent to D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0122] The term "wild type antibody" as used herein with respect to the use of the antibodies of the present invention in comparative assays refers to an antibody that is identical to the test antibody except that it is not inactive. In this context, the term "inactive" refers to a modified Fc region that has reduced or absent C1q binding (i.e., C1q binding is determined by ELISA); a modified Fc region that has reduced or absent Fc-mediated T cell proliferation as determined in a PBMC-based functional assay (i.e., T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay); and / or a modified Fc region that has reduced or absent Fc-mediated CD69 expression as determined in a PBMC-based functional assay. Thus, a wild type antibody contains natural amino acids in the immunoglobulin heavy chain. That is, an antibody that does not contain any amino acid modifications that may alter or reduce the ability of the antibody to interact with, for example, C1q, Fc receptors, etc. Such a wild type antibody would therefore remain an activated antibody that can bind, for example, C1q. Wild-type antibodies and antibodies of the invention may contain other amino acid modifications that do not affect the ability of the antibody to elicit effector functions, such as for purposes of making the antibody a bispecific antibody.

[0123] As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay (ELISA), a test that uses antibodies and a color change to identify a substance. e nzyme- l inked i mmuno s Orbent a The term ELISA refers to a method for assaying a plate surface. A first specific antibody is attached to the plate surface. Proteins from a sample are then added to test for binding to the first specific antibody. A second antibody is added that binds to the antibody from the sample. The second antibody is linked to an enzyme, and in a final step, a substance containing a substrate for the enzyme is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. The concept of ELISA is well known in the art, and various methods of performing ELISA are considered to be part of the methods for evaluating the antibodies of the present invention.

[0124] Specifically, the ability of the antibodies of the invention to bind to C1q was determined by the steps of (i) coating the antibodies onto a 96-well plate, (ii) adding 3% serum, (iii) adding an anti-human C1q antibody, (iv) developing the plate, and (v) measuring OD 405nm Thus, in one embodiment, the antibody comprises an Fc region modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to a wild-type antibody, where C1q binding is determined by (i) coating the antibody to a 96-well plate, (ii) adding 3% serum, (iii) adding an anti-human C1q antibody, (iv) developing the plate, and (v) measuring OD 405nm The method is determined by ELISA, which includes measuring

[0125] As used herein, the term "Fc receptor" or "FcR" refers to a protein found on the surface of certain cells. FcR binds to the Fc region of an antibody. There are several different types of FcR, which are classified based on the type of antibody they recognize. For example, Fcγ (gamma) receptors bind to antibodies of the IgG class.

[0126] As used herein, the term "Fcγ receptor", "Fc gamma receptor" or "FcγR" refers to a group of Fc receptors that belong to the immunoglobulin superfamily and are the Fc receptors most important for inducing phagocytosis of opsonized (coated) microorganisms. This family includes several members with different molecular structures and therefore different antibody affinities: FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), and FcγRIIIb (CD16b).

[0127] 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 the present invention, "Fc binding," "Fc receptor binding," "FcR binding," and "binding of an antibody Fc region to an FcR" refer to the binding of an Fc region to an Fc receptor (FcR) or an effector molecule. The terms "FcγR binding" and "FcγRI binding" refer to the binding of an Fc region to an Fc gamma receptor and an 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 FcRs present on other cells, e.g., monocytes, which leads to non-specific Fc-mediated activation of the T cell. Such non-specific Fc-mediated activation of the T cell would be undesirable. T cells can also be activated by targeted (or target-specific) T cell activation. Such targeted T cell activation would be highly desirable for the treatment of a range of indications, such as cancer. As used herein, the term "targeted T cell activation" refers to the use of bispecific antibodies that include 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, to induce T cells to specific cells, such as tumor cells. Thus, the use of bispecific antibodies in which one binding region binds to CD3 present on a T cell and the other binding region binds to a target specific antigen, for example, on a tumor cell, can facilitate the targeting of T cells to specific cells, such as tumor cells. Since non-specific Fc-mediated T cell activation is still possible, such undesired non-specific Fc-mediated T cell activation by Fc-mediated cross-linking should be avoided and can be abolished by inactivating the Fc region with respect to such activity. This prevents the interaction between the inactive Fc region and the Fc receptor present.

[0128] The antibody of the present invention may contain modifications in the Fc region. When an antibody contains such modifications, it may be an inactive or non-activated antibody. As used herein, the terms "inactive", "inactive" or "non-activated" refer to an Fc region that is at least unable to bind to any Fcγ receptor, unable to induce Fc-mediated cross-linking via FcR, unable to induce FcR-mediated cross-linking of a target antigen via the Fc region, or unable to bind C1q. The inactivity of the Fc region of a humanized or chimeric CD3 antibody is conveniently tested using an antibody in a monospecific format, but the inactive Fc region thus identified can be used in bispecific or other humanized or chimeric multispecific CD3 antibodies.

[0129] For the purpose of therapeutic antibody development, several mutants can be constructed to render the Fc region of the antibody inactive for interaction with Fc gamma receptors and C1q. Examples of such mutants are described herein.

[0130] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that the antibody mediates Fc-mediated T cell proliferation that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% reduced compared to a wild-type antibody, where said T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0131] The term "reduce" in reference to T cell proliferation refers to the ability of the antibodies of the invention to reduce, minimize or completely inhibit T cell proliferation as compared to the proliferation of T cells bound by wild type antibody. The ability of the antibodies to reduce T cell proliferation can be assessed by a PBMC-based functional assay. In one embodiment, the assay is performed on human PBMCs. In another embodiment, the assay is performed on cynomolgus monkey PBMCs. In yet another embodiment, the assay is performed on rhesus monkey PBMCs. Because the antibodies of the invention are cross-reactive, the PBMC-based assays described herein can be performed with PBMCs of any species, e.g., human, cynomolgus monkey or rhesus monkey, to show reduction in T cell proliferation, as long as the PBMCs of the species used are within the cross-reactivity spectrum of the antibody.

[0132] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay used to assess the functional characteristics of an antibody of the invention, such as the ability of said antibody to affect T cell proliferation or CD69 expression, where the only cells present are peripheral blood mononuclear cells. Thus, in one embodiment, PBMCs are incubated with antibodies in the range of 1-1000 ng / mL in 5% (vol / vol) CO. 2 T cell proliferation is measured by a method that includes incubating at 37° C. for 3 days in a humidified incubator, adding a chemical compound such as BrdU that is incorporated into the DNA of proliferating cells, incubating for 5 hours, pelleting the cells, drying the cells, optionally storing the cells at 4° C., coating the cells onto an ELISA plate, incubating with anti-BrdU-peroxidase for 90 minutes at room temperature, developing with 1 mg / mL 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) for approximately 30 minutes, adding 100 μL of 2% oxalic acid to stop the reaction, and measuring absorbance at 405 nm in a suitable microplate reader.

[0133] As used herein, the term "proliferation" refers to cell growth through cell division.

[0134] The term "BrdU" as used herein refers to 5-bromo-2'-deoxyuridine, a thymidine homologue. BrdU is added to cell culture for a limited period (e.g., 4 hours), and it becomes incorporated into the DNA of proliferating cells. After fixing the cells, detection of incorporated BrdU can be performed in an ELISA using anti-BrdU-peroxidase. Therefore, BrdU incorporation is a measure of proliferation.

[0135] In one embodiment, the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% when compared to a wild-type antibody, where the Fc-mediated CD69 expression is measured in a PBMC-based functional assay.

[0136] In particular, when referring to the expression level of the T cell activation marker CD69, the term "reduce" refers to a reduction in the expression level of CD69 compared to the expression level of CD69 when bound to T cells by a wild type antibody that binds to CD3 and interacts with an Fc receptor. The ability of an antibody to reduce CD69 expression can be assessed by a PBMC-based functional assay. Thus, in one embodiment, PBMCs are incubated with antibodies in the range of 1-1000 ng / mL in 5% (vol / vol) CO 2 CD69 expression is measured by a method that includes incubating at 37°C in a humidified incubator for 16-24 hours, washing the cells, staining the cells with mouse anti-human CD28-PE antibody and mouse anti-human CD69-APC antibody at 4°C, and determining CD69 expression on CD28 positive cells by flow cytometry.

[0137] As used herein, the term "CD69" refers to Cluster of Differentiation 69, a human transmembrane C-type lectin protein encoded by the CD69 gene. Activation of T lymphocytes and natural killer (NK) cells induces expression of CD69 both in vivo and in vitro. CD69 functions as a signaling receptor involved in cell activation events such as proliferation, and functions as a signaling receptor in lymphocytes, e.g., natural killer cells, and platelets, and in the induction of specific genes.

[0138] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay used to assess the functional characteristics of an antibody of the invention, such as the ability of the antibody to affect T cell proliferation or CD69 expression, where the only cells present are peripheral blood mononuclear cells. A PBMC-based functional assay, when assessing CD69 expression, may be performed by (i) incubating PBMCs with the antibody in 5% (vol / vol) CO 2 (ii) incubating at 37°C in a humidified incubator for about 16-24 hours; (iii) washing the cells; (iv) staining the cells with mouse anti-human CD28-PE antibody and mouse anti-human CD69-APC antibody at 4°C; and (iv) determining CD69 expression on CD28-positive cells by flow cytometry.

[0139] 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 amino acid positions that can be modified include positions L234, L235 and P331. Combinations thereof, such as L234F / L235E / P331S, can cause a significant decrease in binding to human CD64, CD32A, CD16 and C1q.

[0140] Thus, 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] ). Also, the L234F and L235E amino acid substitutions can result in an Fc region in which interaction with Fc gamma receptors and C1q is blocked (

[29] -

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

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

[32] ). Thus, 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.

[0141] An alternative approach to minimize the interaction of the Fc region with the Fc gamma receptor and C1q is by removing the glycosylation site of the antibody. By mutating position N297 to, for example, Q, A, and E, a glycosylation site that is critical for IgG-Fc gamma receptor interaction is removed. Thus, in one embodiment, the amino acid at the position corresponding to N297 can be G, Q, A, or E (

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

[31] ).

[0142] Alternatively, human IgG2 and IgG4 subclasses appear to be inherently impaired in their interactions with C1q and Fc gamma receptors, but interactions with Fc gamma receptors have also been reported (

[34] -

[35] ). In both isotypes, mutations can be made that block these residual interactions and result 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 at positions corresponding to L234 and G237 in a human IgG2 heavy chain can be A and A, respectively. In one embodiment, the amino acid at position corresponding to L235 in a human IgG4 heavy chain can be E.

[0143] Other approaches to further minimize interactions with Fc gamma receptors and C1q in IgG2 antibodies include those described in

[36] and

[37] .

[0144] The hinge region of an antibody may also be important for interactions with Fc gamma receptors and complement (

[38] -

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

[0145] As used herein, the term "cross-linking" refers to the indirect cross-linking of an antibody Fab arm (monovalent or bivalent) that is bound to a target antigen by an FcR-bearing cell via binding to the antibody Fc region. Thus, an antibody that binds to the target antigen on a target antigen-bearing cell can cross-link another cell that expresses an FcR.

[0146] The term "non-specific killing" as used herein refers to the killing of cells by the cytotoxic function of T cells or other effector cells, which is activated independent of the tumor target antigen. Thus, non-specific killing means that tumor target-bearing cells can be killed, for example, by cytotoxic T cells, rather than by antibodies that bind to the tumor target, for example by inducing CDC.

[0147] A non-activated Fc region can be obtained by modifying one or more of at least five specific amino acid positions in the Fc region. In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0148] Thus, in one embodiment, the antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0149] In one embodiment, in both the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0150] In another embodiment, in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0151] As used herein, the term "amino acid corresponding to position" refers to the amino acid position number in the human IgG1 heavy chain. Unless otherwise stated or contradicted by the context, the amino acids of the constant region sequences are numbered herein according to the Eu numbering index (described in

[27] ). Thus, an amino acid or segment in one sequence "corresponds to" an amino acid or segment in another sequence is one that aligns with said other amino acid or segment and has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain, e.g., when using standard sequence alignment programs such as ALIGN, ClustalW, or others, typically with default settings. Methods for aligning sequences or segments in sequences and thereby determining positions in sequences that correspond to the amino acid positions of the invention are believed to be well known in the art.

[0152] In the present invention, amino acids can be defined as above.

[0153] The term "amino acid is not" or similar expressions with respect to an amino acid in the heavy chain should be understood to mean that the amino acid is any other amino acid except the particular amino acid mentioned. For example, the amino acid at the position corresponding to L234 in the human IgG1 heavy chain is not L means that the amino acid can be either another natural amino acid other than L or a non-natural amino acid.

[0154] In one embodiment in at least one of said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0155] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0156] In one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0157] The term "hydrophobic" as used herein with respect to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at a position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0158] The term "polar" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0159] In another embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is an aliphatic uncharged amino acid, an aromatic amino acid or an acidic amino acid.

[0160] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L, and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L, and V.

[0161] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at a position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of F, T and W.

[0162] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of D and E.

[0163] In certain embodiments, in at least one of the first and second heavy chains, the amino acid at a position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0164] In one embodiment the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain in both said first and second heavy chains is not D.

[0165] In one embodiment, in both the first and the second heavy chain, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0166] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0167] The term "hydrophobic" as used herein with respect to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0168] The term "polar" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T. In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0169] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0170] In another embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is an aliphatic uncharged amino acid, an aromatic amino acid or an acidic amino acid.

[0171] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V.

[0172] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of F, T and W.

[0173] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of D and E.

[0174] In certain embodiments, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0175] In a further embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N.

[0176] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0177] In one embodiment in both said first and second heavy chains the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N.

[0178] In one embodiment, in both the first and the second heavy chain, the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0179] In a further embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0180] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0181] In one embodiment, in at least one of said first and second heavy chains, the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V.

[0182] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0183] The term "hydrophobic" as used herein with respect to amino acid residues refers to amino acid residues selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y, respectively.

[0184] The term "polar" as used herein with respect to amino acid residues refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S and T, respectively.

[0185] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively.

[0186] In one embodiment in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0187] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to L234 and L235 in a human IgG1 heavy chain are not L and L, respectively, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0188] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0189] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y, respectively.

[0190] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively.

[0191] In certain embodiments, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively.

[0192] In another embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are aliphatic uncharged amino acids, aromatic amino acids or acidic amino acids.

[0193] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, G, I and V, respectively.

[0194] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of F, T and W, respectively.

[0195] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of D and E, respectively.

[0196] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0197] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively.

[0198] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0199] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively.

[0200] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0201] In certain embodiments, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0202] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0203] In one embodiment in at least one of said first and second heavy chains the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0204] In one embodiment, in both said first and second heavy chains, at least the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0205] In one embodiment in at least one of said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively.

[0206] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0207] In one embodiment, in at least one of said first and second heavy chains, the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V, and W, and the amino acid corresponding to position D265 is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.

[0208] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0209] The term "hydrophobic" as used herein with respect to amino acid residues refers to amino acid residues selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y, respectively.

[0210] The term "polar" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively, and the amino acid at a position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0211] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively, and the amino acid at a position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0212] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0213] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y, respectively.

[0214] In one embodiment, in both said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively, and the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0215] In certain embodiments, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively, and the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0216] In another embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged amino acids, aromatic amino acids or acidic amino acids.

[0217] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L, and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L, and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, G, I, and V, respectively.

[0218] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are selected from the group consisting of F, T and W, respectively.

[0219] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are selected from the group consisting of D and E, respectively.

[0220] In certain embodiments, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W, and the amino acids at the positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0221] In one embodiment in both said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively.

[0222] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0223] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged amino acids, aromatic amino acids or acidic amino acids.

[0224] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, G, I and V, respectively.

[0225] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are selected from the group consisting of D and E, respectively.

[0226] In certain embodiments, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W, and the amino acids at the positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0227] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively.

[0228] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0229] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, or A, A and A, respectively.

[0230] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0231] In certain embodiments, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0232] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively.

[0233] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A, respectively.

[0234] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A, respectively.

[0235] In another embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, Q, and S, respectively.

[0236] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are F, E, A, Q and S, respectively.

[0237] In one embodiment, the antibody of the invention comprises a VH sequence as set forth in any one of the sequences in the group of SEQ ID NOs: 107;59;245;299;285;55;185;179;237;177 and 293, a VL sequence as set forth in SEQ ID NO: 8, and in at least one or both of the heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively. This provides an embodiment of an anti-CD3 antibody having reduced affinity for human CD3ε compared to a reference antibody comprising the VH and VL sequences as set forth in SEQ ID NOs: 4 and 8, and further comprising a non-activated Fc region.

[0238] In certain embodiments, the antibody of the invention comprises a VH sequence as set forth in any one of the sequences set forth in SEQ ID NOs: 107;59;245;299;285;55;185;179;237;177 and 293, a VL sequence as set forth in SEQ ID NO: 10, and in at least one or both of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively. This provides an embodiment of an anti-CD3 antibody that has reduced affinity for human CD3ε compared to a reference antibody comprising the VH and VL sequences set forth in SEQ ID NOs: 4 and 8, further comprising a non-activated Fc region, and a VL region that allows for enhanced production.

[0239] In another embodiment, an antibody of 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 both of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0240] In one embodiment of the invention, the human IgG1 heavy chain has the IgG1m(f) sequence shown in SEQ ID NO: 407. In yet another embodiment, the amino acids at positions corresponding to positions L234, L235, and D265 in human IgG1m(f) shown in SEQ ID NO: 407 are F, E, and A, respectively.

[0241] In one embodiment of the invention, the human IgG1 heavy chain has the IgG1m(f) sequence shown in SEQ ID NO:409.

[0242] In one aspect, an antibody of the invention comprises a human IgLC2 / IgLC3 constant domain lambda light chain of SEQ ID NO:408.

[0243] In one aspect, the antibody of the present invention may be modified in the light chain (LC) and / or the heavy chain (HC) to increase expression levels and / or production yields. In one embodiment, the antibody of the present invention may be modified in the light chain (LC). Such modifications are known in the art and may 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.

[0244] In one aspect, the antibody of the present invention can be modified in the VH and / or VL regions to alter the affinity of the antibody, for example to reduce or enhance the affinity of the antibody. This can be advantageous in some settings and lead to enhanced efficacy. In particular, the low affinity of the CD3 arm can affect the motility of T cells in circulation and at the tumor site, thus leading to better engagement of T cells with tumor cells. See MolhOj et al, Molecular Immunology 44 (2007). In particular, this can be useful in bispecific formats in which a CD3 antibody is used as one of the binding arms. Modifications that lead to reduced antibody affinity are known in the art, see for example Webster et al. Int J Cancer Suppl. 1988;3:13-6.

[0245] Thus, in one embodiment, an antibody of the invention comprises a light chain variable (VL) region comprising CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 6, GTN, 7, and a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 having CDR sequences selected from one of the group consisting of: a) the CDR sequence shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; and k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V].

[0246] In another aspect, the present invention provides an antibody that binds to human CD3, comprising a binding region comprising a light chain variable (VL) region having the sequence shown in SEQ ID NO: 10, and a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 having a sequence selected from one of the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; and k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]. This provides an embodiment comprising the T41K mutation in the VL region as shown in SEQ ID NO:10, which allows for increased production of said antibody.

[0247] In one aspect, the invention relates to a multispecific antibody comprising at least a first binding region of an antibody according to any aspect or embodiment described herein and one or more binding regions that bind to one or more targets different from the first binding region. Such a multispecific antibody can be a bispecific antibody.

[0248] Thus, in one aspect, the 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 than the first binding region.

[0249] The term "multispecific antibody" refers to an antibody having specificity for at least two different, e.g., at least three, typically non-overlapping, epitopes. Such epitopes may be on the same target or on different targets. When the epitopes are on different targets, such targets may be on the same cell or cell type or on different cells or cell types.

[0250] The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same target or on different targets. If the epitopes are on different targets, such targets may be on the same cell or cell type or on different cells or cell types.

[0251] In one embodiment, the bispecific antibody comprises a first heavy chain and a second heavy chain.

[0252] The embodiments related to the modification of the Fc region and the embodiments related to specific amino acid substitutions are considered to be 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, such as those described with respect to providing an inactive Fc region. In one embodiment, both the first and second heavy chains comprise one or more amino acids modified as defined in any embodiment described herein, such as those described with respect to providing an inactive Fc region. Thus, a 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.

[0253] Examples of bispecific antibody molecules that can be used in the present invention include: (i) single antibodies with two arms containing different antigen binding regions; (ii) single chain antibodies with specificity for two different epitopes, for example by means of two scFvs linked in tandem by an additional peptide linker; (iii) dual-variable domain antibodies (DVD-Ig™) (

[40] ), in which each light and heavy chain contains two variable domains in tandem by a short peptide bond; (iv) chemically linked bispecific (Fab')2 fragments; (v) TandAb®, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each of the target antigens; (vi) flexibodies, which are a combination of scFvs and diabodies resulting in a multivalent molecule; (vii) so-called "dock and lock" antibodies based on the "dimerization and docking domain" in protein kinase A. These include "Dock-and-Lock®" molecules, which when applied to Fab can give trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments, (viii) the so-called Scorpion molecules, which contain, for example, two scFvs fused to either end of a human Fab arm, and (ix) diabodies.

[0254] In one embodiment the bispecific antibody of the invention is a diabody, a cross-body or a bispecific antibody obtained by controlled Fab arm exchange, e.g. a DuoBody® such as those described in the present invention (e.g. as described in

[41] ).

[0255] Examples of different types of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization, (ii) recombinant IgG-like dual targeting molecules, where 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, where a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment, (iv) Fc fusion molecules, where a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof, (v) Fab fusion molecules, where different Fab fragments are fused together or fused to a heavy chain constant domain, an Fc region or a portion thereof, and (vi) ScFv and diabody-based antibodies as well as heavy chain antibodies (e.g. domain antibodies, Nanobodies®) where different single chain Fv molecules, or different diabodies or different heavy chain antibodies (e.g. domain antibodies, Nanobodies®) are fused to each other or to another protein or carrier molecule that is fused to a heavy chain constant domain, an Fc region or a portion thereof.

[0256] Examples of IgG-like molecules with complementary CH3 domain molecules include Triomab® (Trion Pharma / Fresenius Biotech,

[42] ), Knobs-into-Holes (Genentech,

[43] ), CrossMAb (Roche,

[44] ) and electrostatically-matched (Amgen,

[45] -

[46] ; Chugai,

[47] ; Oncomed,

[48] ), LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-bodies and PIG-bodies (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain bodies (SEEDbodies) (EMD Serono,

[49] ), Biclonic (Merus, WO2013157953), FcΔAdp (Regeneron,

[50] ), bispecific IgG1 and IgG2 (Pfizer / Rinat,

[51] ), Azymetric scaffolds (Zymeworks / Merck,

[52] ), mAb-Fv (Xencor,

[53] ), bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody® molecules (Genmab A / S,

[41] ).

[0257] Examples of recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK / Domantis, WO2009058383), Two-in-one antibodies (Genentech, Bostrom, et al 2009. Science 323, 1610-1614), Cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star,

[54] ), Zybodies™ (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), the common light chain approach (Crucell / Merus,

[55] ), κλBody (NovImmune, WO2012023053) and CovX-body® (CovX / Pfizer, Doppalapudi, VR, et al 2007. Bioorg. Med. Chem. Lett. 17,501-506), but are not limited thereto.

[0258] Examples of IgG fusion molecules include Dual Variable Domain (DVD)-Ig™ (Abbott,

[56] ), Dual domain double head antibodies (Unilever; Sanofi Aventis,

[57] ), IgG-like bispecifics (ImClone / Eli Lilly, 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 fusions (Novartis), scFv fusions (Changzhou Adam Biotech Inc,

[59] ) and TvAb (Roche,

[59] ,

[60] ).

[0259] 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 100 th Annual meeting 2009 (Abstract # 5465);Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics,

[62] ,

[63] ) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0260] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).

[0261] Examples of ScFv-based antibodies, diabody-based antibodies and domain antibodies include Bispecific T Cell Engager (BiTE®) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™) (MacroGenics), Single-chain Diabody (Academic, Lawrence FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75), dual targeting Examples of antibodies that can be used include, but are not limited to, dual targeting heavy chain only domain antibodies, and dual targeting nanobodies® (Ablynx, Hmila et al., FASEB J. 2010).

[0262] Furthermore, it is believed that any monospecific antibody that satisfies the assay conditions described herein may form the basis of a bispecific antibody. That is, a bispecific antibody, one of whose binding regions binds to CD3, may be derived from any monospecific CD3 antibody that is tested in a functional assay and satisfies the requirements set forth herein. Such a bispecific antibody may be provided by the method described in

[41] , which is incorporated herein by reference.

[0263] In one aspect, the bispecific antibody of the present invention comprises a first Fc region comprising a first CH3 region and a second Fc region comprising a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different and the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction of the first CH3 region and the second CH3 region, respectively. Further details about these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0264] Thus, in certain embodiments, each of the first and second heavy chains comprises at least the hinge, CH2 and CH3 regions, and in the first heavy chain at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain is substituted, and in the second heavy chain at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain is substituted, and the first and second heavy chains are not substituted at the same position. In this context, the term "substituted" refers to the replacement of the amino acid at a particular amino acid position with another natural or non-natural amino acid. Thus, a "substituted" amino acid at a position that corresponds to a position in a human IgG1 heavy chain means that the amino acid at that particular position is different from the naturally occurring amino acid in an IgG1 heavy chain.

[0265] In one embodiment, in said first heavy chain, the amino acid at a position corresponding to K409 in a human IgG1 heavy chain is not K, L or M, and optionally the amino acid at a position corresponding to F405 in a human IgG1 heavy chain is F; and in said second heavy chain, at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405 and Y407 in a human IgG1 heavy chain has been substituted.

[0266] In one embodiment, in said first heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is not K, L or M, and in said second heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is not F, and optionally the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is K.

[0267] In one embodiment, in said first heavy chain, the amino acid at a position corresponding to F405 in a human IgG1 heavy chain is not F, R or G, and in said second heavy chain, an amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, Y407 and K409 in a human IgG1 heavy chain is substituted.

[0268] In one embodiment, in said first heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is not K, L or M and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is not F.

[0269] In yet another embodiment, in said first heavy chain the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L and in said second heavy chain the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R, or vice versa.

[0270] Thus, in one embodiment in the first heavy chain the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R and in the second heavy chain the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L.

[0271] In yet another embodiment, a humanized or chimeric CD3 antibody of the invention comprises in at least one of the first and second heavy chains an inactivating substitution as disclosed in any one of the above embodiments, such as one or more of L234F, L235E, and D265A, and wherein the amino acid at the position corresponding to F405 is not F. In one embodiment, a humanized or chimeric CD3 antibody of the invention comprises in at least one of the first and second heavy chains an inactivating substitution as disclosed in any one of the above embodiments, such as one or more of L234F, L235E, and D265A, and a further substitution at position K409, such as K409R. In particular, in one embodiment, the humanized or chimeric CD3 antibody of the invention comprises in both the first and second heavy chain an inactivating substitution as disclosed in any one of the above embodiments, such as one or more of L234F, L235E, and D265A, and a substitution at position F405, such as F405L. In one embodiment, the humanized or chimeric CD3 antibody of the invention comprises in both the first and second heavy chain an inactivating substitution as disclosed in any one of the above embodiments, such as one or more of L234F, L235E, and D265A, and a further substitution at position K409, such as K409R. Such antibodies are useful for generating bispecific antibodies.

[0272] Thus, in yet another embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R.

[0273] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P, respectively; in the first heavy chain, the amino acid at a position corresponding to F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at a position corresponding to K409 in a human IgG1 heavy chain is R.

[0274] In an alternative embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at position corresponding to K409 in a human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at position corresponding to F405 in a human IgG1 heavy chain is L.

[0275] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P, respectively; in the first heavy chain, the amino acid at a position corresponding to K409 in a human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at a position corresponding to F405 in a human IgG1 heavy chain is L.

[0276] In another embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R.

[0277] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are F, E, A, N and P, respectively; in the first heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R.

[0278] In an alternative embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L.

[0279] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are F, E, A, N and P, respectively; in the first heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L.

[0280] As described herein, T cell recruitment to a specific target cell, such as a cancer or tumor cell, is a method for killing the target cell. T cell-mediated killing can be achieved by a bispecific antibody that targets CD3 with a first binding region and another target with a second binding region. Thus, in one embodiment, the first binding region is according to any of the embodiments described herein for a humanized or chimeric CD3 antibody, and the second binding region binds to a different target than the first binding region. When the antibody is a bispecific antibody, it should be understood that at least half of the antibody, i.e., one of the pair of heavy and light chains of the antibody, is a humanized or chimeric antibody as described herein. Thus, one half of the bispecific antibody can be a humanized or chimeric antibody of the invention that binds to CD3, and the other half can be humanized, chimeric, fully non-human or fully human that binds to a second target. Thus, in one embodiment, the antibody comprises a first heavy chain and a second heavy chain, a first light chain and a second light chain, wherein 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 second light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and wherein the second binding region binds to a different target. In one embodiment, the antibody comprises a first heavy chain and a second heavy chain, a first light chain and a second light chain, wherein 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 second light chain are humanized or chimeric and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and wherein the second binding region binds to a different CD3 epitope than the first binding region.

[0281] As used herein, the term "disulfide bridge" refers to a covalent bond between two cysteine ​​residues, i.e., this interaction can also be referred to as a Cys-Cys interaction.

[0282] As used herein, the term "target" refers to a molecule to which the binding region of an antibody of the invention binds. When used in reference to antibody binding, it means that the antibody is directed against any antigen encompassed by this term.

[0283] In certain embodiments, 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 second light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein and the second binding region binds to a different target, and in at least one of the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0284] In certain embodiments, 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 second light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a different CD3 epitope than the first binding region, and in at least one of the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0285] In certain embodiments, 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 second light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein and the second binding region binds to a different target, and in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0286] In certain embodiments, 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 second light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a different CD3 epitope than the first binding region, and in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0287] In another aspect, the present invention relates to a method for reducing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 3, comprising the step of introducing a mutation in one of the three CDR sequences of the reference antibody selected from a mutation in one of the positions selected from the group of T31M, T31P, N57, H101, S110 and Y114, wherein the positions are numbered according to the reference sequence of SEQ ID NO: 4.

[0288] The numbering of the amino acids in the VH region and the mutated positions are according to the amino acids in SEQ ID NO: 4. The numbering follows a direct numeric numbering scheme from the first amino acid to 125 in the N-terminal to C-terminal direction. The numeric numbering of the positions corresponding to SEQ ID NO: 4 is shown in Figure 2. Additionally, the CDR regions have been annotated according to the IMGT definition.

[0289] In one embodiment of the invention, the method comprises the step of introducing a T31M or T31P mutation, wherein position T31 is according to SEQ ID NO:4.

[0290] In one embodiment of the invention the method comprises introducing a mutation at position N57. Position N57 is according to SEQ ID NO: 4. In one embodiment the mutation is N57E.

[0291] In one embodiment of the invention the method comprises the step of introducing a mutation at position H101. Position H101 is according to SEQ ID NO: 4. In one embodiment the mutation is H101G or H101N.

[0292] In one embodiment of the invention the method comprises introducing a mutation at position Y114. Position Y114 is according to SEQ ID NO: 4. In one embodiment the mutation is Y114, Y114R or Y114V.

[0293] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH CDR3 region corresponding to a position selected from the group of H101, S110, and Y114.

[0294] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH CDR3 region selected from the group consisting of H101G, H101N, S110A, S110G, Y114M, Y114R, and Y114V.

[0295] In one embodiment of the invention, the method comprises introducing a mutation, wherein the antibody has a K D Value 1.6×10 -8 M~9.9×10 -8 M or 1.0×10 -7 ~9.9×10 -7 M, has binding affinity for the human CD3ε peptide of SEQ ID NO: 402.

[0296] In one embodiment of the invention, the method comprises introducing a mutation, wherein the antibody has a K D Value 1.4×10 -8 ~1.0×10 -8M or 9.9×10 -9 ~1×10 -9 M, has binding affinity for the human CD3ε peptide of SEQ ID NO: 402.

[0297] In one embodiment of the invention, the antibody has a K D Value 1.6×10 -8 M~9.9×10 -8 M or 1.0×10 -7 ~9.9×10 -7 M, has binding affinity for the human CD3ε peptide of SEQ ID NO: 402.

[0298] In another aspect, the present invention relates to a method for enhancing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2, 3, comprising introducing a mutation in VH CDR3 corresponding to position G105, said positions being numbered according to the reference sequence of SEQ ID NO: 4.

[0299] In one embodiment of the invention the method comprises the step of introducing a mutation at position G105. Position G105 is according to SEQ ID NO: 4. In one embodiment the mutation is G105P.

[0300] In one embodiment of the invention, the method comprises the step of introducing up to 5 further mutations, up to 4 further mutations, up to 3 further mutations, up to 2 further mutations, or up to 1 further mutation in the CDRs of the VH region of a reference antibody shown in SEQ ID NO: 1, 2, 3.

[0301] In one embodiment of the invention, the method of enhanced or decreased binding affinity comprises a binding region comprising a heavy chain variable (VH) region, said VH region comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 292 [Y114R]; and k) The CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V].

[0302] In another embodiment of the invention, the method comprises introducing a mutation in the VH region CDR2 region corresponding to N57E. In yet another embodiment of the invention, the method comprises introducing a mutation in the VH region CDR3 region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V. In another aspect, the present invention relates to a method for reducing or enhancing the binding affinity of an antibody to CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region, said VH region comprising a mutation in one of the three CDR sequences of a reference antibody as shown by CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2 and CDR3 SEQ ID NO: 3, and wherein the antibody comprises a mutation in one of the positions selected from the group of: T31M, T31P, N57, H101, G105, S110 and Y114, said positions corresponding to the reference sequence of SEQ ID NO: 4.

[0303] In one embodiment of the present invention, the method comprises introducing a mutation in the VH region CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the present invention, the method comprises introducing a mutation in the VH region CDR2 region corresponding to N57E. In yet another embodiment of the present invention, the method comprises introducing a mutation in the VH region CDR3 region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, or Y114V.

[0304] In yet another aspect, the present invention relates to a method for reducing the binding affinity of an antibody that binds to CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising CDR1, CDR2 and CDR3 having the CDR sequences shown in SEQ ID NOs: 1, 2 and 3, comprising introducing a mutation in one of the VH region CDR1, CDR2 or CDR3 sequences shown in SEQ ID NOs: 1, 2 or 3.

[0305] In one embodiment of the invention, the method comprises introducing a mutation in one of the three CDR regions of the VH region corresponding to one of the following positions: T31, N57, H101, S110, or Y114, wherein said positions correspond to the reference sequence of SEQ ID NO: 4.

[0306] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH region CDR1 sequence corresponding to position T31, wherein said CDR1 sequence is as shown in SEQ ID NO: 1. If the mutation is represented by X, the resulting CDR1 sequence may be presented as GFTFNXYA. In one embodiment, the three CDR sequence of the VH region has the following sequence: In one embodiment the mutation at position T31 in the VH region CDR1 is a T31M or T31P mutation.

[0307] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH region CDR2 sequence corresponding to position N57, wherein said CDR2 sequence is as shown in SEQ ID NO: 2. The mutation is represented by X and the resulting CDR2 sequence is In one embodiment, the three CDR sequences of the VH region are represented by the following sequence: In one embodiment the mutation at position N57 in the VH region CDR2 is an N57E mutation.

[0308] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH region CDR3 sequence corresponding to position H101, wherein said CDR3 sequence is as shown in SEQ ID NO: 3. The mutation is represented by X and the resulting CDR3 sequence is In one embodiment, the three CDR sequences of the VH region are represented by the following sequence: In one embodiment the mutation at position H101 in the VH region CDR3 is a H101G or H101N mutation.

[0309] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH region CDR3 sequence corresponding to position S110, wherein said CDR3 sequence is as shown in SEQ ID NO: 3. The mutation is represented by X and the resulting CDR3 sequence is In one embodiment, the three CDR sequences of the VH region are represented by the following sequence: In one embodiment the mutation at position H101 in the VH region CDR3 is an S110A or S110G mutation.

[0310] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH region CDR3 sequence corresponding to position Y114, wherein said CDR3 sequence is as shown in SEQ ID NO: 3. The mutation is represented by X and the resulting CDR3 sequence is In one embodiment, the three CDR sequences of the VH region are represented by the following sequence: In one embodiment, the mutation at position Y114 in the VH region CDR3 is a Y114M, Y114R, or Y114V mutation.

[0311] In one embodiment of the invention, the method comprises introducing up to three mutations, up to two mutations, or up to one mutation in one or more of the three CDRs of the VH region of a reference antibody shown in SEQ ID NO: 1, 2, 3.

[0312] In one embodiment of the invention, the method comprises introducing up to 10 mutations, up to 9 mutations, up to 8 mutations, up to 7 mutations, up to 6 mutations, up to 5 mutations, up to 4 mutations, up to 3 mutations, up to 2 mutations, or up to 1 mutation in the heavy chain variable framework region of an antibody, which mutations preferably do not alter binding of the antibody to CD3 compared to the same antibody without the mutations.

[0313] In one embodiment of the invention, the method comprises introducing a mutation in the VH region CDR1 sequence selected from T31M or T31P. In another embodiment of the invention, the method comprises introducing a mutation in the VH region CDR2 sequence of N57E. In yet another embodiment of the invention, the method comprises introducing a mutation in the VH region CDR3 sequence selected from the group of H101G, H101N, S110A, S110G, Y114M, Y114R, and Y114V.

[0314] In another aspect, the present invention relates to a method for enhancing the binding affinity of an antibody that binds to CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 having the CDR sequences shown in SEQ ID NOs: 1, 2, and 3, comprising introducing a mutation in one of the VH region CDR1, CDR2, or CDR3 sequences shown in SEQ ID NOs: 1, 2, or 3.

[0315] In one embodiment of the invention, the method comprises the step of introducing a mutation in the VH region CDR3 sequence corresponding to position G105, wherein said CDR3 sequence is as shown in SEQ ID NO: 3. The mutation is represented by X and the resulting CDR3 sequence is In one embodiment, the three CDR sequences of the VH region are represented by the following sequence: In one embodiment the mutation at position G105 in the VH region CDR3 is a G105P mutation.

[0316] Nucleic acid constructs, expression vectors, and host cells In one aspect, the present invention relates to a nucleic acid construct encoding one or more of the sequences shown in Table 1. Thus, the present invention relates to a nucleic acid construct encoding any one of the sequences shown in SEQ ID NOs: 107;221;59;245;299;285;55;185;179;237;177 and 293.

[0317] In yet another aspect, the present invention relates to a nucleic acid construct encoding the sequence of a humanized or chimeric CD3 antibody of the invention, an expression vector comprising the nucleic acid construct of the invention, a host cell comprising said expression vector, and a method for producing said antibody by culturing said host cell under suitable conditions, thereby producing and optionally recovering the antibody. The humanized CD3 antibody may also be referred to as "huCD3".

[0318] In one embodiment, the invention provides an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized or chimeric antibody of the invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody of the invention, or (iii) both (i) and (ii).Accordingly, the expression vector comprises one or more nucleic acid constructs or nucleic acid sequences according to any aspect or embodiment described herein.

[0319] In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding one or more of the heavy and light chain CDR sequences, wherein the VH CDR sequence is TIFF2025038049000015.tif106162, and the VL CDR sequence is selected from the group consisting of: The CDR sequences are selected from the group consisting of those shown in TIFF2025038049000016.tif55162.

[0320] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the heavy chain CDR sequences and the light chain CDR sequences, wherein the CDR sequences of the VL region CDR1, CDR2, CDR3 regions comprise the CDR sequences shown in SEQ ID NO: 6, GTN, 7, and the CDR sequences of the VH region CDR1, CDR2, CDR3 regions comprise the CDR sequences shown in SEQ ID NO: 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; CDR1, CDR2, CDR3 shown in SEQ ID NO: 1, 2, 220; CDR1, CDR2, CDR3 as shown in SEQ ID NO: 1, 2, 236; CDR1, CDR2, CDR3 as shown in SEQ ID NO: 1, 2, 244; CDR1, CDR2, CDR3 as shown in SEQ ID NO: 1, 2, 284; CDR1, CDR2, CDR3 as shown in SEQ ID NO: 1, 2, 292, and CDR1, CDR2, CDR3 as shown in SEQ ID NO: 1, 2, 298.

[0321] In certain embodiments, the expression vector comprises a nucleic acid sequence encoding one or more variants of the above amino acid sequences, the variants having up to 25 amino acid modifications, such as up to 20, such as up to 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative or non-conservative substitutions, or having at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity, to any of the above amino acid sequences. The present invention also relates to nucleic acid sequences that differ from the above-mentioned nucleic acid sequences, but code for the same amino acid sequence as the antibody of the present invention due to the diversity of the genetic code. For example, the nucleic acid sequence may be changed to result in the same amino acid sequence as any of the amino acid sequences described herein. Methods for identifying such additional nucleic acid sequences based on the genetic code are well known to those skilled in the art.

[0322] In yet another embodiment, the expression vector further comprises a nucleic acid sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of an antibody, e.g., a human antibody.

[0323] Expression vectors as described above can be used for the recombinant production of antibodies of the invention.

[0324] In the context of the present invention, an expression vector can be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the humanized or chimeric CD3 antibody is expressed in a naked DNA or RNA vector, such as a linear expression element (e.g., as described in

[64] ), a compacted nucleic acid vector (e.g., as described in

[65] and / or

[66] ), a plasmid vector such as pBR322, pUC19 / 18, or pUC118 / 119, a "midge" minimum size nucleic acid vector (e.g., as described in

[67] ), or a CaP0 vector. 4 - The nucleic acid vectors are included in the precipitating nucleic acid vector constructs, such as those described in

[68] ,

[69] ,

[70] , and

[71] . Such nucleic acid vectors and methods for their use are well known in the art (see, for example,

[72] and

[73] ).

[0325] In one embodiment, a vector is suitable for expression of a humanized or chimeric CD3 antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (

[74] ), pET vectors (Novagen, Madison, WI), etc.

[0326] Additionally or alternatively, the expression vector may 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 constitutive or inducible promoters, such as alpha factor, alcohol oxidase and PGH (reviewed in

[75] and

[76] ).

[0327] The nucleic acid construct and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence capable of targeting a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretion leader or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transport sequences, chloroplast transport sequences), membrane localization / anchor sequences (e.g., membrane permeabilization stop sequences, GPI anchor sequences), and the like, all of which are well known in the art.

[0328] In the expression vectors of the present invention, the nucleic acid encoding the humanized or chimeric CD3 antibody may contain or be associated with any suitable promoter, enhancer, and other expression promoting elements. Examples of such elements include a strong expression promoter (e.g., human CMV IE promoter / enhancer, as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), an effective poly(A) termination sequence, an origin of replication for plasmid production in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid construct and / or vector may also contain an inducible promoter, such as CMV IE, rather than a constitutive promoter (those skilled in the art will appreciate that these terms are descriptive of the degree of gene expression under certain conditions).

[0329] In one embodiment, an expression vector encoding a humanized or chimeric CD3 antibody can be placed into and / or delivered to a host cell or animal by a viral vector.

[0330] Such expression vectors can be used for the recombinant production of humanized or chimeric CD3 antibodies.

[0331] In one aspect, the invention provides a host cell comprising an expression vector of the invention.

[0332] In one aspect, the humanized or chimeric CD3 antibody of any aspect or embodiment described herein is provided using a recombinant eukaryotic, prokaryotic, or microbial host cell that produces the antibody. Thus, the present invention provides a recombinant eukaryotic, prokaryotic, or microbial host cell that produces a humanized or chimeric CD3 antibody or immunoglobulin as defined herein. Examples of host cells include yeast, bacterial, 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 cellular genome that comprises a sequence that codes for the expression of a humanized or chimeric CD3 antibody described herein. In another embodiment, the host cell comprises a non-integrated nucleic acid sequence, such as a plasmid, cosmid, phagemid, or linear expression element, that comprises a sequence that codes for the expression of a humanized or chimeric CD3 antibody described herein.

[0333] The term "recombinant host cell" (or simply "host cell") as used herein refers to a cell into which an expression vector or a nucleic acid construct or sequence has been introduced. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny of such cells. Since certain modifications may occur over successive generations, either by mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still encompassed within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, eukaryotic host cells such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells, prokaryotic cells such as E. coli, and other eukaryotic hosts such as plant cells and fungi.

[0334] In yet another aspect, the present invention relates to a method for producing a humanized or chimeric CD3 antibody of the present invention, the method comprising: a) culturing the host cell of the invention as described above, and b) recovering and / or purifying the antibody of the invention from the culture medium Includes.

[0335] In yet another aspect, the nucleotide sequence encoding the humanized or chimeric CD3 antibody sequence encodes a second moiety, such as a therapeutic polypeptide. Exemplary therapeutic polypeptides are described elsewhere herein. In one embodiment, the invention relates to a method for producing a humanized or chimeric CD3 antibody fusion protein, the method comprising: a) culturing a host cell containing an expression vector comprising such a nucleotide sequence; and b) recovering and / or purifying the humanized or chimeric CD3 antibody fusion protein from the culture medium Includes.

[0336] composition In one aspect, the invention provides a composition comprising an antibody or bispecific antibody according to any of the aspects and embodiments described herein.

[0337] In one aspect, the invention provides a pharmaceutical composition comprising an antibody or bispecific antibody as defined in any one of the aspects and embodiments described herein and a pharma- ceutically acceptable carrier.

[0338] The pharmaceutical compositions can be formulated according to conventional techniques, e.g., those disclosed in

[77] , using pharma- ceutically acceptable carriers or diluents and any other known adjuvants and excipients.

[0339] Pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, should be suitable for the humanized or chimeric antibody of the present invention and the selected mode of administration. Suitability of carriers and other components of the pharmaceutical composition is determined based on the lack of significant negative effect (e.g., no significant effect (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on the desired biological properties of the selected compound of the present invention or pharmaceutical composition with respect to antigen binding.

[0340] Pharmaceutical compositions of the invention may also include diluents, bulking agents, salts, buffers, detergents (e.g., non-ionic detergents such as Tween®-20 or Tween®-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0341] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The dosage level selected will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention or its amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and other factors well known in the medical art.

[0342] The pharmaceutical composition can be administered by any suitable route and manner. Suitable routes for administering the humanized or chimeric antibodies of the invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

[0343] In one embodiment, the pharmaceutical compositions of the present invention are administered parenterally.

[0344] As used herein, the expressions "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0345] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion, hi a preferred embodiment, the pharmaceutical composition is administered subcutaneously.

[0346] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with the humanized or chimeric antibodies of the present invention.

[0347] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical art.

[0348] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for extemporaneously preparing sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Unless conventional media or agents are incompatible with active compounds, their use in the pharmaceutical compositions of the present invention is contemplated.When referring to "active compounds", it is also contemplated to refer to the humanized or chimeric antibodies of the present invention.

[0349] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0350] The pharmaceutical compositions of the present invention may also contain pharma- ceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0351] The pharmaceutical compositions of the present invention may also contain isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0352] The pharmaceutical compositions of the invention may also contain one or more adjuvants appropriate for the chosen route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers that may enhance the shelf life or effectiveness of the pharmaceutical composition. The humanized or chimeric antibodies of the invention may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including, for example, implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with 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] ).

[0353] In one embodiment, the humanized or chimeric antibody of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Conventional media or agents are contemplated for use in the pharmaceutical compositions of the present invention, unless they are incompatible with the active compound. Other active or therapeutic compounds can also be incorporated into the composition.

[0354] Injectable pharmaceutical compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the components as listed above as necessary, and then carrying out sterilization precision filtration.Generally, dispersion is prepared by incorporating active compound into a sterile medium that contains a basic dispersion medium and other necessary components, such as those listed above.In the case of sterile powder for preparing sterile injectable solution, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which give the powder of active ingredient and any additional desired components from the solution that has been previously sterilized and filtered.

[0355] Sterile injectable solution can be prepared by incorporating the required amount of active ingredient into a suitable solvent with one or combination of the above-listed ingredients as required, and then carrying out sterilization precision filtration.Generally, dispersion is prepared by incorporating active compound into a sterile medium that contains a basic dispersion medium and other necessary ingredients, such as those listed above.In the case of sterile powder for preparing sterile injectable solution, examples of preparation methods are vacuum drying and freeze-drying (lyophilization), which give the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0356] therapeutic use In another aspect, the present invention relates to a humanized or chimeric antibody of the invention or a pharmaceutical composition as defined in any aspect or embodiment described herein, for use as a medicament.

[0357] In another aspect, the invention relates to a humanized or chimeric antibody or a pharmaceutical composition of the invention as defined in any aspect or embodiment described herein for use in the treatment of a disease.

[0358] In one aspect of the invention, the bispecific antibodies, compositions and pharmaceutical compositions are for use in the treatment of a disease.

[0359] In one aspect of the invention, the bispecific antibody, composition, pharmaceutical composition is for use in the treatment of a disease, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

[0360] The humanized or chimeric antibody or pharmaceutical composition of the present invention can be used to treat any cancer in which the effector mechanism of cytotoxic T cells is desired.For example, the humanized or chimeric antibody can be administered to cultured cells in vitro or ex vivo, or administered to a human subject, for example, in vivo, to treat or prevent disorders such as cancer, inflammatory disorders, or autoimmune disorders.The term "subject" as used herein is typically a human who responds to the humanized or chimeric antibody or pharmaceutical composition.Subjects can include, for example, human patients with disorders that can be corrected or improved by modulating target function or directly or indirectly leading to cell death.

[0361] In another aspect, the present invention provides a method for treating or preventing a disorder, such as cancer, in which the recruitment of T cells contributes to the treatment or prevention, comprising administering to a subject in need thereof a therapeutically effective amount of a humanized or chimeric antibody or pharmaceutical composition of the present invention. In this method, typically, an amount of a humanized or chimeric antibody effective to treat or prevent the disorder is administered to the subject.

[0362] In one particular aspect, the present invention relates to a method for treating cancer comprising administering to a subject in need thereof a humanized or chimeric antibody or a pharmaceutical composition of the invention as defined in any of the aspects and embodiments described herein.

[0363] In another aspect, the invention relates to a use or method as defined in any aspect or embodiment described herein, wherein the humanized or chimeric antibody is a bispecific antibody that specifically binds both CD3 and a cancer-specific target or a target 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 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), low-grade or high-grade B-cell lymphoma, chronic lymphocytic leukemia or acute lymphocytic leukemia.

[0364] The effective dosage and dosage regimen of the humanized or chimeric antibody depends on the disease or condition being treated and can be determined by one skilled in the art.

[0365] A physician having ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician can start the dosage of humanized or chimeric antibody used in the pharmaceutical composition at a level lower than the dosage required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, the appropriate dosage of the composition of the present invention is the amount of humanized or chimeric antibody that is the minimum dosage effective to produce a therapeutic effect in a particular dosage regimen. Such an effective dosage will generally depend on the factors mentioned above.

[0366] 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 predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of a humanized or chimeric antibody to inhibit cell growth or induce cytotoxicity in an in vitro assay known to those skilled in the art. A therapeutically effective amount of a therapeutic compound, i.e., a therapeutic humanized or chimeric antibody or pharmaceutical composition of the present invention, can reduce tumor size or otherwise ameliorate symptoms in a subject. One skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0367] An exemplary non-limiting range for a therapeutically effective amount of a humanized or chimeric antibody of the present invention is about 0.001-30 mg / kg, such as about 0.001-20 mg / kg, such as about 0.001-10 mg / kg, such as about 0.001-5 mg / kg, such as about 0.001-2 mg / kg, such as about 0.001-1 mg / kg, such as 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.

[0368] Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, eg, near the target site.

[0369] The dosage regimen in the above-mentioned treatment methods and uses is adjusted to give the optimum desired response (e.g., therapeutic response).For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as required by the therapeutic situation.

[0370] In one embodiment, the efficacy of treatment is monitored, eg, at predetermined time points, during therapy.

[0371] If desired, the effective daily dose of the pharmaceutical composition may be divided into two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In another embodiment, the humanized or chimeric antibody or pharmaceutical composition is administered by slow continuous infusion over an extended period of time, e.g., 24 hours or more, to minimize any undesirable side effects.

[0372] While it is possible for the humanized or chimeric antibodies of the invention to be administered alone, it is preferable to administer the humanized or chimeric antibodies as a pharmaceutical composition as described above.

[0373] An effective dose of a humanized or chimeric antibody of the invention can also be administered using a dosing period of once a week, once every two weeks, or once every three weeks. The dosing period can be limited, for example, to 8 weeks, 12 weeks, or until clinical progression is established. Alternatively, an effective dose of a humanized or chimeric antibody of the invention can be administered every two weeks, every three weeks, or every four weeks.

[0374] In one embodiment, the humanized or chimeric antibody is administered in mg / m 2 The humanized or chimeric antibody may be administered by infusion in a weekly dose calculated as: Dose (mg / kg) x 70:1.8. Such dosage may be based on the mg / kg dosage above, for example, according to the following formula: Dose (mg / kg) x 70:1.8. Such administration may be repeated, for example, 1-8 times, for example, 3-5 times. Administration may be by continuous infusion over a period of 2-24 hours, for example, 2-12 hours. In one embodiment, to reduce toxic side effects, the humanized or chimeric antibody may be administered by slow continuous infusion over an extended period of time, for example, 24 hours or more.

[0375] In one embodiment, the humanized or chimeric antibody can be administered in a weekly amount calculated as a fixed dose, up to 8 times, e.g., 4-6 times, when administered once a week. Such a regimen can be repeated one or more times, as necessary, e.g., after 6 months or 12 months. Such fixed dosages can be based on the mg / kg dosages mentioned above, e.g., assuming a body weight of 70 kg. The dosage can be determined or adjusted by measuring the amount of the humanized or chimeric antibody of the invention in the blood after administration, e.g., by taking a biological sample and using an anti-idiotypic antibody targeting the binding region of the humanized or chimeric antibody of the invention.

[0376] In one embodiment, the humanized or chimeric antibody can be administered in a maintenance therapy, for example, once a week for a period of six months or more.

[0377] Humanized or chimeric antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of occurrence of certain events in the progression of cancer, and / or reduce the risk of recurrence if the cancer is in remission.

[0378] Parenteral compositions can be formulated into dosage unit form to facilitate administration and ensure uniformity of dosage.Dosage unit form as used herein refers to a physically discrete unit suitable as a unitary dosage to the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect together with necessary pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by or directly depends on (a) the characteristics specific to active compound and the specific therapeutic effect to be achieved, and (b) the inherent constraints in the art when formulating such active compound for the treatment of susceptibility in an individual.

[0379] Humanized or chimeric antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of certain events in the progression of cancer, and / or reduce the risk of recurrence if the cancer is in remission. This can be particularly useful in patients where other biological factors make it difficult to localize a tumor known to be present.

[0380] Diagnostic Uses The humanized or chimeric antibodies of the present invention can also be used for diagnostic purposes, using compositions comprising the humanized or chimeric antibodies described herein. Thus, the present invention provides diagnostic methods and compositions using the humanized or chimeric antibodies described herein. Such methods and compositions can be used for purely diagnostic purposes, e.g., to detect or identify disease, but also for purposes such as monitoring the progress of therapeutic treatment, monitoring the progression of disease, assessing post-treatment status, monitoring recurrence of disease, and assessing the risk of developing disease.

[0381] In one aspect, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, the method comprising the step of administering to a subject a humanized or chimeric antibody of the invention, a composition of the invention, or a pharmaceutical composition of the invention, optionally wherein the humanized or chimeric antibody is labeled with a detectable agent.

[0382] In one aspect, the humanized or chimeric antibody of the present invention is used ex vivo, for example, in the diagnosis of a disease in which cells expressing a specific target of interest to which the humanized or chimeric antibody binds are indicative of the disease or involved in a pathogenesis process, by detecting the level of the target in a sample taken from a patient or the level of cells expressing the target of interest on their cell surface. This can be achieved, for example, by contacting a test sample, optionally together with a control sample, with a humanized or chimeric antibody of the present invention under conditions that allow binding of the antibody to the target. Complex formation can then be detected (for example, using ELISA). If a control sample is used together with the test sample, the level of the humanized or chimeric antibody or antibody-target complex is analyzed in both samples, and a statistically significantly higher level of the humanized or chimeric antibody or antibody-target complex in the test sample indicates the target in the test sample being present at a higher level compared to the control sample.

[0383] Examples of conventional immunoassays in which the humanized or chimeric antibodies of the present invention can be used include, but are not limited to, ELISA, RIA, FACS assay, plasmon resonance assay, chromatographic assay, tissue immunohistochemistry, Western blot, and / or immunoprecipitation.

[0384] Thus, in one embodiment, the invention relates to a method of diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, the method comprising the step of administering to a subject an antibody, bispecific antibody, composition or pharmaceutical composition according to any aspect or embodiment described herein, optionally wherein the antibody is labeled with a detectable label.

[0385] In one aspect, the present invention relates to a method for detecting the presence of a target or a cell expressing a target in a sample, the method comprising: contacting the sample with a humanized or chimeric antibody of the invention under conditions that allow binding of the humanized or chimeric antibody to a target in the sample; and Analyzing whether a complex has been formed The sample is typically a biological sample.

[0386] In one embodiment, the sample is a tissue sample known or suspected to contain a specific target and / or cells expressing said target. For example, in situ detection of target expression can be achieved by removing a histological specimen from a patient and applying the humanized or chimeric antibody of the present invention to such specimen. The humanized or chimeric antibody can be applied by applying or overlaying the humanized or chimeric antibody to the specimen, which is then detected using appropriate means. It is then possible to determine not only the presence of the target or target-expressing cells, but also the distribution of the target or target-expressing cells in the examined tissue (e.g., in connection with the assessment of the spread of cancer cells). Those skilled in the art will readily appreciate that by using the present invention, any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0387] In the above assays, the humanized or chimeric antibody can be labeled with a detectable substance so that the bound antibody can be detected. Alternatively, the bound (primary) specific humanized or chimeric antibody can be detected with an antibody labeled with a detectable substance that binds to the primary specific humanized or chimeric antibody. Furthermore, the above assays can use diagnostic compositions comprising an antibody or bispecific antibody according to any aspect or embodiment described herein. Thus, in one aspect, the present invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to any aspect or embodiment described herein.

[0388] The level of target in a sample can also be estimated by a competitive immunoassay that utilizes a target standard labeled with a detectable substance and an unlabeled target-specific humanized or chimeric antibody. In this type of assay, the biological sample, the labeled target standard and the target-specific humanized or chimeric antibody are combined to determine the amount of labeled target standard bound to the unlabeled target-specific humanized or chimeric antibody. The amount of target in the biological sample is inversely proportional to the amount of labeled target standard bound to the target-specific humanized or chimeric antibody.

[0389] Suitable labels for target-specific humanized or chimeric antibodies, secondary antibodies and / or target standards used in in vitro diagnostic techniques include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; examples of luminescent materials include luminol; examples of suitable radioactive materials include fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. 125 I, 131 I, 35 S, and 3 Examples include H.

[0390] In one aspect, the target-specific humanized or chimeric antibodies of the present invention are used for in vivo imaging of target-expressing tissues, such as tumors. For in vivo methods, e.g., (Fab') 2 Antibody fragments, such as Fab and Fab' fragments, are particularly advantageous as they have rapid distribution kinetics.

[0391] In vivo imaging can be performed by any suitable technique. For example, 99 Tc, 131 I, 111 Using a target-specific humanized or chimeric antibody (e.g., an antibody or fragment) labeled with In or other gamma-emitting isotopes, the accumulation or distribution of the target-specific antibody in target-expressing tissues, such as tumors, can be imaged with a gamma scintillation camera (e.g., an Elscint Apex 409 ECT device), typically using a low-energy high-resolution collimator or a low-energy universal collimator. 89 Zr, 76 Br, 18 Labeling with F or other positron-emitting radionuclides can also be used to image the distribution of target-specific humanized or chimeric antibodies or antibody fragments in tumors using positron emission tomography (PET). Images obtained using such techniques can be used to assess the biodistribution of targets in patients, mammals, or tissues, for example when the targets are used as biomarkers for the presence of cancer / tumor cells. Variations on this technique can include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques. Traditional immunoscintigraphy methods and principles are described, for example, in

[79] ,

[80] , and

[81] . Additionally or alternatively, such images can also serve as the basis for surgical techniques to remove tumors. Furthermore, such in vivo imaging techniques allow for the identification and localization of tumors in situations where a patient is confirmed to have a tumor (e.g., by the presence of other biomarkers, metastases, etc.), but the tumor cannot be identified by traditional analytical techniques. All of these methods are features of the present invention.

[0392] The in vivo imaging and other diagnostic methods provided by the present invention are particularly useful for detecting micrometastases in human patients (e.g., patients who have not been previously diagnosed with cancer or who are in recovery / remission from cancer).

[0393] In one embodiment, the invention provides an in vivo imaging method in which a target-specific humanized or chimeric antibody of the invention is conjugated to a radiopaque substance that facilitates detection, the conjugated humanized or chimeric antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled humanized or chimeric antibody in the host is assayed. By this technique and other diagnostic methods described herein, the invention provides a method for screening for the presence of disease-associated cells in a human patient or a biological sample taken from a human patient and / or for assessing the distribution of a target-specific humanized or chimeric antibody prior to target-specific ADC therapy.

[0394] For diagnostic imaging, radioisotopes can be attached to target-specific humanized or chimeric antibodies either directly or indirectly through intermediate functional groups. Useful intermediate functional groups include ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example,

[82] ).

[0395] Besides radioisotopes and radio-opaque substances, diagnostic methods can also be performed using target-specific antibodies conjugated to dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancing agents for magnetic resonance imaging (MRI), such as paramagnetic ions (see, e.g.,

[83] , which describes MRI techniques and the preparation of antibodies conjugated to MRI enhancing agents). Such diagnostic / detection agents can be selected from agents and fluorescent compounds used in MRI. To load a target-specific humanized or chimeric antibody with a radiometal or paramagnetic ion, it may be necessary to react it with a reagent that has a long tail, to which multiple chelating groups for binding the ions can be attached. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains with pendant groups to which chelating groups can be attached, such as, for example, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and the like, which are known to be useful for this purpose. Chelates can be coupled to target-specific humanized or chimeric antibodies using standard chemistries.

[0396] The present invention thus provides diagnostic target-specific humanized or chimeric antibodies, which are conjugated to an imaging agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound contrast enhancement agent) or to a radionuclide, which may be, for example, a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope.

[0397] In one aspect, the invention relates to a diagnostic composition comprising an antibody or bispecific antibody of the invention.

[0398] In yet another aspect, the present invention provides a kit for detecting the presence of a target antigen or a cell expressing a target antigen in a sample, comprising: a target-specific humanized or chimeric antibody of the present invention, and Instructions for use of the kit The present invention relates to a kit comprising:

[0399] Thus, in one aspect, the present invention comprises: a) contacting a sample with an antibody or bispecific antibody of the invention under conditions that allow for the formation of a complex between the antibody or bispecific antibody and CD3; and b) analyzing whether a complex is formed The present invention provides a kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising:

[0400] 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 can include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, where the enzymatic reaction produces a product that can be visualized. In one embodiment, the present invention provides a diagnostic kit, comprising one or more target-specific humanized or chimeric antibodies of the present invention, labeled or unlabeled, in a suitable container, incubation reagents for indirect assays, and, depending on the nature of the label, a substrate or derivatizing agent for detection in such assays. Control reagents and instructions can also be included.

[0401] Diagnostic kits can also be provided for use with target-specific humanized or chimeric antibodies, such as labeled target-specific antibodies, to detect the presence of a target in a tissue sample or a host. In such diagnostic kits, as well as in kits for therapeutic use as described elsewhere herein, the target-specific humanized or chimeric antibodies can typically be provided in a container in lyophilized form, either alone or together with additional antibodies specific for a target cell or target peptide. Typically, a pharma- ceutical 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, etc. (typically in a separate container for mixing), and additional reagents (also typically in separate containers) are also included. Certain kits also include a second antibody capable of binding to the target-specific humanized or chimeric antibody, typically in a separate container. The second antibody is typically conjugated to a label and formulated in a manner similar to the target-specific humanized or chimeric antibodies of the invention. Using the methods described above and elsewhere herein, target-specific humanized or chimeric antibodies can be used to define subsets of cancer / tumor cells and characterize such cells and associated tumor tissue.

[0402] Anti-idiotype antibodies In yet another aspect, the present invention relates to an anti-idiotypic antibody that binds to a humanized or chimeric antibody of the invention described herein. In one embodiment, the present invention relates to an anti-idiotypic antibody that binds to an antibody of any one of the claims of the invention or to a bispecific antibody of the invention.

[0403] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants generally associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genotype as the source of the CD3 monoclonal antibody with the monoclonal antibody for which the anti-Id is to be prepared. The immunized animal is typically able to recognize and respond to the idiotypic determinants of the immunized antibody by producing antibodies (anti-Id antibodies) that respond to these idiotypic determinants. Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the present invention.

[0404] The anti-Id antibody can also be used as an "immunogen" to induce an immune response in yet another animal to produce a so-called anti-anti-Id antibody. The anti-anti-Id antibody can be epitopically identical to the original monoclonal antibody that induced the anti-Id antibody. Thus, by using antibodies against the idiotypic determinants of the monoclonal antibody, it is possible to identify other clones expressing antibodies with the same specificity. The anti-Id antibody can be altered (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, for example by techniques as described elsewhere herein for the CD3-specific antibodies of the invention. For example, the monoclonal anti-Id antibody can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice will typically contain anti-anti-Id antibodies with binding properties similar, if not identical, to the original / parent CD3 antibody.

[0405] array (Table 1) TIFF2025038049000017.tif92170TIFF2025038049000018.tif237170TIFF2025038049000019.tif234170TIFF20250380490 00020.tif234170TIFF2025038049000021.tif234170TIFF2025038049000022.tif234170TIFF2025038049000023.tif23417 0TIFF2025038049000024.tif234170TIFF2025038049000025.tif234170TIFF2025038049000026.tif234170TIFF202503804 9000027.tif234170TIFF2025038049000028.tif234170TIFF2025038049000029.tif234170TIFF2025038049000030.tif2341 70TIFF2025038049000031.tif234170TIFF2025038049000032.tif234170TIFF2025038049000033.tif234170TIFF20250380 49000034.tif234170TIFF2025038049000035.tif234170TIFF2025038049000036.tif234170TIFF2025038049000037.tif23 4170TIFF2025038049000038.tif234170TIFF2025038049000039.tif234170TIFF2025038049000040.tif234170TIFF2025038049000041.tif234170TIFF2025038049000042.tif237170TIFF2025038049000043.tif252170CDR regions were annotated according to the IMGT definition. EXAMPLES

[0406] Example 1: Generation of humanized CD3 antibodies and non-activating antibody mutants Humanization of CD3 antibody Humanization of the murine CD3 antibody (US8,236,308, herein referred to as IgG1-CD3) was performed by Antitope (Cambridge, UK) using their modified germline humanization (CDR grafting) technology (EP0629240). Using this technology, one different VH chain (SEQ ID NO:4) and two different VL chains (SEQ ID NO:8, 10) were designed. By combining the one VH with the two VL chains, two different antibodies were generated. These humanized variants are referred to herein as huCD3. Thus, a humanized variant comprising a VH and a VL of the present invention is for example referred to as IgG1-huCD3-H1L1, which means that the particular variant is an IgG1 isotype, a humanized CD3, and comprises a VH amino acid sequence named "H1" and defined in SEQ ID NO:4, and a VL amino acid sequence named "L1" and defined in SEQ ID NO:8. Thus, for example, H1 would refer to the heavy chain variable region, VH1, and L1 would refer to the light chain variable region, VL1.

[0407] Specifically, in the examples described herein, the variants IgG1-huCD3-H1L1 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:4 and the VL1 sequence shown in SEQ ID NO:8), IgG1-huCD3-L1-T41K (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:4 and the VL sequence shown in SEQ ID NO:10).

[0408] b12 antibody In some examples, the HIV-1 gp120 specific antibody, antibody b12 (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3)812-23), was used as a negative control and is designated "IgG1-b12".

[0409] Expression Antibodies were expressed as IgG1,κ or IgG1,λ with or without inactivating mutations as described below and with mutations in the CH3 domain that allow for the generation of bispecific antibodies by the methods described below. Plasmid DNA mixtures encoding both the heavy and light chains of the antibodies were transiently transfected into Freestyle HEK293F cells (Invitrogen, USA) using 293fectin (Invitrogen, USA) essentially as described by the manufacturer.

[0410] Antibody purification Culture supernatants were filtered through a 0.2 μm dead-end filter, loaded onto a 5 mL MabSelect SuRe column (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH (pH 3). The eluate was immediately neutralized with 2 M Tris-HCl (pH 9) and dialyzed overnight against 12.6 mM NaH2P04, 140 mM NaCl, pH 7.4 (B. Braun). Alternatively, following purification, the eluate was loaded onto a Hi Prep desalting column and the antibody was exchanged into 12.6 mM NaH2P04, 140 mM NaCl, pH 7.4 (B. Braun) buffer. After dialysis or buffer exchange, samples were sterile filtered through a 0.2 μm dead-end filter. Purity was determined by SDS-PAGE and concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2–8 °C.

[0411] Example 2: Construction of a mutant library Point mutations were generated by random mutagenesis performed using the Quick change mutagenesis kit (Stratagene, according to the manufacturer's instructions) and the HC (p33HGTE-huCD3-H1) and LC (p33L-huCD3-L1-T41K) expression plasmids as templates. The HC plasmid encodes the monovalent UniBody-TE format described in WO2011110642. Each selected position was randomized by using primers containing an NNS codon at the selected position (N=G, A, T, or C, and S=G or C). The mutant library was transformed into OneShot DH5α (Invitrogen) according to the manufacturer's instructions.

[0412] Colony picking and LEE PCR 96 clones for each mutated position were individually picked in 50 μL LEE (Linear Expression Element) PCR buffer (5 μL 10× AccuPrime PCR Buffer 1, 44.6 μL water (B. Braun), 0.1 μL CMV P f (MAR5) and 0.1 μL Tk pA r (MAR1) primers (100 μM stock), 0.2 μL Accuprime Taq (Invitrogen)) to amplify the expression cassette from the expression plasmid (from promoter to polyA). LEE PCR was performed by incubating the mixture at 94°C for 2 min, [94°C for 35 s, 55°C for 30 s, 68°C for 5 min]×35, 72°C for 10 min, and storing at 4°C until further use.

[0413] Each library of 96 colonies ( 12 ) was sequenced using Sanger sequencing (Beckman Coulter Genomics, UK).

[0414] Table 2 Primer sequences for LEE PCR TIFF2025038049000044.tif20137

[0415] Example 3: Expression of mutant libraries and IgG quantification 1.11 μL HC and 1.11 μL LC LEE PCR products of each mutant (12×96 in total) were diluted in 2.78 μL water. 5 μL of DNA dilutions were used to transfect a single well in a 96-well plate.

[0416] 0.4 μL ExpiFectamine™ 293 (Invitrogen, USA) and 4.6 μL Opti-MEM (Gibco, USA) per well were mixed and incubated at room temperature for 5 min. Then, the Fectin / Opti-MEM mix was added to 5 μL DNA dilution and incubated at room temperature for 30 min. Finally, 8.3 μL Fectin / Opti-MEM / DNA mix was added to 117.5 μL Expi293F™ cells. During all procedures, the plate containing Expi293F™ cells was shaken to keep the cells in suspension. After transfection, the cells were incubated at 37° C. / 8% CO2 for 5 days.

[0417] Five days after transfection, the supernatants were harvested and the antibody concentrations in the supernatants were measured by biolayer interferometry using Octet RED (ForteBio, USA).

[0418] Example 4: Generation of a CD3 / TCR-LC13 screening library Freestyle 293-F cells (Invitrogen, USA) were co-transfected with expression constructs encoding the human TCR 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). In these sequences, the signal peptide sequence has been omitted. Transfection was performed according to the manufacturer's instructions (Invitrogen, USA). One day after transfection, the cells were frozen until further use.

[0419] Example 5: Screening of affinity mutants Homogeneous Assay (Dose Response) Based on sequence data, mutants were selected whose sequence tracking showed high PHRED scores and no multiple mutations, and when available, multiple overlapping clones per mutation were selected.

[0420] Binding of recombinantly produced UniBody molecules in cell culture supernatants was determined by homogeneous antigen-specific binding assays using Fluorometric Micro volume Assay Technology (FMAT; Applied Biosystems, Foster City, CA, USA). In the assay study design, samples were analyzed in a dose response for antibody or monovalent antibody molecule binding to CD3 / TCR-LC13 (Freestyle 293-F cells transiently expressing human CD3 and human T cell receptor (TCR); produced as described above) and Freestyle 293-F wild-type cells (negative control not expressing human TCR). IgG levels for sample normalization prior to dose response binding were measured using an Octet instrument (Fortebio, Menlo Park, USA).

[0421] A dilution series of samples was added to the cells and allowed to bind to CD3. Binding of 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 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 Cellular Detection System (8200 CDS) and total fluorescence across sample concentrations was used as the readout. Samples were declared positive if the counts were higher than 50 and the counts x fluorescence (total fluorescence) was at least 3-fold higher than the negative control.

[0422] Heatmap From the homogeneous dose-response screen, the binding curves were fitted using a four-parameter sigmoidal model. From the fits, the maximum binding was determined for every mutant. The average maximum binding per mutant was calculated and plotted as the ratio between the average maximum over wt binding, as shown in Figure 1.

[0423] Alignment Selected HC mutants generated in these libraries are aligned and depicted diagrammatically in FIG. The CDR regions are annotated according to the IMGT definition. Sequence numbering is annotated according to the direct numbering scheme.

[0424] Example 6-2 - Generation of bispecific antibodies by MEA-induced Fab arm exchange Bispecific antibodies of the present invention may be made by use of the methods disclosed in WO2011131746 and WO2013060867 (Genmab).

[0425] As an example, a mutation at position F405L may be introduced into one parent antibody of IgG1 isotype and a mutation at position K409R may be introduced into the other parent antibody of IgG1 isotype.

[0426] These two parent antibodies, each at a final concentration of 0.5 mg / mL (equimolar), may be incubated under reducing conditions with 25 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL Tris-EDTA (TE) for 90 min at 37° C. The reduction reaction is stopped by removing the reducing agent 2-MEA using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol.

[0427] The bispecific antibody may be filtered through a 0.2 μm dead-end filter and the absorbance at 280 nm (A280) of the bispecific antibody may be measured to determine its final concentration.

[0428] Example 7: Binding Data For all binding assays described below, a panel of selected heavy chain variants of huCD3-H1L1 were tested in a variety of formats.

[0429] Table 3. Affinity variants of huCD3-H1L1 in selected monovalent antibody-TE formats TIFF2025038049000045.tif110128

[0430] Determination of Octet-binding affinity of CD3 affinity mutants in monovalent antibody-TE format The affinity of a panel of selected affinity VH mutants (Table 3) was determined using Biolayer Interferometry on a ForteBio Octet HTX. CD3 affinity mutants in monovalent antibody-TE format (2 μg / mL) were loaded onto an Anti-human Fc Capture (AHC) biosensor (ForteBio, Portsmouth, UK; Cat. No. 18-5060) for 600 s, aiming for a loading response of 0.4 nm. Antibodies in UniBody-TE format were used to specifically measure monovalent interaction affinities between CD3 affinity mutants and the CD3ε27-GSKa ligand. After a baseline (150 s), the association (1000 s) and dissociation (1000 s) of CD3ε27-GSKa (100 and 1000 nM) were determined. The CD3ε27-GSKa protein consists of the human CD3ε peptide (amino acids 1-27) fused to the N-terminus of kappa LC (SEQ ID NO: 402). For calculations, the theoretical molecular weight of CD3ε27-GSKa based on the amino acid sequence was used, i.e., 27.1 kDa. The experiments were performed at 30°C with shaking at 1000 rpm.

[0431] Data were analyzed with ForteBio Data Analysis Software v8.1 using a 1:1 model and a global full fit with an association time of 1000 s and a dissociation time of 200 s. Data traces were corrected by subtracting a reference curve (CD3ε27-CD3 affinity mutant without GSKa), aligning the Y-axis to the last 5 s of baseline, and applying interstep correction and Savitzky-Golay filtering.

[0432] Table 4. Equilibrium dissociation constants (KD) for selected mutants TIFF2025038049000046.tif82128nd=Undecided

[0433] T cell binding of affinity mutants of humanized CD3 (UniTE-huCD3-H1L1-LT41K) by flow cytometry (FACS) T cell binding of purified humanized CD3 (IgG1-huCD3-H1L1) antibody VH affinity variants was determined using fluorescence-activated cell sorting on a FACSCanto 752 (BD Biosciences). T cells were isolated from buffy coat fractions of anticoagulated human donor blood samples and resuspended at 1.8×10E6 cells / mL in PBS / 0.1% BSA / 0.02% azide. 50 μL of T cell suspension and 50 μL of antibody dilution were combined on ice in a 96-well plate, incubated at 4° C. for 30 minutes, and washed twice with PBS / 0.1% BSA / 0.02% azide. Then, 50 μL of secondary antibody, R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1 / 200 in PBS / 0.1% BSA / 0.02% azide, was added for staining, and the mixture was incubated for 30 min at 4° C., and then washed twice with PBS / 0.1% BSA / 0.02% azide. The cells were resuspended in 120 μL PBS / 0.1% BSA / 0.02% azide, and PE geometric mean fluorescence intensity was measured. Binding curves were analyzed using nonlinear regression (sigmoidal dose response with variable slope) with GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA), and apparent affinities (KD) were derived from the concentration at half-maximal binding. Figure 4 shows the binding curves of affinity variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K), and Figure 5 shows the binding curves of low affinity variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K).

[0434] Table 5. Summary of binding data for CD3 affinity mutants in monovalent antibody-TE format. TIFF2025038049000047.tif140128

[0435] For all binding assays described below, a panel of selected preferred heavy chain variants was tested (see Table 5).

[0436] Determination of Octet binding affinity of IgG1-huCD3-H1L1-FEAL affinity mutants The affinity of selected CD3 affinity variants in the IgG1-huCD3-H1L1-FEAL format was determined using biolayer interferometry on a ForteBio Octet HTX (ForteBio, UK) (Table 6). An Anti-human Fc capture biosensor (cat: 18-5060, ForteBio, UK) was loaded with hIgG (1 μg / mL) for 600 s. After a baseline (200 s), the association (1000 s) and dissociation (2000 s) of CD3E27-GSKa were determined using a CD3E27-GSKa concentration range of 27.11 μg / mL to 0.04 μg / mL (1000 nM to 1.4 nM) in 3-fold dilution steps (sample diluent, cat: 18-5028, ForteBio, UK). For calculations, the theoretical molecular weight of CD3E27-GSKa based on the amino acid sequence was used, i.e., 27.11 kDa. Experiments were performed at 30° C. with shaking at 1000 rpm. Each antibody was tested in at least two independent experiments (Table 6).

[0437] Data were analyzed with ForteBio Data Analysis Software v8.1 using a 1:1 model and a global full fit with an association time of 1000 s and a dissociation time of 100 s. Data traces were corrected by subtracting a reference curve (antibody without CD3E27-GSKa), aligning the Y-axis to the last 10 s of baseline, and applying stepwise correction and Savitzky-Golay filtering. Data traces with responses shorter than 0.05 nm were excluded from the analysis.

[0438] (Table 6) TIFF2025038049000048.tif75162

[0439] Determination of T cell binding affinity of IgG1-huCD3-H1L1-FEAL affinity mutants T cells from donor buffy coats (Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (cat: 15021C.1, Stemcell Technologies, France) according to the manufacturer's instructions. Briefly, 50 μL of T cell isolation cocktail was added to 1 mL of buffy coat and incubated for 20 min at room temperature. The buffy coat was then diluted (1:3, v / v) with PBS (cat: 3623140, B.Braun, Germany) and gently transferred into a 50 mL falcon tube (cat: 227261, Greiner bio-one, The Netherlands) filled with 15 mL of lymphocyte separation medium (cat: 17-829E, Lonza, Switzerland). The tubes were centrifuged at 1200×g for 20 min at room temperature without brake. T cells were collected from the density medium and washed twice with PBS.

[0440] 2x10E6 T cells / mL were resuspended in FACS buffer and 50 μL were transferred into a round bottom 96 well plate (cat: 650101, Greiner bio-one, The Netherlands). 50 μL of 5-fold diluted antibody solution was added starting at 5 μg / mL and incubated for 30 min at 4°C. The 96 well plate was centrifuged at 300×g for 5 min at 4°C and the supernatant was discarded. The cells were washed twice on ice with ice cold FACS buffer and 100 μL / well of 1:200 diluted secondary antibody (anti-IgG Fcγ-PE(fab)'2, cat: 109-116-098, Jackson Immuno Research, UK) was added, incubated for 30 min and washed twice with FACS buffer. Fluorescence intensity was measured on a FACS Canto and the geometric mean was calculated by FlowJo V10 software. Graphs were generated using GraphPad (V6.04), see Figure 5.

[0441] Example 9: In vitro cytotoxicity screening of CD3 affinity mutants Cytotoxicity of CD3 affinity mutants against solid tumor cell lines (Alamar Blue assay) T cells from donor buffy coats (Sanquin, Amsterdam, The Netherlands) were isolated using RosetteSep human T cell enrichment cocktail (cat: 15021C.1, Stemcell Technologies, France) according to the manufacturer's instructions. NCI-N87 (25.000 cells / well) (Figure 6A), SKOV3 (16.000 cells / well) (Figure 6B), and MDA-MB-231 (16.000 cells / well) (Figure 6C) cells were seeded in flat-bottom 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 in the following ratios: NCI-N87 cells:T cells, 1:3; SKOV3 cells:T cells, 1:4; MDA-MB-231 cells:T cells, 1:8. Afterwards, antibody solutions were added in 10-fold dilutions and the plates were incubated at 37°C for 2 days. The supernatant was then discarded and the attached cells were washed twice with PBS. 150 μL of a 10% Alamar Blue (Cat: DAL1100, Life Technologies, Netherlands) solution prepared in RPMI-1640 (Cat: BE12-115F, Lonza, Switzerland) medium containing 10% donor bovine serum with iron (Cat: 10371-029, Life Technologies, Netherlands) was added to the wells and incubated at 37°C for 3-5 hours. The absorbance was measured with an Envision multilabel plate reader (PerkinElmer, USA). Staurosporine (Cat: S6942, Sigma-Aldrich, USA) treated cells were set as 100% dead and untreated cells were set as 0% dead. Viable cells were calculated by subtracting staurosporine-treated cells from all groups and the percentage relative to the untreated group was plotted. Graphs were generated using GraphPad (v6.04). See Figure 6.

[0442] Cytotoxicity of CD3 affinity mutants against blood cell lines (chromium release assay) 5x10E6 Daudi cells / mL were incubated in complete culture medium containing 100μCi chromium for 1 hour at 37°C under shaking conditions. Cells were then washed twice in PBS and resuspended in 5 mL complete cell culture medium (RPMI 1640 with 10% donor bovine serum with iron). 5.000 Daudi cells were seeded in round-bottom 96-well plates. T cells from donor buffy coats (purchased from Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (catalog: 15021C.1, Stemcell Technologies, France) according to the manufacturer's instructions. T cells were added to Daudi cells at a ratio of 1:10 (tumor cells:T cells) followed by 2-fold dilution of antibody solution. Plates were incubated at 37°C for 24 hours. After 24 hours, the plates were centrifuged for 3 minutes at 300×g and the supernatants were harvested and the radioactivity was measured, see FIG.

[0443] (Table 7) TIFF2025038049000049.tif118128

[0444] Example 10: Tumor efficacy of CD3 x HER2 bispecific antibodies in a (human PBMC + NCI-N87 cells) co-implant model in NOD-SCID mice The in vivo antitumor efficacy of several CD3×HER2 bispecific antibodies was evaluated in a subcutaneous NCI-N87 co-implant model ( FIG. 8 ). Humanized WT CD3 (huCD3-FEAL) and four different CD3 affinity mutants (N57E, H101K, S110A, Y114M) were used as the CD3 arm of the bispecific antibodies. In all cases, the HER2 targeting arm was the same (Herceptin-FEAR). BisG1-huCD3-FEAL×1014-Herceptin-FEAR BisG1-huCD3-N57E-FEAL×1014-Herceptin-FEAR BisG1-huCD3-H101K-FEAL×1014-Herceptin-FEAR BisG1-huCD3-S110A-FEAL×1014-Herceptin-FEAR BisG1-huCD3-Y114M-FEAL×1014-Herceptin-FEAR

[0445] 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).

[0446] Mice were sorted into treatment groups (n=4 per treatment group). On day 0, a mixture containing HLA-A matched hPBMC (5×10E6, Sanquin) and NCI-N87 (5×10E6) cells in 200 μL PBS / 0.1% BSA was transfected into each female NOD-SCID mouse (NOD.CB-17-Prkdc scid Mice (16-19 years, Charles River, 6-11 weeks of age) were inoculated subcutaneously (sc) in the right flank of mice. Immediately after tumor cell injection, a single intravenous dose (150 μL) of five different CD3×HER2 antibodies was administered at two different concentrations (0.5 and 0.05 mg / kg) for all bispecific antibodies. Tumor volumes were determined at least twice per week. Tumor volumes (mm 3 ) is calculated by 0.52 x (length) x (width) from the caliper (PLEXX) measurement. 2 It was calculated as:

[0447] NCI-N87 cells (ATCC number CRL-5822, gastric carcinoma arising 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 CellSTACK culture chambers, harvested in log phase, and counted by trypan blue exclusion.

[0448] For each study, hPBMCs were isolated from buffy coats (Sanquin) by Ficoll density centrifugation from human HLA-A matched donors for NCI-N87 (HLA-A-01,23). Isolated cells were frozen in nitrogen and thawed before use. All cells were washed in PBS / 0.1% BSA, filtered through a cell strainer, and resuspended in PBS / 0.1% BSA to a concentration of 50×10E6 cells / mL.

[0449] The results are shown in Figure 8. Figures 6A-B show the mean tumor volume over time after treatment. Figures 6C-D show dot plot representations of mean NCI-N87 tumor volumes at day 44. Statistical analysis (Mann-Whitney) on tumor volumes on day 44, the last day when all groups were still intact, revealed significant tumor growth inhibition (p<0.05) for BisG1-huCD3-FEAL×1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEAL×1014-Herceptin-FEAR, and BisG1-huCD3-Y114M-FEAL×1014-Herceptin-FEAR compared to control (PBMC) at a dose of 0.05 mg / kg, but no inhibition for BisG1-huCD3-N57E-FEAL×1014-Herceptin-FEAR and BisG1-huCD3-H101K-FEAL×1014-Herceptin-FEAR. At a dose of 0.5 mg / kg, BisG1-huCD3-FEAL×1014-Herceptin-FEAR and all CD3 arm affinity mutants showed significant tumor growth (p<0.05) inhibition compared to the PBS (PBMC) control group, except for BisG1-huCD3-H101K-FEAL×1014-Herceptin-FEAR.

[0450] BisG1-huCD3-FEAL×1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEAL×1014-Herceptin-FEAR, and BisG1-huCD3-Y114M-FEAL×1014-Herceptin-FEAR significantly (p<0.05) reduced NCI-N87 tumor volume at doses of 0.05 and 0.5 mg / kg. BisG1-huCD3-N57E-FEAL×1014-Herceptin-FEAR significantly (p<0.05) reduced NCI-N87 tumor volume only at a dose of 0.5 mg / kg. BisG1-huCD3-H101K-FEAL×1014-Herceptin-FEAR had no effect on NCI-N87 tumor growth at both doses tested.

[0451] List of references TIFF2025038049000050.tif158162TIFF2025038049000051.tif235163TIFF2025038049000052.tif229163

Claims

1. A humanized or chimeric antibody that binds to human CD3, the antibody comprising a binding region comprising a heavy chain variable (VH) region, the VH region comprising a mutation in one of three CDR sequences of a reference antibody having CDR sequences as shown in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2, and CDR3 SEQ ID NO: 3, the mutation being at one of the positions selected from the group consisting of T31M, T31P, N57, H101, G105, S110, and Y114, the positions being numbered according to the reference sequence of SEQ ID NO:

4.

2. The antibody of claim 1, wherein the antibody has a binding affinity to human CD3 that is reduced or enhanced 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. An antibody described in any one of claims 1 to 2, comprising a T31M or T31P mutation.

4. The antibody of any one of claims 1 to 2, comprising a mutation at position N57.

5. 5. The antibody of any one of claims 1, 2, and 4, comprising an N57E mutation.

6. 3. The antibody of claim 1, comprising a mutation at position H101.

7. 7. The antibody of any one of claims 1, 2, and 6, comprising an H101G or H101N mutation.

8. 3. The antibody of claim 1, comprising a mutation at position G105.

9. 9. The antibody of any one of claims 1, 2, and 8, comprising a G105P mutation.

10. 3. The antibody of claim 1, comprising a mutation at position Y114.

11. 11. The antibody of any one of claims 1, 2, and 10, comprising a Y114M, Y114R, or Y114V mutation.

12. The VH region is a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, and 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V]; and l) CDR1, CDR2, and CDR3 sequences having at least 90% or at least 95% amino acid sequence identity across the three CDR sequences to any one of the three CDR sequences shown in a) to k), except that the CDR1, CDR2, and CDR3 sequences do not have the sequences shown in SEQ ID NOs: 1, 2, and 3. The antibody of any one of claims 1 to 2, comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of:

13. The VH region is a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, and 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R]; k) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V], and l) CDR1, CDR2 and CDR3 sequences as defined in a) to k), which have up to 5 further mutations or substitutions, up to 4 further mutations or substitutions, up to 3 further mutations or substitutions, up to 2 further mutations or substitutions or up to 1 further mutation or substitution across the three CDR sequences, which mutations or substitutions preferably do not alter the binding affinity to human CD3. The antibody of any one of claims 1 to 2, comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of:

14. The antibody of claim 13, wherein the further mutations or substitutions are conservative, physical, or functional amino acids.

15. The VH region is a) the VH sequence shown in SEQ ID NO: 55 [T31M]; b) the VH sequence shown in SEQ ID NO: 59 [T31P]; c) the VH sequence shown in SEQ ID NO: 107 [N57E]; d) the VH sequence shown in SEQ ID NO: 177 [H101G]; e) the VH sequence shown in SEQ ID NO: 185 [H101N]; f) the VH sequence shown in SEQ ID NO: 221 [G105P]; g) the VH sequence shown in SEQ ID NO: 237 [S110A]; h) the VH sequence shown in SEQ ID NO: 245 [S110G]; i) the VH sequence shown in SEQ ID NO: 285 [Y114M]; j) the VH sequence shown in SEQ ID NO: 293 [Y114R]; k) the VH sequence shown in SEQ ID NO: 299 [Y114V], and l) a VH sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to any one of the sequences set forth in a) to k). The antibody of any one of claims 1 to 2, having a VH sequence selected from the group consisting of:

16. The VH region is a) the VH sequence shown in SEQ ID NO: 55 [T31M]; b) the VH sequence shown in SEQ ID NO: 59 [T31P]; c) the VH sequence shown in SEQ ID NO: 107 [N57E]; d) the VH sequence shown in SEQ ID NO: 177 [H101G]; e) the VH sequence shown in SEQ ID NO: 185 [H101N]; f) the VH sequence shown in SEQ ID NO: 221 [G105P]; g) the VH sequence shown in SEQ ID NO: 237 [S110A]; h) the VH sequence shown in SEQ ID NO: 245 [S110G]; i) the VH sequence shown in SEQ ID NO: 285 [Y114M]; j) the VH sequence shown in SEQ ID NO: 293 [Y114R], and k) the VH sequence shown in SEQ ID NO: 299 [Y114V] 3. The humanized or chimeric antibody of claim 1, having a VH sequence selected from the group consisting of:

17. The antibody of any one of the preceding claims, wherein the binding region comprises a light chain variable (VL) region, the VL region comprising CDR1, CDR2, and CDR3 regions having the CDR sequences shown in SEQ ID NO: 6, GTN, and SEQ ID NO:

7.

18. The binding domain comprises a light chain variable (VL) domain, the VL domain comprising: a) the VL sequence shown in SEQ ID NO: 8; b) the VL sequence shown in SEQ ID NO: 10; or c) a VL sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to any one of the sequences set forth in a) to b). The antibody of any one of the preceding claims, selected from the group consisting of:

19. The VL region comprises CDR1, CDR2, and CDR3 regions having the sequences shown in SEQ ID NO: 6, GTN, and SEQ ID NO: 7, and the VH region comprises a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, and 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R]; and k) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V] The antibody of claim 17, comprising CDR1, CDR2, and CDR3 regions having CDR sequences selected from the group consisting of:

20. The antibody of any one of the preceding claims, wherein the human CD3 is human CD3ε as defined in SEQ ID NO:

399.

21. The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. D Value 1.6×10 -8 M~9.9×10 -8 M or 1.0×10 -7 ~9.9×10 -7 The antibody according to any one of the preceding claims, which corresponds to M.

22. The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. D Value 1.4×10 -8 ~1.0×10 -8 M or 9.9×10 -9 ~1×10 -9 The antibody according to any one of the preceding claims, which corresponds to M.

23. The antibody of any one of the preceding claims, which is a humanized antibody.

24. The antibody of any one of the preceding claims, which is a full-length antibody.

25. 10. The antibody of any one of the preceding claims, which is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

26. The antibody of any one of the preceding claims, which is monovalent, bivalent or multivalent.

27. The antibody of any one of the preceding claims, comprising an Fc region comprising a first and a second immunoglobulin heavy chain.

28. 10. The antibody of claim 9, wherein the antibody comprises a first and a second immunoglobulin heavy chain, and in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

29. 29. The antibody of claim 28, wherein in at least one of the first and second immunoglobulin heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D.

30. 29. The antibody of claim 28, wherein in at least one of the first and second heavy chains the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N.

31. 29. The antibody of claim 28, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

32. 32. The antibody of claim 28, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E; or A and A, respectively.

33. 33. The antibody of claim 32, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

34. 33. The antibody of claim 32, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

35. 28. The antibody of claim 27, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively.

36. 36. The antibody of claim 35, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively.

37. 37. The antibody of claim 35, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

38. 37. The antibody of any one of claims 35 to 36, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are A, A, and A, respectively.

39. 29. The antibody of claim 28, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, Q, and S, respectively.

40. A bispecific antibody comprising a first binding region of an antibody according to any one of claims 1 to 39 and a second binding region that binds to a target different from the first antigen-binding region.

41. 41. The bispecific antibody of claim 40, comprising a first and a second heavy chain.

42. 42. The bispecific antibody of any one of claims 40 to 41, wherein each of the first and second heavy chains comprises at least a hinge region, a CH2 and a CH3 region, and wherein in the first heavy chain at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain is substituted, and in the second heavy chain at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy chain is substituted, and wherein the first and the second heavy chains are not substituted at the same positions.

43. 43. The bispecific antibody of claim 42, wherein the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L in the first heavy chain and the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R in the second heavy chain, or vice versa.

44. 44. The bispecific antibody of any one of claims 40 to 43, wherein the first binding region is according to any one of claims 1 to 27 and the second binding region binds to a different target than the first binding region.

45. A method for reducing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, 2, 3, comprising introducing a mutation selected from a mutation at one of the positions selected from the group of T31M, T31P, N57, H101, S110, and Y114 in one of the three CDR sequences of the reference antibody, wherein the positions are numbered according to the reference sequence of SEQ ID NO:

4.

46. 46. ​​The method of claim 45, wherein the mutation is a T31M or T31P mutation.

47. 46. ​​The method of claim 45, comprising introducing a mutation corresponding to position N57 in the VH CDR2 region.

48. 48. The method of claim 47, wherein the mutation at position N57 is an N57E mutation.

49. 46. ​​The method of claim 45, comprising introducing mutations corresponding to positions selected from the group of H101, S110, and Y114 in the VH CDR3 region.

50. 50. The method of claim 49, wherein the mutation in the VH CDR3 region is selected from the group consisting of H101G, H101N, S110A, S110G, Y114M, Y114R, and Y114V.

51. 1. A method for enhancing the binding affinity of an antibody that binds to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 3, comprising introducing a mutation corresponding to position G105 in the VH CDR3, said positions being numbered according to the reference sequence of SEQ ID NO:

4.

52. 52. The method of claim 51, wherein the mutation at position G105 is a G105P mutation.

53. The method of any one of claims 45 to 52, comprising introducing up to 5 further mutations, up to 4 further mutations, up to 3 further mutations, up to 2 further mutations, or up to 1 further mutation in the CDRs of the VH region of the reference antibody shown in SEQ ID NO: 1, 2, 3.

54. The antibody having enhanced or decreased binding affinity comprises a binding region comprising a heavy chain variable (VH) region, the VH region comprising: a) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 54, 2, 3 [T31M]; b) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 58, 2, 3 [T31P]; c) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 106, and 3 [N57E]; d) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, 176 [H101G]; e) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 184 [H101N]; f) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 220 [G105P]; g) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 236 [S110A]; h) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 244 [S110G]; i) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 284 [Y114M]; j) the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 292 [Y114R]; and k) CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, 298 [Y114V] The method of any one of claims 45 to 53, comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of:

55. The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. D Value 1.6×10 -8 M~9.9×10 -8 M or 1.0×10 -7 ~9.9×10 -7 The method according to any one of claims 45 to 54, wherein said method corresponds to M.

56. The binding affinity of the antibody to the human CD3ε peptide of SEQ ID NO: 402 was determined by biolayer interferometry. D Value 1.4×10 -8 ~1.0×10 -8 M or 9.9×10 -9 ~1×10 -9 The method according to any one of claims 45 to 54, wherein said method corresponds to M.

57. The method of any one of claims 45 to 57, wherein the human CD3ε is expressed on T cells.

58. The method of any one of claims 45 to 57, wherein the human CD3ε is in isolated form, e.g., an isolated human CD3ε peptide.

59. 45. A nucleic acid construct encoding one or more amino acid sequences according to any one of claims 1 to 44.

60. (i) a nucleic acid sequence encoding the heavy chain sequence of the humanized or chimeric antibody of any one of claims 1 to 16; (ii) a nucleic acid sequence encoding the light chain sequence of a humanized or chimeric antibody according to any one of claims 17 to 18; or (iii) Both (i) and (ii) An expression vector comprising:

61. A host cell comprising the expression vector of claim 60.

62. 62. The host cell of claim 61, which is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell.

63. A composition comprising an antibody according to any one of claims 1 to 27 or a bispecific antibody according to claims 28 to 44.

64. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 27 or a bispecific antibody according to any one of claims 28 to 44, and a pharma- ceutically acceptable carrier.

65. An antibody according to any one of claims 1 to 44, a composition according to claim 63 or a pharmaceutical composition according to claim 64 for use as a medicament.

66. An antibody according to any one of claims 1 to 44, a composition according to claim 63 or a pharmaceutical composition according to claim 64 for use as a medicament in the treatment of cancer, an infectious disease or an autoimmune disease.

67. An antibody according to any one of claims 1 to 27, a bispecific antibody according to claims 28 to 44, a composition according to claim 63 or a pharmaceutical composition according to claim 64 for use in the treatment of a disease.

68. Use of an antibody according to any one of claims 1 to 27 or a bispecific antibody according to claims 28 to 44 for the preparation of a medicament for the treatment of a disease.

69. 69. The use of claim 68 for the treatment of a disease which is cancer, an infectious disease, or an autoimmune disease.

70. A method for treating a disease comprising administering to a subject in need thereof an antibody according to any one of claims 1 to 27, a bispecific antibody according to claims 28 to 44, a composition according to claim 63, or a pharmaceutical composition according to claim 64.

71. 71. The method of claim 70, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

72. 65. A method for diagnosing a disease characterized by the involvement or accumulation of CD3 expressing cells, comprising administering to a subject an antibody according to any one of claims 1 to 27, a composition according to claim 63, or a pharmaceutical composition according to claim 64, wherein optionally the antibody is labeled with a detectable agent.

73. A method for producing an antibody according to any one of claims 1 to 27, comprising the steps of: a) culturing a host cell according to any one of claims 61 to 62; and b) purifying the antibody from the culture medium The method comprising:

74. A diagnostic composition comprising an antibody according to any one of claims 1 to 27 or a bispecific antibody according to any one of claims 28 to 44.

75. 1. A method for detecting the presence of CD3 antigen or CD3 expressing cells in a sample, comprising: a) contacting said sample with an antibody according to any one of claims 1 to 27 or a bispecific antibody according to any one of claims 28 to 44 under conditions allowing the formation of a complex between said antibody or bispecific antibody and CD3; and b) analyzing whether or not a complex is formed The method comprising:

76. A kit for detecting the presence of CD3 antigen or CD3 expressing cells in a sample, comprising: i) an antibody according to any one of claims 1 to 27 or a bispecific antibody according to any one of claims 28 to 44; and ii) Instructions for use of the kit.

77. An anti-idiotypic antibody that binds to the antibody of any one of claims 1 to 27 or the bispecific antibody of any one of claims 28 to 44.

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