CD3-targeting antibodies and uses thereof

Abstract

The present disclosure relates to antibodies targeting CD3 and uses thereof. Specifically, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD3, nucleic acids encoding the antibodies or antigen-binding fragments, nucleic acid vectors comprising the nucleic acids, host cells comprising the nucleic acids or nucleic acid vectors, pharmaceutical compositions comprising the foregoing, methods for preparing and using the same.

Description

[Technical field]

[0001] The present application relates to the field of biomedicine, in particular to antibodies or antigen-binding fragments thereof that target CD3, as well as methods for their preparation and use. [Background technology]

[0002] The TCR / CD3 complex is the main regulator of T cell function and specificity in humans and other mammals, and plays a crucial role in antigen recognition and intracellular signaling pathways. CD3 is a protein complex composed of four different chains (CD3ε, CD3γ, CD3ζ, and CD3δ) and includes three dimers, CD3ε / γ (or CD3e / g), CD3ε / δ (or CD3e / d), and CD3ζ / ζ (or CD3z / z), all of which contain the immunoreceptor tyrosine-based activation motif (ITAM) required for signaling initiation. TCR is a heterodimer of covalently linked α and β chains ("TCRαβ") that mainly recognizes antigens presented by MHC molecules, but its intracellular region is very short and needs to form a TCR / CD3 complex with CD3 to transmit stimulatory signals into the cell (Dong, D., Zheng, L., Lin, J. et al., Nature 573, 546-552 (2019)).

[0003] Currently, antibody drugs targeting CD3 have already been widely used in clinical practice. In 1986, the first monoclonal antibody drug approved and launched by the FDA was CD3 Muromonab (also known as OKT3), which was used to treat acute rejection after organ transplantation. Later, other monoclonal antibodies targeting CD3, such as Otelixizumab, Teplizumab, and Visilizumab, were successively developed for the treatment of Crohn's disease, ulcerative colitis, type I diabetes, and other diseases. In recent years, due to the important role of CD3 in processes such as T cell activation and proliferation, bispecific or multispecific antibodies targeting human CD3 and tumor-associated antigens (TAA) have also become the focus of research in anti-tumor therapy.

[0004] The difficulty in developing an antibody targeting CD3 (which in this disclosure can be used interchangeably as "CD3 antibody", "anti-CD3 antibody", and "antibody that specifically binds to CD3") is that it is difficult to obtain better immune responses and serum titers, resulting in a low number of positive clones after fusion. Currently available CD3 antibodies include OKT3. After the end of treatment with OKT3, T cell function usually returns to normal within one week, but the side effects at the beginning of OKT3 use, such as OKT3 flu-like syndrome and cytokine storm, were also quite significant. In addition, OKT3 does not bind to CD3 derived from cynomolgus monkeys, which caused inconvenience in the selection of cynomolgus monkeys as a primate animal in preclinical safety evaluation studies.

[0005] The present disclosure provides a novel anti-CD3 antibody capable of binding to human and cynomolgus CD3 with low side effects, providing a new potential treatment for diseases including cancer and autoimmune diseases. The antibody or antigen-binding fragment thereof of the present disclosure has excellent biological activity (e.g., high affinity). It can bind to human CD3e / g (hCD3e / g), human CD3e / d (hCD3e / d), and cynomolgus CD3e / g (cCD3e / g), thereby making it possible to select a disease model of the primate cynomolgus monkey (cynomolgus, cyno) for pharmacological and toxicological experiments in preclinical safety evaluation research, providing great convenience to preclinical pharmacological, toxicological, and other research. Furthermore, the antibody or antigen-binding fragment thereof of the present disclosure can effectively activate the NFAT downstream signal pathway of human T cell line Jurkat cells, which is advantageous for exerting subsequent biological activity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dong, D., Zheng, L., Lin, J. et al., Nature 573, 546-552(2019) Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to CD3 and uses thereof. [Means for solving the problem]

[0008] In a first aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds to CD3, wherein the heavy chain comprises CDR1, CDR2 and CDR3, (a) the heavy chain CDR1 comprises a sequence having at least 80% identity to NYYMH (SEQ ID NO:1), DNYIH (SEQ ID NO:2), DYTVN (SEQ ID NO:3), or DYTIH (SEQ ID NO:4); (b) the heavy chain CDR2 comprises a sequence having at least 80% identity to WTYPGNNNIKYNEKFKG (SEQ ID NO: 18), WIYPGSVNIKYNEKFKD (SEQ ID NO: 6), or YINPFNSYTKYNQKFKD (SEQ ID NO: 22), and (c) the heavy chain CDR3 comprises a sequence having at least 80% identity to DGYGYYFFDY (SEQ ID NO: 8), DISRYYFDY (SEQ ID NO: 9), SVSTY (SEQ ID NO: 10), or SVSIY (SEQ ID NO: 11).

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain CDR1 comprising a sequence selected from NYYMH (SEQ ID NO: 1), DNYIH (SEQ ID NO: 2), DYTVN (SEQ ID NO: 3), DYTIH (SEQ ID NO: 4), or a sequence in which one amino acid is added, substituted, and / or deleted compared to any of SEQ ID NOs: 1 to 4; (b) WTYPGX 1 X 2 X 3 IKYNEKFKG (SEQ ID NO: 5), WIYPGSVNIKYNEKFKD (SEQ ID NO: 6), or a sequence having a total of 3 or less different amino acids added, substituted, and / or deleted from SEQ ID NO: 6, or YINPFX 4 X 5 YTKYNQKFKD (SEQ ID NO: 7), wherein X 1 , X 2 and X 3 are each independently N or Q, 4 is N or S, and X 5 a heavy chain CDR2 which is S or D; (c) a heavy chain CDR3 comprising a sequence selected from DGYGYYFFDY (SEQ ID NO: 8), DISRYYFDY (SEQ ID NO: 9), SVSTY (SEQ ID NO: 10), SVSIY (SEQ ID NO: 11), or a sequence in which a total of two or less amino acids different from any of SEQ ID NOs: 8 to 11 have been added, substituted, and / or deleted; (d)KSSQSLLNX 6 RTRKNYLA (SEQ ID NO: 12) or KSSQSLLDX 7 DX8 KTYLN (SEQ ID NO: 13), wherein X 6 is N, Q or S, and X 7 is S, A or G, and X 8 a light chain CDR1 that is G or A; (e) a light chain CDR2 comprising a sequence selected from WASTRES (SEQ ID NO: 14) or LVSKLNS (SEQ ID NO: 15); and (f)KQSX 9 X 10 LRT (SEQ ID NO: 16) or WQGTHFPRT (SEQ ID NO: 17), wherein X 9 is Y or F, and X 10 and a light chain CDR3 that is T or I.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1) and WTYPGX 1 X 2 X 3 IKYNEKFKG (SEQ ID NO: 5), 1 , X 2 and X 3 a heavy chain CDR2, each independently N or Q; a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8); and KSSQSLLNX 6 RTRKNYLA (SEQ ID NO: 12), 6 comprises a light chain CDR1 which is N or Q, a light chain CDR2 which comprises, or consists of, WASTRES (SEQ ID NO: 14), and a light chain CDR3 which comprises, or consists of, KQSYTLRT (SEQ ID NO: 31); or (2) a heavy chain CDR1 comprising or consisting of DNYIH (SEQ ID NO: 2), a heavy chain CDR2 comprising or consisting of WIYPGSVNIKYNEKFKD (SEQ ID NO: 6), a heavy chain CDR3 comprising or consisting of DISRYYFDY (SEQ ID NO: 9), a light chain CDR1 comprising or consisting of KSSQSLLNSRTRKNYLA (SEQ ID NO: 26), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSFILRT (SEQ ID NO: 32); or (3) A heavy chain CDR1 comprising or consisting of DYTVN (SEQ ID NO: 3), a heavy chain CDR2 comprising or consisting of YINPFNSYTKYNQKFKD (SEQ ID NO: 22), a heavy chain CDR3 comprising or consisting of SVSTY (SEQ ID NO: 10), and KSSQSLLDX 7 DX 8 KTYLN (SEQ ID NO: 13), 7 is S and X 8 comprises a light chain CDR1 which is G or A, a light chain CDR2 which comprises, or consists of, LVSKLNS (SEQ ID NO: 15), and a light chain CDR3 which comprises, or consists of, WQGTHFPRT (SEQ ID NO: 17); or (4) A heavy chain CDR1 comprising or consisting of DYTIH (SEQ ID NO: 4), a heavy chain CDR2 comprising or consisting of YINPFSDYTKYNQKFKD (SEQ ID NO: 23), a heavy chain CDR3 comprising or consisting of SVSIY (SEQ ID NO: 11), and KSSQSLLDX 7 DX 8 KTYLN (SEQ ID NO: 13), 7 is G or A, and X 8 comprises a light chain CDR1 which is G, a light chain CDR2 which comprises, or consists of, LVSKLNS (SEQ ID NO: 15), and a light chain CDR3 which comprises, or consists of, WQGTHFPRT (SEQ ID NO: 17).

[0011] In some embodiments, the heavy chain CDR2 comprises a sequence selected from WTYPGNNNIKYNEKFKG (SEQ ID NO: 18), WTYPGQNNIKYNEKFKG (SEQ ID NO: 19), WTYPGNQNIKYNEKFKG (SEQ ID NO: 20), WTYPGQNQIKYNEKFKG (SEQ ID NO: 21), WIYPGSVNIKYNEKFKD (SEQ ID NO: 6), YINPFNSYTKYNQKFKD (SEQ ID NO: 22), and YINPFSDYTKYNQKFKD (SEQ ID NO: 23). In some embodiments, the light chain CDR1 comprises a sequence selected from KSSQSLLNNRTRKNYLA (SEQ ID NO:24), KSSQSLLNQRTRKNYLA (SEQ ID NO:25), KSSQSLLNSRTRKNYLA (SEQ ID NO:26), KSSQSLLDSDGKTYLN (SEQ ID NO:27), KSSQSLLDSDAKTYLN (SEQ ID NO:28), KSSQSLLDGDGKTYLN (SEQ ID NO:29), and KSSQSLLDADGKTYLN (SEQ ID NO:30). In some embodiments, the light chain CDR3 comprises a sequence selected from KQSYTLRT (SEQ ID NO:31), KQSFILRT (SEQ ID NO:32), or WQGTHFPRT (SEQ ID NO:17).

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) A heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1), a heavy chain CDR2 comprising or consisting of WTYPGNNNIKYNEKFKG (SEQ ID NO: 18), a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8), a light chain CDR1 comprising or consisting of KSSQSLLNNRTRKNYLA (SEQ ID NO: 24), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (2) a heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1); a heavy chain CDR2 comprising or consisting of WTYPGNNNIKYNEKFKG (SEQ ID NO: 18); a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8); a light chain CDR1 comprising or consisting of KSSQSLLNQRTRKNYLA (SEQ ID NO: 25); a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14); and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (3) A heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1), a heavy chain CDR2 comprising or consisting of WTYPGQNNIKYNEKFKG (SEQ ID NO: 19), a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8), a light chain CDR1 comprising or consisting of KSSQSLLNNRTRKNYLA (SEQ ID NO: 24), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (4) A heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1), a heavy chain CDR2 comprising or consisting of WTYPGQNNIKYNEKFKG (SEQ ID NO: 19), a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8), a light chain CDR1 comprising or consisting of KSSQSLLNQRTRKNYLA (SEQ ID NO: 25), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (5) A heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1), a heavy chain CDR2 comprising or consisting of WTYPGNQNIKYNEKFKG (SEQ ID NO: 20), a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8), a light chain CDR1 comprising or consisting of KSSQSLLNNRTRKNYLA (SEQ ID NO: 24), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (6) A heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1), a heavy chain CDR2 comprising or consisting of WTYPGNQNIKYNEKFKG (SEQ ID NO: 20), a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8), a light chain CDR1 comprising or consisting of KSSQSLLNQRTRKNYLA (SEQ ID NO: 25), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (7) A heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1), a heavy chain CDR2 comprising or consisting of WTYPGQNQIKYNEKFKG (SEQ ID NO: 21), a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8), a light chain CDR1 comprising or consisting of KSSQSLLNNRTRKNYLA (SEQ ID NO: 24), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (8) A heavy chain CDR1 comprising or consisting of NYYMH (SEQ ID NO: 1), a heavy chain CDR2 comprising or consisting of WTYPGQNQIKYNEKFKG (SEQ ID NO: 21), a heavy chain CDR3 comprising or consisting of DGYGYYFFDY (SEQ ID NO: 8), a light chain CDR1 comprising or consisting of KSSQSLLNQRTRKNYLA (SEQ ID NO: 25), a light chain CDR2 comprising or consisting of WASTRES (SEQ ID NO: 14), and a light chain CDR3 comprising or consisting of KQSYTLRT (SEQ ID NO: 31); or (9) A heavy chain CDR1 comprising or consisting of DYTVN (SEQ ID NO: 3), a heavy chain CDR2 comprising or consisting of YINPFNSYTKYNQKFKD (SEQ ID NO: 22), a heavy chain CDR3 comprising or consisting of SVSTY (SEQ ID NO: 10), a light chain CDR1 comprising or consisting of KSSQSLLDSDGKTYLN (SEQ ID NO: 27), a light chain CDR2 comprising or consisting of LVSKLNS (SEQ ID NO: 15), and a light chain CDR3 comprising or consisting of WQGTHFPRT (SEQ ID NO: 17), or (10) A heavy chain CDR1 comprising or consisting of DYTVN (SEQ ID NO: 3), a heavy chain CDR2 comprising or consisting of YINPFNSYTKYNQKFKD (SEQ ID NO: 22), a heavy chain CDR3 comprising or consisting of SVSTY (SEQ ID NO: 10), a light chain CDR1 comprising or consisting of KSSQSLLDSDAKTYLN (SEQ ID NO: 28), a light chain CDR2 comprising or consisting of LVSKLNS (SEQ ID NO: 15), and a light chain CDR3 comprising or consisting of WQGTHFPRT (SEQ ID NO: 17), or (11) A heavy chain CDR1 comprising or consisting of DYTIH (SEQ ID NO: 4), a heavy chain CDR2 comprising or consisting of YINPFSDYTKYNQKFKD (SEQ ID NO: 23), a heavy chain CDR3 comprising or consisting of SVSIY (SEQ ID NO: 11), a light chain CDR1 comprising or consisting of KSSQSLLDADGKTYLN (SEQ ID NO: 30), a light chain CDR2 comprising or consisting of LVSKLNS (SEQ ID NO: 15), and a light chain CDR3 comprising or consisting of WQGTHFPRT (SEQ ID NO: 17), or (12) A heavy chain CDR1 comprising or consisting of DYTIH (SEQ ID NO: 4), a heavy chain CDR2 comprising or consisting of YINPFSDYTKYNQKFKD (SEQ ID NO: 23), a heavy chain CDR3 comprising or consisting of SVSIY (SEQ ID NO: 11), a light chain CDR1 comprising or consisting of KSSQSLLDGDGKTYLN (SEQ ID NO: 29), a light chain CDR2 comprising or consisting of LVSKLNS (SEQ ID NO: 15), and a light chain CDR3 comprising or consisting of WQGTHFPRT (SEQ ID NO: 17).

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises: QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVRQATGQGLEWMGWTYPGNNNIKYNEKFKGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARDGYGYYFFDYWGQGTLVTVSS (SEQ ID NO: 33), QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVRQATGQGLEWMGWTYPGQNNIKYNEKFKGRVTMTRNTSSSTAYMELSSLRSEDTAVYYCARDGYGYYFFDYWGQGTLVTVSS (SEQ ID NO: 34), QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVRQATGQGLEWMGWTYPGNQNIKYNEKFKGKVTMTRNTSISTAYMELSSLRSEDSAVYYCARDGYGYYFFDYWGQGTIVTVSS (SEQ ID NO: 35), QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVRQATGQGLEWMGWTYPGQNQIKYNEKFKGKVTMTRNTSSSTAYMELSSLRSEDSAVYYCARDGYGYYFFDYWGQGILVTVSS (SEQ ID NO: 36), QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVRQAPGQRLEWMGWTYPGNNNIKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDGYGYYFFDYWGQGTLVTVSS (SEQ ID NO: 37), QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVRQAPGQRLEWIGWTYPGNNNIKYNEKFKGRVTITADTSASTAYMELSSLRSEDTAVYYCARDGYGYYFFDYWGQGTLVTVSS (SEQ ID NO: 38), QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVRQAPGQRLEWIGWTYPGNNNIKYNEKFKGRATITADTSASTAYMELSSLRSEDTAVYYCARDGYGYYFFDYWGQGTLVTVSS (SEQ ID NO: 39), QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYMHWVKQAPGQRLEWIGWTYPGNNNIKYNEKFKGRATITADTSASTAYMELSSLRSEDTAVYYCARDGYGYYFFDYWGQGTLVTVSS (SEQ ID NO: 40), QVQLVQSGAEVKKPGASVKVSCKASGYTFTDNYIHWVRQATGQGLEWMGWIYPGSVNIKYNEKFKDRVTMTRNTSISTAYMELSSLRSEDTAVYYCARDISRYYFDYWGQGTLVTVSS (SEQ ID NO: 41), QVQLVQSGAEVKKPGASVKVSCKASGSTFTDNYIHWVRQATGQGLEWMGWIYPGSVNIKYNEKFKDKVTLTRNTSISTAYMELSSLRSEDSAVYYCARDISRYYFDYWGQGTLVTVSS (SEQ ID NO: 42), QVQLVQSGAEVVKPGASVKVSCKASGYTFTDNYIHWVRQRTGQGLEWMGWIYPGSVNIKYNEKFKDRVTMTADTSISTAYMQLSSLSSEDTAVYYCARDISRYYFDYWGQGTLVTVSS (SEQ ID NO: 43), QVQLVQSGAELVKPGASVKVSCKASGSTFTDNYIHWVRQRTGQGLEWMGWIYPGSVNIKYNEKFKDRVTLTADTSITTAYMQLSSLSSEDTAIYYCARDISRYYFDYWGQGTLVTVSS (SEQ ID NO: 44), QVQLVQSGAEVKKPGASVKVSCKASGSTFTDNYIHWVRQAPGQRLEWMGWIYPGSVNIKYNEKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISRYYFDYWGQGTTVTVSS (SEQ ID NO: 45), QVQLVQSGAEVKKPGASVKVSCKASGSTFTDNYIHWVRQAPGQRLEWIGWIYPGSVNIKYNEKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARDISRYYFDYWGQGTTVTVSS (SEQ ID NO: 46), QVQLVQSGAEVKKPGASVKVSCKASGSTFTDNYIHWVRQAPGQRLEWIGWIYPGSVNIKYNEKFKDRATITADTSASTAYMELSSLRSEDTAVYYCARDISRYYFDYWGQGTTVTVSS (SEQ ID NO: 47), QVQLVQSGAEVKKPGASVKVSCKASGSTFTDNYIHWVKQAPGQRLEWIGWIYPGSVNIKYNEKFKDRATITADTSATTAYMELSSLRSEDTAVYYCARDISRYYFDYWGQGTTVTVSS (SEQ ID NO: 48), QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYTVNWVRQAPGQGLEWMGYINPFNSYTKYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCANSVSTYWGQGTLVTVSS (SEQ ID NO: 49), QVQLVQSGAEVAKPGSSVKVSCKASGYTFTDYTVNWVRQRPGQGLEWMGYINPFNSYTKYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCANSVSTYWGQGTLVTVSS (SEQ ID NO:50), QVQLVQSGAEVAKPGSSVKVSCKASGYTFTDYTVNWVRQRPGQGLEWMGYINPFNSYTKYNQKFKDRVTITADESTSTAYMELSSLTSEDTAVYYCANSVSTYWGQGTLVTVSS (SEQ ID NO:51), QVQLVQSGAEVAKPGSSVKVSCKTSGYTFTDYTVNWVRQRPGQGLEWMGYINPFNSYTKYNQKFKDRVTLTADESTSTAYMELSSLTSEDTAVYYCANSVSTYWGQGTLVTVSS (SEQ ID NO:52), QVQLVQSGAEVAKPGSSVKVSCKTSGYTFTDYTVNWVRQRPGQGLEWMGYINPFNSYTKYNQKFKDRVTLTADKSTSTAYMQLSSLTSEDTAVYYCANSVSTYWGQGTLVTVSS (SEQ ID NO:53), QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTVNWVRQAPGQRLEWMGYINPFNSYTKYNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARSVSTYWGQGTTVTVSS (SEQ ID NO:54), QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTVNWVRQAPGQRLEWIGYINPFNSYTKYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCANSVSTYWGQGTTVTVSS (SEQ ID NO:55), QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTVNWVRQAPGQRLEWIGYINPFNSYTKYNQKFKDRATITADTSASTAYMELSSLRSEDTAVYFCANSVSTYWGQGTTVTVSS (SEQ ID NO:56), QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTVNWVKQAPGQRLEWIGYINPFNSYTKYNQKFKDRATITADTSASTAYMELSSLRSEDTAVYFCANSVSTYWGQGTTVTVSS (SEQ ID NO: 57), or a sequence having at least 85% identity to the above sequence.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises: DIQMTQSPSSLSASVGDRVTITCKSSQSLLNNRTRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYTLRTFGGGTKVEIK (SEQ ID NO:58), DIVMTQSPSSLSASVGDRVTITCKSSQSLLNQRTRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYTLRTFGGGTKVEIK (SEQ ID NO:59), DIQMTQSPSSLSASVGDRVTMTCKSSQSLLNNRTRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYTLRTFGGGTKVEIK (SEQ ID NO: 60), DIVMTQSPSSLSASVGDRVTMTCKSSQSLLNQRTRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFTGSGSGTDFTLTISSLQPEDFATYYCKQSYTLRTFGGGTKVEIK (SEQ ID NO:61), DIVMTQSPDSLAVSLGERATINCKSSQSLLNNRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYTLRTFGGGTKLEIK (SEQ ID NO:62), DIVMTQSPDSLAVSLGERATINCKSSQSLLNNRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCKQSYTLRTFGGGTKLEIK (SEQ ID NO:63), DIVMTQSPDSLAVSLGERATMSCKSSQSLLNNRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYTLRTFGGGTKLEIK (SEQ ID NO:64), DIVMTQSPDSLAVSLGERATMSCKSSQSLLNNRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCKQSYTLRTFGGGTKLEIK (SEQ ID NO:65), DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSFILRTFGGGTKVEIK (SEQ ID NO:66), DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCKQSFILRTFGGGTKVAIK (SEQ ID NO: 67), DIQMSQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDFATYYCKQSFILRTFGGGTKVAIK (SEQ ID NO: 68), DIVMSQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDFATYYCKQSFILRTFGGGTKLAIK (SEQ ID NO:69), DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGGGTKLEIK (SEQ ID NO: 70), DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGGGTKLEIK (SEQ ID NO:71), DIVMTQSPDSLAVSLGERATMNCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGGGTKLEIK (SEQ ID NO:72), DIVMTQSPDSLAVSLGERATMNCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGGGTKLEIK (SEQ ID NO:73), DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLNSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKVEIK (SEQ ID NO:74), DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKLNSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKVEIK (SEQ ID NO: 75), DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIQLVSKLNSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKVEIK (SEQ ID NO:76), DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLNSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO:77), DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIQLVSKLNSGVPDRFSGSGSGTDFTLKISRVEAEDVGVFYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO:78), DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIQLVSKLNSGVPDRFSGSGSGTDFTLKISRVEAEDVGVFYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO:79), DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIQLVSKLNSGVPDRFSGSGSGTDFTLKISRVEAEDVGVFYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 80), DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDAKTYLNWLLQRPGQSPKRLIQLVSKLNSGVPDRFTGSGSGTDFTLKISRVEAEDLGIFYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO:81), DVVMTQTPLTLSVTIGQPASISCKSSQSLLDADGKTYLNWLLQRPGQSPKRLIQLVSKLNSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 82), or a sequence having at least 85% identity to the foregoing sequence.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following group of heavy chain variable regions and light chain variable regions: (1) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61; or (2) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65; or (3) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69; or (4) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73; or (5) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76; or (6) A heavy chain variable region having an amino acid sequence selected from SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57, and a light chain variable region having an amino acid sequence selected from SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of any one of the following (1) to (18): (1) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 38 and a light chain variable region whose amino acid sequence is SEQ ID NO: 62; or (2) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 38 and a light chain variable region whose amino acid sequence is SEQ ID NO: 63; or (3) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 38 and a light chain variable region whose amino acid sequence is SEQ ID NO: 64; or (4) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 39 and a light chain variable region whose amino acid sequence is SEQ ID NO: 63; or (5) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 39 and a light chain variable region whose amino acid sequence is SEQ ID NO: 64; or (6) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 39 and a light chain variable region whose amino acid sequence is SEQ ID NO: 65; or (7) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 46 and a light chain variable region whose amino acid sequence is SEQ ID NO: 71; or (8) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 46 and a light chain variable region whose amino acid sequence is SEQ ID NO: 72; or (9) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 47 and a light chain variable region whose amino acid sequence is SEQ ID NO: 70; or (10) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 47 and a light chain variable region whose amino acid sequence is SEQ ID NO: 72; or (11) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 48 and a light chain variable region whose amino acid sequence is SEQ ID NO: 70; or (12) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 48 and a light chain variable region whose amino acid sequence is SEQ ID NO: 71; or (13) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 50 and a light chain variable region whose amino acid sequence is SEQ ID NO: 76; or (14) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 55 and a light chain variable region whose amino acid sequence is SEQ ID NO: 79; or (15) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 55 and a light chain variable region whose amino acid sequence is SEQ ID NO: 80; or (16) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 56 and a light chain variable region whose amino acid sequence is SEQ ID NO: 79; or (17) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 56 and a light chain variable region whose amino acid sequence is SEQ ID NO: 80; or (18) The antibody comprises a heavy chain variable region whose amino acid sequence is SEQ ID NO:57, and a light chain variable region whose amino acid sequence is SEQ ID NO:79.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a single chain antibody, a bibody, a triple chain antibody, a quadruple chain antibody, a Fab fragment, a F(ab') 2 fragment, scFv fragment, Fv fragment, Fab' fragment, domain antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an IgA antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof binds to human CD3 and / or cynomolgus CD3.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof binds to CD3e / d and / or CD3e / g.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof (i) significantly activates human total T cells, upregulates CD25 expression, and / or induces IFN-γ release, and / or (ii) inhibits over 4×10 -8 K less than M D It binds to human CD3 at 100 ng / s.

[0021] In a second aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that competes with or cross-blocks binding to CD3, in particular CD3e / d or CD3e / g, with the antibody or antigen-binding fragment according to the first aspect.

[0022] In a third aspect, the present disclosure relates to an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to the first or second aspect.

[0023] In a fourth aspect, the present disclosure relates to a nucleic acid carrier (vector) comprising the isolated nucleic acid according to the third aspect.

[0024] In a fifth aspect, the present disclosure relates to a host cell comprising the isolated nucleic acid according to the third aspect or the nucleic acid vector according to the fourth aspect.

[0025] In a sixth aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first or second aspect, an isolated nucleic acid according to the third aspect or a nucleic acid vector according to the fourth aspect, and a pharma- ceutically acceptable carrier.

[0026] In a seventh aspect, the present disclosure relates to a method for producing an antibody or antigen-binding fragment thereof according to the first or second aspect, comprising incubating a host cell according to the fifth aspect under conditions permissive for expression of said antibody or antigen-binding fragment thereof.

[0027] In an eighth aspect, the present disclosure relates to the use of an antibody or antigen-binding fragment thereof according to any one of the first or second aspect, an isolated nucleic acid according to the third aspect, a nucleic acid vector according to the fourth aspect or a pharmaceutical composition according to the sixth aspect in the preparation of a medicament for inhibiting an immune response or for activating T cells.

[0028] In a ninth aspect, the present disclosure relates to the use of an antibody or antigen-binding fragment thereof according to any one of the first or second aspect, an isolated nucleic acid according to the third aspect, a nucleic acid vector according to the fourth aspect or a pharmaceutical composition according to the sixth aspect in the preparation of a medicament for treating cancer or an autoimmune disease.

[0029] In a tenth aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof according to any one of the first or second aspect, an isolated nucleic acid according to the third aspect, a nucleic acid vector according to the fourth aspect or a pharmaceutical composition according to the sixth aspect for use in inhibiting an immune response or activating T cells or treating cancer or an autoimmune disease.

[0030] In an eleventh aspect, the present disclosure relates to a method of inhibiting an immune response or activating T cells or treating cancer or an autoimmune disease comprising administering to an individual in need thereof an inhibitory, activating or therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of the first or second aspect, the isolated nucleic acid according to the third aspect, the nucleic acid vector according to the fourth aspect or the pharmaceutical composition according to the sixth aspect.

[0031] In some embodiments, the cancer is melanoma (e.g., metastatic malignant melanoma), kidney cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, colon cancer, endocrine system cancer, thyroid cancer, cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), bladder cancer, renal pelvis cancer, central nervous system tumor, glioma, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and combinations thereof.

[0032] In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, insulin-dependent diabetes mellitus, autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, Wegener's granulomatosis, Crohn's disease, ulcerative colitis, lupus such as systemic lupus erythematosus, atherosclerosis, chronic obstructive pulmonary disease, liver cirrhosis, renal transplant fibrosis, renal transplant nephropathy, pulmonary fibrosis, and combinations thereof.

[0033] These and other aspects will be described in more detail in the present disclosure.Each of the above aspects provided may include multiple embodiments provided herein.Therefore, it is anticipated that all embodiments relating to one element or combination of elements may be included in each aspect described, and all such combinations of the above aspects and embodiments are expressly contemplated. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 shows the results of serum titers against immunogens in mouse serum detected by ELISA. [Diagram 2] FIG. 1 shows the results of binding between CD3 antibodies and human CD3e / g detected by ELISA. [Diagram 3] FIG. 1 shows the results of binding between CD3 antibodies and cynomolgus monkey CD3e / g detected by ELISA. [Figure 4] FIG. 1 shows the results of binding between CD3 antibodies and human CD3e / d detected by ELISA. [Figure 5(a)] FIG. 1 shows the results of binding between CD3 antibody and Jurkat cells detected by FACS. [Figure 5(b)] FIG. 1 shows the results of binding between CD3 antibody and Jurkat cells detected by FACS. [Figure 5(c)] FIG. 1 shows the results of binding between CD3 antibody and Jurkat cells detected by FACS. [Figure 5(d)] FIG. 1 shows the results of binding between CD3 antibody and Jurkat cells detected by FACS. [Figure 5(e)] FIG. 1 shows the results of binding between CD3 antibody and Jurkat cells detected by FACS. [Figure 6(a)] FIG. 1 shows the effect of CD3 antibody on CD25 expression detected by FACS. [Figure 6(b)] FIG. 1 shows the effect of CD3 antibody on CD25 expression detected by FACS. [Figure 6(c)]FIG. 1 shows the effect of CD3 antibody on CD25 expression detected by FACS. [Figure 6(d)] FIG. 1 shows the effect of CD3 antibody on CD25 expression detected by FACS. [Figure 6(e)] FIG. 1 shows the effect of CD3 antibody on CD25 expression detected by FACS. [Figure 6(f)] FIG. 1 shows the effect of CD3 antibody on CD25 expression detected by FACS. [Figure 6(g)] FIG. 1 shows the effect of CD3 antibodies on CD25 expression detected by FACS. [Figure 6(h)] FIG. 1 shows the effect of CD3 antibody on CD25 expression detected by FACS. [Figure 7(a)] FIG. 1 shows the effect of CD3 antibody on IFN-γ secretion detected by FACS. [Figure 7(b)] FIG. 1 shows the effect of CD3 antibody on IFN-γ secretion detected by FACS. [Figure 7(c)] FIG. 1 shows the effect of CD3 antibody on IFN-γ secretion detected by FACS. [Figure 7(d)] FIG. 1 shows the effect of CD3 antibody on IFN-γ secretion detected by FACS. [Figure 7(e)] FIG. 1 shows the effect of CD3 antibody on IFN-γ secretion detected by FACS. [Figure 7(f)] FIG. 1 shows the effect of CD3 antibody on IFN-γ secretion detected by FACS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art, and unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0036] In general, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present application are generally carried out according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. References include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to manufacturer's instructions as commonly accomplished in the art or as described herein. Generally, the nomenclature used in connection with, and the laboratory methods and techniques of, analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, compounding and delivery, and treatment of patients.

[0037] It should be understood that this disclosure is not limited to the particular methodology, approaches, and reagents described herein, as such may vary, and the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosure.

[0038] Unless otherwise indicated in or within the operating examples, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as being modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±5%, e.g., 1%, 2%, 3%, or 4%.

[0039] definition The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the present invention. Unless the context clearly indicates otherwise, the open phrases "comprise" and "contain" are to be interpreted as meaning that unmentioned components or method steps may be included, but the open phrases also cover cases consisting only of the recited components and method steps (i.e., cover the closed phrase "consisting of").

[0040] As used throughout, ranges are used as a shorthand for describing each and every value within a range. Any value within the range, particularly integer values, can be selected as the end points of the range. For example, the range "at least 80% identity" is used to describe all values ​​within the range, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, etc., and includes all subranges, such as at least 85%, at least 90%, at least 95%, etc.

[0041] As used herein, "antibody" refers to monomers and multimers. Intact antibodies (including multimers) or antibody fragments carrying antigen-binding fragments of the antibody can be used. The antibody may be human, humanized, chimeric, monoclonal, polyclonal, or recombinant. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0042] "Fab fragment" is V L , V H , C L and C H A monovalent fragment having the F(ab') 2 The "Fab fragment" is a bivalent fragment having two Fab fragments linked by a disulfide bond at the hinge region.

[0043] Single chain antibodies (scFv) have the following V L Area and V H A diabody is a bivalent antibody comprising two polypeptide chains, each of which comprises a V domain connected by a linker, e.g., a synthetic sequence of amino acid residues, to form a continuous protein chain, said linker being long enough to allow the protein chain to fold back on itself to form a monovalent antigen-binding site (see, e.g., Bird et al., Science 242:423-26 (1988), and Huston et al., 1988, Proc Natl Acad Sci USA 85:5879-83 (1988)). A diabody is a bivalent antibody comprising two polypeptide chains, each of which comprises a V domain connected by a linker. H and V L Domains are included and the linker is too short to allow pairing between the two domains on the same chain, so that each domain can pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc Natl Acad Sci USA 90:6444-48(1993), and Poljaket al., Structure 2:1121-23(1994)). If the two polypeptide chains of a bibody are identical, the bibody produced by their pairing has two identical antigen-binding sites. Polypeptide chains with different sequences can be used to produce bibodies (diabodies) with two different antigen-binding sites. Similarly, tribodies (or "three-chain antibodies") and tetrabodies (or "four-chain antibodies") are antibodies that contain three and four polypeptide chains, respectively, which may be the same or different, forming three and four antigen-binding sites, respectively, which may be the same or different.

[0044] The complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody may use the system identifiers described in Sequences of Proteins of Immunological Interest, 5th ed., by Kabat et al., US Department of Health and Public Service, PHS, NIH, NIH Publication No. 91-3242, 1991. If necessary, the CDRs may be redefined according to alternative naming schemes such as Chothia (see Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883; or Honegger & Pluckthun, 2001, J. Mol. Biol. 309:657-670). One or more CDRs may be incorporated covalently or non-covalently into a molecule to convert it into an antigen-binding protein such as an antibody. An antigen binding protein may incorporate the CDRs as part of a larger polypeptide chain, may covalently link the CDRs to another polypeptide chain, or may incorporate the CDRs non-covalently, which enable the antigen binding protein to specifically bind to a particular antigen of interest.

[0045] The CDR of an antibody can be defined by various methods in the art, and methods and techniques for identifying CDRs in variable regions and antibody variable region amino acid sequences in antibody molecules are also well known in the art and can be used to recognize CDRs in specific antibody variable region amino acid sequences disclosed herein. In this application, the amino acid sequences of the listed CDRs are all shown according to the Kabat definition rules (the sequences in the claims of this application are also shown according to the Kabat definition rules). Those skilled in the art should understand that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or a region thereof (e.g., variable region) include the complementarity determining region defined by any of the above known schemes described by this disclosure. The scope claimed in this disclosure is the sequence shown based on the Kabat definition rules, but the corresponding amino acid sequences according to other CDR definition rules should also be included in the scope of protection of the present invention.

[0046] The "antibody or antigen-binding fragment thereof" described herein may also include an antibody heavy chain constant region and / or an antibody light chain constant region. For example, the antibody heavy chain constant region is a mouse-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and the antibody light chain constant region is a mouse-derived light chain antibody constant region or a human-derived antibody light chain constant region. More preferably, the antibody heavy chain constant region is a human-derived antibody heavy chain constant region, for example, the heavy chain constant region of a human-derived IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 antibody, and the antibody light chain constant region is a human-derived antibody light chain κ or λ chain constant region.

[0047] The term "humanized antibody" includes antibodies having variable and constant regions from human species-based immunoglobulin sequences in which CDR sequences from another species have been grafted onto human framework sequences.

[0048] The term "nucleic acid" includes single- and double-stranded nucleotide polymers. The nucleotides that make up a polynucleotide may be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, selenophosphates, diselenophosphates, thioaniline phosphates, and phosphoramidoesters.

[0049] The term "carrier (vector)" refers to any molecule or entity (eg, nucleic acid, plasmid, phage, or virus) for transferring protein-encoding information to a host cell.

[0050] The term "host cell" refers to a cell that has been transformed with a nucleic acid sequence or that can be transformed with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present. Host cells can be prokaryotic (e.g., E. coli) or eukaryotic (e.g., unicellular eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or hybridomas.

[0051] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of the sequences. In general, identity refers to the number or percentage of identical positions shared by two amino acid or nucleic acid sequences, taking into account the number of gaps and the length of each gap that must be introduced to achieve optimal alignment of the two sequences. When an amino acid sequence is described as being at least 85%, or at least 90%, or at least 95% identical to another amino acid sequence, the amino acid sequence may differ by conservative substitutions, including where all substitutions are conservative substitutions.

[0052] As used herein, a "conservative substitution" is considered in the art to be the substitution of one amino acid for another amino acid having similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO97 / 09433, p. 10, March 13, 1997; Lehninger, Biochemistry, 2nd ed., Worth Publishers, Inc. NY:NY (1975), p. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

[0053] Naturally occurring amino acids can be divided into the following class groups based on common side chain properties: 1) Hydrophobic: Norleucine (Nle), Met, Ala, Val, Leu, Ile; 2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) Acidic: Asp, Glu; 4) Alkaline: His, Lys, Arg; 5) Residues that affect chain orientation: Gly, Pro; and 6) Aromaticity: Trp, Tyr, Phe.

[0054] Conservative substitutions may involve exchanging a member of one of these class groups with another member of the same class group. Conservative amino acid substitutions may involve unnatural amino acid residues, which are usually incorporated by chemical peptide synthesis without recourse to synthesis in living systems. These include peptide analogs and other reverse or inverted forms of amino acid moieties. Non-conservative substitutions may involve exchanging a member of one of the above class groups with a member from another class group. Such replacement residues may be introduced into antibody regions that are homologous to human antibodies or into non-homologous regions of the molecule.

[0055] An "effective amount" is typically an amount sufficient to reduce the severity and / or frequency of a symptom, eliminate a symptom and / or its underlying etiology, prevent the onset of a symptom and / or its underlying etiology, and / or ameliorate or treat damage caused by or associated with a disease (e.g., cancer or an autoimmune disease). In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to ameliorate in some way a disease state (e.g., cancer or an autoimmune disease) or symptom, a specific condition or symptom associated with said disease state, or an amount that prevents, inhibits, slows or reverses the progression of a disease state or any other deleterious symptoms associated with a disease. The therapeutically effective amount of the antibody or antigen-binding protein thereof of the present disclosure used, a nucleic acid encoding said antibody or antigen-binding fragment, a nucleic acid vector comprising said nucleic acid, a host cell comprising said nucleic acid or nucleic acid vector, or a pharmaceutical composition comprising the foregoing will depend, for example, on the context and subject of the treatment. One of skill in the art will appreciate that appropriate dosage levels for treatment will vary in part depending on the molecule being delivered, the indication for the antibody or antigen binding protein thereof being used, the route of administration, and the patient's physical size (weight, body surface area, or organ size) and / or condition (age and general health). In certain embodiments, the clinician can adjust the dosage and tailor the route of administration to achieve the optimal therapeutic effect.

[0056] The terms "subject," "patient," and "individual" may be used interchangeably herein and refer to warm-blooded animals, such as mammals. This term includes, but is not limited to, domestic animals, rodents (e.g., rats and mice), primates, and humans. Preferably, this term refers to humans.

[0057] As used herein, "EC 50 The term "half-maximal" refers to the concentration that produces 50% of the maximal effect.

[0058] The terms "CD3 antibody," "anti-CD3 antibody," and "antibody that specifically binds to CD3" can be used interchangeably and refer to an antibody that specifically binds to human or other mammalian CD3. In some embodiments, the antibody is at least 1×10 -4 Less than 1×10 -5 Less than 1×10 -6 Less than 1×10 -7 Less than 1×10 -8 Less than 1×10 -9 Less than 1×10 -10 Less than or 1×10 -11 K under M D The term "CD3e / g antibody" refers to an antibody that can specifically bind to the CD3e / g subunit of a human or other mammal. In some embodiments, the CD3e / g antigen has a sequence as set forth in SEQ ID NO: 83 and / or SEQ ID NO: 84. In some embodiments, the antibody is capable of specifically binding to the CD3 antigen at a concentration of 1×10 -4 Less than 1×10 -5 Less than 1×10 -6 Less than 1×10 -7 Less than 1×10 -8 Less than 1×10 -9 Less than 1×10 -10 Less than or 1×10 -11 K under M D The term "CD3e / d antibody" refers to an antibody that can specifically bind to the CD3e / d subunit of a human or other mammal. In some embodiments, the antibody has a specific binding affinity of 1×10-4 Less than 1×10 -5 Less than 1×10 -6 Less than 1×10 -7 Less than 1×10 -8 Less than 1×10 -9 Less than 1×10 -10 Less than or 1×10 -11 K under M D In some preferred embodiments, the antibody is capable of specifically binding to the CD3e / d antigen at a concentration of 1×10 -6 Less than 1×10 -7 Less than 1×10 -8 Less than 1×10 -9 Less than 1×10 -10 Less than or 1×10 -11 K under M D The antibody can specifically bind to the CD3e / g antigen and / or the CD3e / d antigen at a specific value.

[0059] "K D The term "equilibrium dissociation constant" is a commonly used parameter to characterize the affinity of an antibody. Its calculation formula is K D = [antibody][antigen] / [antibody-antigen], which indicates the degree of dissociation of the antibody-antigen complex at equilibrium, K D The larger the K, the more dissociation occurs, and the weaker the affinity between the antibody and the antigen. D The smaller the dissociation, the stronger the affinity between the antibody and the antigen. In some preferred embodiments, the antibody has a molecular weight of 4×10 -8 K under M D It binds to human CD3 at 100 ng / s.

[0060] As used herein, in phrases such as "significantly activates all T cells, upregulates expression of CD25, and / or induces release of IFN-γ," "significantly" means that at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells are activated, or the amount that upregulates expression of CD25 is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about It can mean about 90%, or at least about 95%, or that the amount that induces the release of IFN-γ is at least about 5000 pg / mL, about 6000 pg / mL, about 7000 pg / mL, about 8000 pg / mL, about 9000 pg / mL, about 10000 pg / mL, about 11000 pg / mL, about 12000 pg / mL, about 13000 pg / mL, about 14000 pg / mL, about 15000 pg / mL, about 16000 pg / mL, about 17000 pg / mL, about 18000 pg / mL, about 19000 pg / mL, or about 20000 pg / mL. In some embodiments, "significantly" can mean that the amount of total T cell activation, the amount that upregulates CD25 expression, and / or the amount that induces IFN-γ release is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold or more than a negative control, i.e., an isotype control or a blank control. In some preferred embodiments, "significantly" can refer to the amount of total T cell activation, the amount that upregulates CD25 expression, and / or the amount that induces IFN-γ release is at least about 3-fold that of a negative control, i.e., an isotype control or a blank control.

[0061] The term "isotype control" refers to an antibody that maintains similar properties to the test antibody but lacks a specific target. In flow cytometry experiments, the isotype control serves as a negative control to distinguish specific antibody signals from non-specific background staining, and to eliminate interfering factors to ensure the accuracy of experimental results.

[0062] antibody The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to CD3 (e.g., CD3e, particularly CD3e / d and / or CD3e / g). In some embodiments, the CD3 is human CD3 and / or cynomolgus CD3.

[0063] In general, an antibody or antigen-binding fragment thereof of the disclosure will generally comprise one or more CDRs described herein (eg, 1, 2, 3, 4, 5, or 6 CDRs).

[0064] In some embodiments, the heavy chain CDR1 comprises a sequence having at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with any of SEQ ID NOs: 1 to 4, or a sequence having a total of three or less different amino acids (e.g., one, two, and three) added, substituted, and / or deleted therefrom. In some embodiments, the heavy chain CDR2 comprises a sequence having at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with any of SEQ ID NOs: 5 to 7 and 18 to 23, or a sequence having a total of three or less different amino acids (e.g., one, two, and three) added, substituted, and / or deleted therefrom. In some embodiments, the heavy chain CDR3 comprises a sequence having at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to any of SEQ ID NOs: 8 to 11, or a sequence having a total of no more than three (e.g., 1, 2, and 3) different amino acids added, substituted, and / or deleted therefrom.

[0065] In some embodiments, the light chain CDR1 comprises a sequence having at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with any of SEQ ID NOs: 12 to 13 and 24 to 30, or a sequence having a total of three or less different amino acids (e.g., one, two, and three) added, substituted, and / or deleted therefrom. In some embodiments, the light chain CDR2 comprises a sequence having at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with any of SEQ ID NOs: 14 to 15, or a sequence having a total of three or less different amino acids (e.g., one, two, and three) added, substituted, and / or deleted therefrom. In some embodiments, the light chain CDR3 comprises a sequence having at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to any of SEQ ID NOs: 16-17 and 31-32, or a sequence having a total of no more than three (e.g., 1, 2, and 3) different amino acids added, substituted, and / or deleted therefrom.

[0066] In some embodiments, the substitution is a conservative substitution.

[0067] In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region and / or a light chain variable region. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 33-57, or a sequence having at least 85% identity (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to any of the foregoing sequences. In some embodiments, the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 58-82, or a sequence having at least 85% identity (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to any of the foregoing sequences.

[0068] In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:33 and a light chain variable region as set forth in SEQ ID NO:58. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:33 and a light chain variable region as set forth in SEQ ID NO:59. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:33 and a light chain variable region as set forth in SEQ ID NO:60. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:33 and a light chain variable region as set forth in SEQ ID NO:61. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:34 and a light chain variable region as set forth in SEQ ID NO:58. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:34 and a light chain variable region as set forth in SEQ ID NO:59. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:34 and a light chain variable region as set forth in SEQ ID NO:60. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:34 and a light chain variable region as set forth in SEQ ID NO:61. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:35 and a light chain variable region as set forth in SEQ ID NO:58. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:35 and a light chain variable region as set forth in SEQ ID NO:59. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:35 and a light chain variable region as set forth in SEQ ID NO:60. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:35 and a light chain variable region as set forth in SEQ ID NO:61.In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:36 and a light chain variable region as set forth in SEQ ID NO:58. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:36 and a light chain variable region as set forth in SEQ ID NO:59. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:36 and a light chain variable region as set forth in SEQ ID NO:60. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:36 and a light chain variable region as set forth in SEQ ID NO:61. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:37 and a light chain variable region as set forth in SEQ ID NO:62. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:37 and a light chain variable region as set forth in SEQ ID NO:63. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:37 and a light chain variable region as set forth in SEQ ID NO:64. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:37 and a light chain variable region as set forth in SEQ ID NO:65. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:38 and a light chain variable region as set forth in SEQ ID NO:62. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:38 and a light chain variable region as set forth in SEQ ID NO:63. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:38 and a light chain variable region as set forth in SEQ ID NO:64. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:38 and a light chain variable region as set forth in SEQ ID NO:65.In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:39 and a light chain variable region as set forth in SEQ ID NO:62. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:39 and a light chain variable region as set forth in SEQ ID NO:63. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:39 and a light chain variable region as set forth in SEQ ID NO:64. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:39 and a light chain variable region as set forth in SEQ ID NO:65. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:40 and a light chain variable region as set forth in SEQ ID NO:62. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:40 and a light chain variable region as set forth in SEQ ID NO:63. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 40 and a light chain variable region as set forth in SEQ ID NO: 64. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 40 and a light chain variable region as set forth in SEQ ID NO: 65.

[0069] In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:41 and a light chain variable region as set forth in SEQ ID NO:66. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:41 and a light chain variable region as set forth in SEQ ID NO:67. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:41 and a light chain variable region as set forth in SEQ ID NO:68. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:41 and a light chain variable region as set forth in SEQ ID NO:69. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:42 and a light chain variable region as set forth in SEQ ID NO:66. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:42 and a light chain variable region as set forth in SEQ ID NO:67. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:42 and a light chain variable region as set forth in SEQ ID NO:68. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:42 and a light chain variable region as set forth in SEQ ID NO:69. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:43 and a light chain variable region as set forth in SEQ ID NO:66. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:43 and a light chain variable region as set forth in SEQ ID NO:67. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:43 and a light chain variable region as set forth in SEQ ID NO:68. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:43 and a light chain variable region as set forth in SEQ ID NO:69.In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:44 and a light chain variable region as set forth in SEQ ID NO:66. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:44 and a light chain variable region as set forth in SEQ ID NO:67. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:44 and a light chain variable region as set forth in SEQ ID NO:68. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:44 and a light chain variable region as set forth in SEQ ID NO:69.

[0070] In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:70. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:71. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:72. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:73. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:46 and a light chain variable region as set forth in SEQ ID NO:70. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:46 and a light chain variable region as set forth in SEQ ID NO:71. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:46 and a light chain variable region as set forth in SEQ ID NO:72. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:46 and a light chain variable region as set forth in SEQ ID NO:73. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:47 and a light chain variable region as set forth in SEQ ID NO:70. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:47 and a light chain variable region as set forth in SEQ ID NO:71. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:47 and a light chain variable region as set forth in SEQ ID NO:72. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:47 and a light chain variable region as set forth in SEQ ID NO:73.In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:48 and a light chain variable region as set forth in SEQ ID NO:70. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:48 and a light chain variable region as set forth in SEQ ID NO:71. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:48 and a light chain variable region as set forth in SEQ ID NO:72. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:48 and a light chain variable region as set forth in SEQ ID NO:73.

[0071] In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:49 and a light chain variable region as set forth in SEQ ID NO:74. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:49 and a light chain variable region as set forth in SEQ ID NO:75. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:49 and a light chain variable region as set forth in SEQ ID NO:76. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:50 and a light chain variable region as set forth in SEQ ID NO:74. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:50 and a light chain variable region as set forth in SEQ ID NO:75. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:50 and a light chain variable region as set forth in SEQ ID NO:76. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:51 and a light chain variable region as set forth in SEQ ID NO:74. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:51 and a light chain variable region as set forth in SEQ ID NO:75. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:51 and a light chain variable region as set forth in SEQ ID NO:76. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:52 and a light chain variable region as set forth in SEQ ID NO:74. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:52 and a light chain variable region as set forth in SEQ ID NO:75. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:52 and a light chain variable region as set forth in SEQ ID NO:76.In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:53 and a light chain variable region as set forth in SEQ ID NO:74. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:53 and a light chain variable region as set forth in SEQ ID NO:75. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:53 and a light chain variable region as set forth in SEQ ID NO:76.

[0072] In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:54 and a light chain variable region as set forth in SEQ ID NO:77. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:54 and a light chain variable region as set forth in SEQ ID NO:78. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:54 and a light chain variable region as set forth in SEQ ID NO:79. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:54 and a light chain variable region as set forth in SEQ ID NO:80. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:55 and a light chain variable region as set forth in SEQ ID NO:77. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:55 and a light chain variable region as set forth in SEQ ID NO:78. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:55 and a light chain variable region as set forth in SEQ ID NO:79. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:55 and a light chain variable region as set forth in SEQ ID NO:80. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:56 and a light chain variable region as set forth in SEQ ID NO:77. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:56 and a light chain variable region as set forth in SEQ ID NO:78. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:56 and a light chain variable region as set forth in SEQ ID NO:79. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:56 and a light chain variable region as set forth in SEQ ID NO:80.In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:57 and a light chain variable region as set forth in SEQ ID NO:77. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:57 and a light chain variable region as set forth in SEQ ID NO:78. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:57 and a light chain variable region as set forth in SEQ ID NO:79. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:57 and a light chain variable region as set forth in SEQ ID NO:80.

[0073] In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 85 and a light chain variable region as set forth in SEQ ID NO: 82. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 89 and a light chain variable region as set forth in SEQ ID NO: 81.

[0074] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure (i) significantly activate human total T cells, upregulate CD25 expression and / or induce IFN-γ release, and / or (ii) inhibit or inhibit 4×10 -8 Less than (e.g., 1 × 10 -8 Less than 1×10 -9 Less than 1×10 -10 Less than or 1×10 -11 (less than) M's K D The antibody binds to human CD3 (e.g., CD3e, particularly CD3e / d and / or CD3e / g) at low antibody levels.

[0075] Also, antibodies that compete with the antibodies or antigen-binding fragments thereof disclosed herein, or cross-block the binding of the antibodies disclosed herein to CD3, or are themselves cross-blocked by the antibodies disclosed herein, such that they are prevented from binding to CD3, may be used in the present invention. In some cases, these competing, cross-blocking, or cross-blocked antibodies have epitopes that cross and / or overlap with the antibodies or antigen-binding fragments thereof disclosed herein. In some cases, these competing, cross-blocking, or cross-blocked antibodies have the same epitope as the antibodies or antigen-binding fragments thereof disclosed herein.

[0076] Competing antibodies, cross-blocking antibodies, and cross-blocked antibodies can be identified using any suitable method known in the art, including competitive ELISA or BIACORE® assays, where the binding of the competing or cross-blocking antibody to human CD3 inhibits the binding of an antibody disclosed herein, or vice versa.

[0077] In some embodiments, the competing or cross-blocking antibody is competed or cross-blocked to greater than 80%, greater than 85%, greater than 90%, or greater than 95%. In some embodiments, the competing, cross-blocking, or cross-blocking antibody is a chimeric antibody, a fully human antibody, or a humanized antibody.

[0078] The antibodies or antigen-binding fragments thereof disclosed herein can also be used to construct chimeric antigen receptors or genetically modified cells, the chimeric antigen receptors comprising the antibodies or antigen-binding fragments thereof disclosed herein above. The genetically modified cells are preferably eukaryotic cells, more preferably isolated human cells, and even more preferably immune cells such as T cells, or NK cells, such as the NK92 cell line.

[0079] The term "chimeric antigen receptor" or "CAR" is meant to include an extracellular domain capable of binding to an antigen (extracellular binding domain), a hinge domain, a transmembrane domain, and a polypeptide that transmits a cytoplasmic signal to the domain (i.e., an intracellular signal domain). The hinge domain is believed to be a part of the extracellular antigen binding region to provide flexibility. An intracellular signal domain refers to one that generates a signal that can promote immune effector function of the cell of the CAR (e.g., a CART cell) through a specific signaling pathway, by a protein that generates a second messenger to transmit information intracellularly to regulate cellular activity, or by a protein that functions as an effector in response to such a messenger. Intracellular signal domains include signaling domains and can also include costimulatory intracellular domains derived from costimulatory molecules.

[0080] The term "immune cell" refers to a cell capable of eliciting an immune response, and "immune cell" and other grammatical variations thereof can refer to immune cells of any origin. "Immune cells" include, for example, leukocytes (white cells), lymphocytes (T cells, B cells, natural killer (NK) cells), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) derived from hematopoietic stem cells (HSCs) produced in the bone marrow. The term "immune cell" may be human or non-human. For example, immune cells may be derived from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, and allogeneic NK cells, or may be derived from cell lines, such as NK cell lines prepared by EBV virus infection, NK cells induced to differentiate from embryonic stem cells or iPSCs, and NK92 cell lines.

[0081] The antibodies or antigen-binding fragments thereof disclosed herein can also be used to construct an antibody drug conjugate comprising a cytotoxic agent and an antibody or antigen-binding fragment thereof of the present disclosure described above. In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent, and the antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic drug or a cell proliferation inhibitor (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, maytansinoid alkaloids (such as DM-1 and DM-4), diketones, serine, mitomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, purinomicin, epirubicin, cyclophosphamide, and analogs thereof).

[0082] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the present disclosure. These pharmaceutical compositions comprise a therapeutically effective amount of the antibody or antigen-binding fragment thereof and one or more additional components, such as a physiologically acceptable carrier, excipient, or diluent. In some embodiments, these additional components may include buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and / or preservatives, etc.

[0083] The pharmaceutical composition may also contain, as active ingredients, a therapeutically effective amount of an antibody or an antigen-binding fragment thereof and one or more other therapeutic agents. In combination therapy, the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned antibody-drug conjugate, etc., and / or additional therapeutic or diagnostic agents may each be administered as a single agent within any time range suitable for carrying out the intended treatment or diagnosis. Thus, these single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially in sequence.

[0084] In some embodiments, the other therapeutic agent may include one or more inhibitors selected from B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3 kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the other therapeutic agent is an indoleamine-2,3-dioxygenase-1 (IDO1) inhibitor (e.g., Epacadostat).

[0085] In some embodiments, the other therapeutic agent may include one or more inhibitors selected from the group consisting of a HER3 inhibitor, an LSD1 inhibitor, an MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an inhibitor of the activated hedgehog signaling pathway, and a drug that selectively degrades the estrogen receptor.

[0086] In some embodiments, the other therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, pemetrexed disodium (Almira), ceritinib, sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IM The therapeutic agents may include one or more therapeutic agents selected from A-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0087] In some embodiments, the other therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapeutic agent, a CXCL9 targeted therapeutic agent, a CXCL10 targeted therapeutic agent, a CCL5 targeted therapeutic agent, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0088] The pharmaceutical composition can be administered according to known methods. The route of administration can be, for example, oral administration or injection via intravenous, intraperitoneal, intracerebral (intracemdrally), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes. It can be administered via sustained release systems or implantable devices. In certain embodiments, the composition can be administered by bolus injection or continuously by infusion or by implantable devices.

[0089] Preparation method The antibodies or antigen-binding fragments thereof of the disclosure can be prepared by any method known in the art for preparing antibodies (e.g., chemical synthesis or recombinant expression techniques).

[0090] Therapeutic antibodies such as monoclonal antibodies can be developed by various techniques and routes, such as hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. Preparation of monoclonal antibodies by hybridoma technology is still the mainstream method for preparing therapeutic monoclonal antibodies.

[0091] The traditional hybridoma preparation technology was established by Kohler and Milstein 40 years ago (Kohler and Milstein 1975, Nature 256: 495), and is now widely used in the preparation and production of many related monoclonal antibodies, such as scientific research, diagnosis, and therapy. Although the basic method is still used today, changes, improvements, and innovations have been made in many aspects, including the use of animals of different lineages such as transgenic animals, the introduction of electrofusion technology, and the use of efficient screening technology devices such as the ClonePix device, making the use of hybridoma technology more diverse and efficient. For monoclonal antibodies prepared from normal animals such as mice, the heavy and light chain variable region genes of the antibody can be cloned by normal molecular biology methods, and the variable region genes can be grafted to the genes of human-derived antibody constant regions to form human-mouse chimeric antibodies (Cabilly et al., U.S. Patent No. 4,816,567), which greatly reduces immunogenicity when used in humans. Furthermore, the CDR domains of the variable regions of mouse-derived antibodies can be grafted onto the structure of human-derived antibodies, reducing mouse-derived antibody components to less than 5%, greatly improving the safety of antibodies used in humans. Antibodies obtained by this method are called humanized antibodies, and are currently the major products in the antibody drug market (U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0092] Recombinant expression of an antibody requires the construction of an expression carrier (vector) containing a nucleic acid encoding the antibody. The method for preparing said nucleic acid is a common method in the art, and may include, for example, obtaining a nucleic acid molecule encoding said antibody by gene cloning technology or obtaining a nucleic acid molecule encoding said antibody by artificial total sequence synthesis.

[0093] Once the nucleic acid encoding the antibody is obtained, a vector for producing the antibody can be produced by recombinant DNA technology. The vector can be obtained by a method conventional in the art, for example, by constructing the nucleic acid molecule described in this application by connecting it to an appropriate expression vector. The expression vector can be any of a variety of vectors conventional in the art, as long as it can carry the aforementioned nucleic acid molecule. The vector includes eukaryotic cell expression vectors and / or prokaryotic cell expression vectors.

[0094] The expression vector is transferred to a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody of the present disclosure. The host cell may be a variety of host cells conventional in the art, so long as the recombinant expression vector can stably replicate and the carried nucleic acid can be effectively expressed. The host cell may be a prokaryotic cell (e.g., E. coli cell) and / or a eukaryotic cell (e.g., HEK293 cell or CHO cell).

[0095] Uses of the invention The antibodies or antigen-binding fragments thereof, isolated nucleic acids, nucleic acid vectors, host cells, antibody-drug conjugates, and pharmaceutical compositions of the present disclosure can be used to treat a variety of conditions, including, for example, various forms of cancer, infections, autoimmune or inflammatory diseases, and / or fibrotic diseases. The antibodies or antigen-binding fragments thereof, isolated nucleic acids, nucleic acid vectors, host cells, antibody-drug conjugates, and pharmaceutical compositions of the present disclosure may be used to inhibit an immune response or activate T cells.

[0096] In some embodiments, there is provided the use of the antibody or antigen-binding fragment thereof, isolated nucleic acid, nucleic acid vector, host cell, antibody-drug conjugate, and pharmaceutical composition of the present disclosure in the preparation of a medicament or kit for inhibiting an immune response or activating T cells. In some embodiments, there is provided the use of the antibody or antigen-binding fragment thereof, isolated nucleic acid, nucleic acid vector, host cell, antibody-drug conjugate, and pharmaceutical composition of the present disclosure in the preparation of a medicament or kit for preventing, treating, or alleviating a disease. In some embodiments, the disease is cancer or an autoimmune disease.

[0097] In some embodiments, the cancer includes, but is not limited to, melanoma, kidney cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, colon cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, bladder cancer, renal pelvis cancer, central nervous system tumor, glioma, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, or T-cell lymphoma.

[0098] In some embodiments, the autoimmune disease includes, but is not limited to, rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, insulin-dependent diabetes mellitus, autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, Wegener's granulomatosis, Crohn's disease, ulcerative colitis, lupus such as systemic lupus erythematosus, atherosclerosis, chronic obstructive pulmonary disease, liver cirrhosis, renal transplant fibrosis, renal transplant nephropathy, or pulmonary fibrosis.

[0099] The present invention will be further described below with reference to examples, but the present invention is not limited to the scope of these examples. Experimental methods for which no specific conditions are specified in the following examples are selected according to normal methods and conditions or according to product instructions. EXAMPLES

[0100] Working Example

[0101] Example 1 Preparation of CD3 antibody

[0102] (1) Preparation of immunogen CD3e / g is a soluble ligand, and CD3e / g recombinant protein, which is a product of CD3e / g protein and human constant region, was designed as an immunogen, and the specific sequence of CD3e / g protein is shown in Table 1.

[0103] [Table 1]

[0104] (2) Preparation of hybridoma cells and screening of antibodies The immunogens were used to immunize 6-8 week-old SJL mice (purchased from SHANGHAI SLAC Co., Ltd.), and the mice were raised under SPF conditions. During the first immunization, the immunogen was emulsified with complete Freund's adjuvant, and then 0.25 ml was injected into the tail vein end. That is, 50 μg of the immunogen was injected into each mouse. During the booster immunization, the immunogen was emulsified with incomplete Freund's adjuvant, and then 0.25 ml was injected into the tail vein end. That is, 50 μg of the immunogen was injected into each mouse. There was a 2-week interval between the first booster immunization and the first booster immunization, and a 3-week interval between each booster immunization thereafter. One week after each booster immunization, blood was collected, and the antibody titer and specificity of the immunogen in the serum was detected using ELISA, and the results are shown in Figure 1 and Table 2. From Figure 1 and Table 2, it can be seen that the serum of mice after immunization with CD3e / g recombinant protein shows various degrees of binding to the immunogen, indicating antigen-antibody reaction, among which the highest dilution is about 1000. Here, the blank control is 1% (w / w) BSA, the batch refers to the mouse serum on the 7th day after the second boost immunization, and the data in the table are OD 450nm value.

[0105] [Table 2]

[0106] Before the immunization step was completed, each selected mouse was intraperitoneally injected with 100 μg of CD3e / g recombinant protein for the final immunization. After 3 days, the mice were sacrificed and spleen cells were collected. 4 OH was added to a final concentration of 1% (w / w) to dissolve the red blood cells mixed with the spleen cells, and a suspension of spleen cells was obtained. The cells were washed three times by centrifugation at 1000 rpm using DMEM-based medium, and then mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC#CRL-1581) at a ratio of 5:1 in terms of live cell number, and cell fusion was performed using a highly efficient electric fusion method (see METHODS IN ENZYMOLOGY, VOL. 220). The fused cells were diluted in DMEM medium containing 20% ​​fetal bovine serum and 1×HAT. Here, the percentages are mass %. Next, 1×10 cells were added per well. 5 Add 200 μl of each solution to a 96-well cell culture plate and incubate in 5% CO 2 After 14 days, the cell fusion plate supernatants were screened using ELISA plates coated with the antigen CD3e / g, and positive clones with an OD value of more than 2 in the ELISA results were amplified onto a 24-well plate and incubated in DMEM (purchased from Invitrogen, catalog number 11995-065) containing 10% (w / w) HT fetal bovine serum at 37°C and 5% (v / v) CO. 2 After 3 days of culture, the culture medium expanded in the 24-well plate is centrifuged, the supernatant is collected, and the antibody subtype analysis is performed on the supernatant, and the binding activity to the antigen is determined by ELISA.

[0107] Based on the screening results of the 24-well plate, the OD of the ELISA detection 450nm Select >2 hybridoma cells as qualified positive clones, subclone the qualified hybridoma cells in 96-well plates by limiting dilution, and culture in DMEM medium (purchased from Invitrogen) containing 10% (w / w) FBS at 37°C and 5% (v / v) CO2 After 10 days, the subclones were pre-screened by ELISA, and single positive monoclones were expanded to 24-well plates for further culture. After 3 days, ELISA was used to determine positive antigen binding, and biological activity was evaluated in a CD3e / g receptor ligand binding experiment (the evaluation standard was OD in the ELISA experiment). 450nm value > 2).

[0108] Based on the detection results of the 24-well plate samples, clones were selected and cultured in DMEM medium (purchased from Invitrogen) containing 10% (w / w) FBS at 37°C and 5% (v / v) CO 2 Clones selected under these conditions are expanded and frozen in liquid nitrogen to obtain hybridoma cells of the present disclosure, which can be used for subsequent antibody production and purification.

[0109] Example 2 Production and purification of CD3 antibodies

[0110] The hybridoma cells obtained in Example 1 were inoculated into a T-75 cell culture flask and subcultured for three generations using a production medium (Hybridoma serum free medium, purchased from Invitrogen). When the cells were in good growth condition, they were inoculated into a cell culture spinner flask. 500 ml of the production medium was added to each 2-liter culture spinner flask, and 1.0 × 10 5 The cells were seeded at a density of 1000 / ml. The culture spinner flasks were placed on a flask rotator at a rotation speed of 3 rpm in a 37°C incubator. After 14 days of continuous rotation, the cell culture fluid was collected, filtered to remove the cells, and filtered through a 0.45 μm filter membrane until the culture supernatant was clear. The clarified culture supernatant can be purified immediately or frozen at -30°C.

[0111] The antibodies in the clarified hybridoma cell culture supernatant (300 mL) were purified using a 2 mL protein A column (purchased from GE Healthcare). The protein A column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2), and then the clarified culture supernatant was loaded onto the protein A column, with the flow rate controlled at 3 mL / min. After loading the sample, the protein A column was washed with equilibration buffer, and the volume of the equilibration buffer was four times the bed volume of the protein A column. The CD3e / g antibody bound to the protein A column was eluted using an eluent (0.1 M glycine hydrochloride buffer, pH 2.5), and the elution status (A 280 The UV absorption peak was monitored. The eluted antibody was collected, and 10% 1.0M Tris-HCl buffer was added to neutralize the pH, where the percentage is vol. %, and immediately dialyzed overnight against PBS phosphate buffer, and the next day the liquid was replaced once and dialysis was continued for 3 hours. The CD3e / g antibody after dialysis was collected, sterile filtered through a 0.22 μm filter, and sterile stored to obtain purified CD3e / g antibody.

[0112] Example 3 Antigen-binding activity of CD3 antibody

[0113] Enzyme-linked immunosorbent assay (ELISA) was used to detect the antigen-antibody binding sites.

[0114] The purified CD3e / g antibody obtained in Example 2 was subjected to a binding reaction with CD3e / g (see Table 1 for the sequence) and CD3e / d protein (purchased from Sinobiological, CAT:CT038-H2508H-B).

[0115] Human CD3e / g protein, cynomolgus monkey CD3e / g protein (see Table 1 for sequences), or human CD3e / d protein were diluted in PBS to a final concentration of 5.0 μg / mL, and then 100 μl was added per well to a 96-well ELISA plate. The plate was sealed with plastic film and incubated overnight at 4° C. The next day, the plate was washed twice with plate washing solution [PBS+0.01% (v / v) Tween 20] and blocked for 2 hours at room temperature by adding blocking solution [PBS+0.01% (v / v) Tween 20+1% (w / w) BSA]. The blocking solution was discarded, and 100 μl of purified CD3e / g antibody obtained in Example 2 was added per well. After 2 hours of incubation at 37° C., the plate was washed three times with plate washing solution [PBS+0.01% (v / v) Tween 20]. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma) was added and incubated at 37°C for 2 hours, after which the plate was washed three times with plate washing solution [PBS + 0.01% (v / v) Tween 20]. 100 μl of TMB substrate was added per well and incubated at room temperature for 30 minutes, after which 100 μl of stop solution (1.0 N HCl) was added per well. A was measured using an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device). 450nm The values ​​were read out, and FIG. 2 and Table 3 show the binding activity of CD3e / g antibody to hCD3e / g detected by ELISA, and FIG. 3 and Table 4 show the binding activity of CD3e / g antibody to cyno CD3e / g detected by ELISA, where the IgG control was mouse IgG, and the data in the table are OD 450nm where HIT3a is purchased from BD pharmigen, catalog number 555336, SP34 is purchased from BD pharmigen, catalog number 551916, OKT3 is purchased from Biolegend.

[0116] 2 and 3, Tables 3 and 4 show that the antibody of the present disclosure can bind to human CD3e / g (hCD3e / g), and the binding affinity of the antibody of the present disclosure to human CD3e / g is better than that of OKT3. At the same time, the antibody of the present disclosure can bind to cynomolgus monkey CD3 (cyno CD3e / g), but OKT3 has no binding affinity to cynomolgus monkey CD3e / g or does not bind significantly to it.

[0117] FIG. 4 and Table 5 show that the antibodies of the present disclosure can also bind to human CD3e / d (hCD3e / d) and that the binding affinity of the antibodies of the present disclosure to human CD3e / d is superior to that of SP34.

[0118] [Table 3]

[0119] [Table 4]

[0120] [Table 5]

[0121] Example 4 Humanization of antibodies

[0122] The heavy and light chain variable region (VH and VL) sequences of the mouse-derived anti-human CD3 antibody were used to search for the corresponding heavy and light chain variable region sequences of human antibodies in the IMGT database. By using MOE software to compare the gene sequences of the heavy and light chain variable regions of the mouse-derived antibody and the heavy and light chain variable regions of the searched human antibodies, the germline genes of the heavy and light chain variable regions having high homology with the mouse-derived antibody were selected as templates, respectively, and the CDRs of the mouse-derived antibody were grafted onto the corresponding human-derived templates, respectively, to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the analysis of the structures of the heavy and light chain variable regions of the selected human antibody and the structure of the mouse-derived antibody, important amino acids in the frame sequence were back-mutated to the amino acids corresponding to the mouse-derived antibody to maintain the affinity of the original mouse-derived antibody, thereby obtaining a humanized anti-CD3 monoclonal antibody. Here, the amino acid residues of the CDR regions are determined and annotated according to the Kabat numbering system.

[0123] The light and heavy chain variable regions of the above mouse-derived antibody are connected to the light and heavy chain constant regions of a human-derived antibody to form a chimeric antibody, and the chimeric antibody corresponding to the mAb001 antibody is named CAb001, and other antibodies are also inferred by analogy.

[0124] 3.1 Humanization of mouse anti-mAb001

[0125] 3.1.1 mAb001 humanization plan 1

[0126] (1) Selection of mAb001 humanized framework The humanized light chain template of mouse-derived antibody mAb001 is IGKV1-39 IGKV1-39*01 X59315 V-KAPPA, and the humanized heavy chain template is IGHV1-8 IGHV1-8*01 M99637 VH, and after humanization, the humanized antibody hAb001 is obtained, and the sequences of the humanized variable regions are as follows:

[0127] >hAb001 VH1-CDR graft (SEQ ID NO:33) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQATGQGLEWMG WTYPGNNNIKYNEKFKG RVTMTRNTSISTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS >hAb001 VL1-CDR graft (SEQ ID NO:58) DIQMTQSPSSLSASVGDRVTITC KSSQSLLNNRTRKNYLA WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC KQSYTLRT FGGGTKVEIK

[0128] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where underlined are CDR sequences and non-underlined are FR sequences.

[0129] (2) The design of the hAb001 back mutations is as shown in the table below.

[0130] TIFF2025510266000006.tif68168

[0131] NOTE: For example, Q3V represents a mutation, numbered according to the natural order of the amino acid sequence, that changes the Q at position 3 back to a V. "Grafting" refers to the transplantation of a murine antibody CDR into a human species-based FR region sequence.

[0132] (3) The combination of hAb001 humanized sequences is as follows:

[0133] TIFF2025510266000007.tif42168

[0134] Note: This table shows the sequences obtained by combining various mutations. As shown in hAb001-6, the humanized antibody hAb001-6 has four back mutations in the light chain mAb001VL_hum2 and the heavy chain mAb001VH_hum2. The rest are inferred.

[0135] (4) The specific sequence of hAb001 humanization is as follows:

[0136] > mAb001VL_hum1 (SEQ ID NO:58) DIQMTQSPSSLSASVGDRVTITC KSSQSLLNNRTRKNYLA WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC KQSYTLRT FGGGTKVEIK > mAb001VL_hum2 (SEQ ID NO:59) DIVMTQSPSSLSASVGDRVTITC KSSQSLLNQRTRKNYLA WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC KQSYTLRT FGGGTKVEIK > mAb001VL_hum3 (SEQ ID NO:60) DIQMTQSPSSLSASVGDRVTMTC KSSQSLLNNRTRKNYLA WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC KQSYTLRT FGGGTKVEIK > mAb001VL_hum4 (SEQ ID NO:61) DIVMTQSPSSLSASVGDRVTMTC KSSQSLLNQRTRKNYLA WYQQKPGKAPKLLIY WASTRES GVPSRFTGSGSGTDFLTISSLQPEDFATYYC KQSYTLRT FGGGTKVEIK > mAb001VH_hum1 (SEQ ID NO:33) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQATGQGLEWMG WTYPGNNNIKYNEKFKG RVTMTRNTSISTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS > mAb001VH_hum2 (SEQ ID NO:34) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQATGQGLEWMG WTYPGQNNIKYNEKFKG RVTMTRNTSSSTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS > mAb001VH_hum3 (SEQ ID NO:35) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQATGQGLEWMG WTYPGNQNIKYNEKFKG KVTMTRNTSISTAYMELSSLRSEDSAVYYCAR DGYGYYFFDY WGQGTIVTVSS > mAb001VH_hum4 (SEQ ID NO:36) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQATGQGLEWMG WTYPGQNQIKYNEKFKG KVTTMTRNTSSSTAYMELSSLRSEDSAVYYCAR DGYGYYFFDY WGQGILVTVSS

[0137] 3.1.2 mAb001 humanization plan 2 (1) Selection of mAb001 humanized framework The humanized light chain template of mouse-derived antibody mAb001 is IGKV4-1 IGKV4-1*01 Z0023 V-KAPPA, the humanized heavy chain template is IGHV1-3 IGHV1-3*01 X62109 VH, after humanization the humanized antibody hAb001 is obtained, and the sequences of the humanized variable regions are as follows:

[0138] >hAb001 VH2-CDR graft (SEQ ID NO:37) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQAPGQRLEWMG WTYPGNNNIKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS >hAb001 VL2-CDR graft (SEQ ID NO:62) DIVMTQSPDSLAVSLGERATINC KSSQSLLNNRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC KQSYTLRT FGGGTKLEIK

[0139] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where underlined are CDR sequences and non-underlined are FR sequences.

[0140] (2) The design of the hAb001 back mutations is as shown in the table below.

[0141] TIFF2025510266000008.tif61166

[0142] NOTE: For example, S69T represents a mutation, numbered according to the natural order of the amino acid sequence, of the S at position 69 back to a T. "Grafting" refers to the transplantation of a murine antibody CDR into a human species-based FR region sequence.

[0143] (3) The combination of hAb001 humanized sequences is as follows:

[0144] TIFF2025510266000009.tif42167

[0145] Note: This table shows the sequences obtained by combining various mutations. As shown in hAb001-22, the humanized antibody hAb001-22 has three back mutations in the light chain mAb001VL_hum6 and the heavy chain mAb001VH_hum6. The rest are inferred.

[0146] (4) The specific sequence of hAb001 humanization is as follows:

[0147] > mAb001VL_hum5 (SEQ ID NO:62) DIVMTQSPDSLAVSLGERATINC KSSQSLLNNRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC KQSYTLRT FGGGTKLEIK > mAb001VL_hum6 (SEQ ID NO:63) DIVMTQSPDSLAVSLGERATINC KSSQSLLNNRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFLTISSLQAEDVAVYYC KQSYTLRT FGGGTKLEIK > mAb001VL_hum7 (SEQ ID NO:64) DIVMTQSPDSLAVSLGERATMSC KSSQSLLNNRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC KQSYTLRT FGGGTKLEIK > mAb001VL_hum8 (SEQ ID NO:65) DIVMTQSPDSLAVSLGERATMSC KSSQSLLNNRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFLTISSLQAEDVAVYYC KQSYTLRT FGGGTKLEIK > mAb001VH_hum5 (SEQ ID NO:37) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQAPGQRLEWMG WTYPGNNNIKYNEKFKG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS > mAb001VH_hum6 (SEQ ID NO:38) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQAPGQRLEWIG WTYPGNNNIKYNEKFKGRVTITADTSASTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS > mAb001VH_hum7 (SEQ ID NO:39) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVRQAPGQRLEWIG WTYPGNNNIKYNEKFKG RATITADTSASTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS > mAb001VH_hum8 (SEQ ID NO: 40) QVQLVQSGAEVKKPGASVKVSCKASGYSFT NYYMH WVKQAPGQRLEWIG WTYPGNNNIKYNEKFKG RATITADTSASTAYMELSSLRSEDTAVYYCAR DGYGYYFFDY WGQGTLVTVSS

[0148] 3.2 Humanization of mouse anti-mAb007

[0149] 3.2.1 Humanization Scheme 1 of Mouse Anti-mAb007

[0150] (1) Selection of mAb007 humanized framework The humanized light chain template of the mouse-derived antibody mAb007 is IGKV1-27 IGKV1-27*01 X63398 V-KAPPA F, and the humanized heavy chain template is IGHV1-8 IGHV1-8*01 M99637 VH F, and after humanization, the humanized antibody hAb007 is obtained, and the sequences of the humanized variable regions are as follows:

[0151] >hAb007 VH1-CDR graft (SEQ ID NO:41) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DNYIH WVRQATGQGLEWMG WIYPGSVNIKYNEKFKD RVTMTRNTSISTAYMELSSLRSEDTAVYYCAR DISRYYFDY WGQGTLVTVSS >hAb007 VL1-CDR graft (SEQ ID NO:66) DIQMTQSPSSLSASVGDRVTITC KSSQSLLNSRTRKNYLA WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC KQSFILRT FGGGTKVEIK

[0152] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where underlined are CDR sequences and non-underlined are FR sequences.

[0153] (2) The design of the hAb007 back mutations is as shown in the table below.

[0154] TIFF2025510266000010.tif81165

[0155] NOTE: For example, A49S represents a mutation, numbered according to the natural order of the amino acid sequence, of the A at position 49 back to an S. "Grafting" refers to the transplantation of a murine antibody CDR into a human species-based FR region sequence.

[0156] (3) The combination of hAb007 humanized sequences is as follows:

[0157] TIFF2025510266000011.tif42167

[0158] Note: This table shows the sequences obtained by combining various mutations. As shown in hAb007-5, the humanized antibody hAb007-5 has four back mutations in the light chain mAb007VL_hum1 and the heavy chain mAb007VH_hum2. The rest are inferred.

[0159] (4) The specific sequence of hAb007 humanization is as follows:

[0160] > mAb007VL_hum1 (SEQ ID NO:66) DIQMTQSPSSLSASVGDRVTITC KSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC KQSFILRT FGGGTKVEIK > mAb007VL_hum2 (SEQ ID NO:67) DIQMTQSPSSLSASVGDRVTITC KSSQSLLNSRTRKNYLA WYQQKPGKSPKLLIY WASTRES GVPDRFTGSGSGTDFLTISSLQPEDFATYYC KQSFILRT FGGGTKVAIK > mAb007VL_hum3 (SEQ ID NO:68) DIQMSQSPSSLSASVGDRVTITC KSSQSLLNSRTRKNYLA WYQQKPGQSPKLLIY WASTRES GVPDRFTGSGSGTDFLTISSLQAEDFATYYC KQSFILRT FGGGTKVAIK > mAb007VL_hum4 (SEQ ID NO:69) DIVMSQSPSSLSASVGDRVTITC KSSQSLLNSRTRKNYLA WYQQKPGQSPKLLIY WASTRES GVPDRFTGSGSGTDFLTISSVQPEDFATYYC KQSFILRT FGGGTKLAIK > mAb007VH_hum1 (SEQ ID NO: 41) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DNYIH WVRQATGQGLEWMG WIYPGSVNIKYNEKFKD RVTMTRNTSISTAYMELSSLRSEDTAVYYCAR DISRYYFDY WGQGTLVTVSS > mAb007VH_hum2 (SEQ ID NO: 42) QVQLVQSGAEVKKPGASVKVSCKASGSTFT DNYIH WVRQATGQGLEWMG WIYPGSVNIKYNEKFKD KVTLTRNTSISTAYMELSSLRSEDSAVYYCAR DISRYYFDY WGQGTLVTVSS > mAb007VH_hum3 (SEQ ID NO: 43) QVQLVQSGAEVVKPGASVKVSCKASGYTFT DNYIH WVRQRTGQGLEWMG WIYPGSVNIKYNEKFKD RVTMTADTSISTAYMQLSSLSSEDTAVYYCAR DISRYYFDY WGQGTLVTVSS > mAb007VH_hum4 (SEQ ID NO:44) QVQLVQSGAELVKPGASVKVSCKASGSTFT DNYIH WVRQRTGQGLEWMG WIYPGSVNIKYNEKFKD RVTLTADTSITTAYMQLSSLSSEDTAIYYCAR DISRYYFDY WGQGTLVTVSS

[0161] 3.2.2 Humanization method 2 of mouse anti-mAb007

[0162] (1) Selection of mAb007 humanized framework The humanized light chain template of the mouse-derived antibody mAb007 is IGKV4-1 IGKV4-1*01 Z0023 V-KAPPA, and the humanized heavy chain template is IGHV1-3 IGHV1-3*01 X62109 VH, and after humanization, the humanized antibody hAb007 is obtained, and the sequences of the humanized variable regions are as follows:

[0163] >hAb007 VH2-CDR graft (SEQ ID NO:45) QVQLVQSGAEVKKPGASVKVSCKASGSTFT DNYIH WVRQAPGQRLEWMG WIYPGSVNIKYNEKFKD RVTITRDTSASTAYMELSSLRSEDTAVYYCAR DISRYYFDY WGQGTTVTVSS >hAb007 VL2-CDR graft (SEQ ID NO:70) DIVMTQSPDSLAVSLGERATINC KSSQSLLNSRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC KQSFILRT FGGGTKLEIK

[0164] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where underlined are CDR sequences and non-underlined are FR sequences.

[0165] (2) The design of the hAb007 back mutations is as shown in the table below.

[0166] TIFF2025510266000012.tif61167

[0167] NOTE: For example, S69T represents a mutation, numbered according to the natural order of the amino acid sequence, of the S at position 69 back to a T. "Grafting" refers to the transplantation of a murine antibody CDR into a human species-based FR region sequence.

[0168] (3) The combination of hAb007 humanized sequences is as follows:

[0169] TIFF2025510266000013.tif41165

[0170] Note: This table shows the sequences obtained by combining various mutations. As shown in hAb007-22, the humanized antibody hAb007-22 has three back mutations in the light chain mAb007VL_hum6 and the heavy chain mAb007VH_hum6. The rest are inferred.

[0171] (4) The specific sequence of hAb007 humanization is as follows:

[0172] > mAb007VL_hum5 (SEQ ID NO:70) DIVMTQSPDSLAVSLGERATINC KSSQSLLNSRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC KQSFILRT FGGGTKLEIK > mAb007VL_hum6 (SEQ ID NO:71) DIVMTQSPDSLAVSLGERATINC KSSQSLLNSRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFLTISSLQAEDVAVYYC KQSFILRT FGGGTKLEIK > mAb007VL_hum7 (SEQ ID NO:72) DIVMTQSPDSLAVSLGERATMNC KSSQSLLNSRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC KQSFILRT FGGGTKLEIK > mAb007VL_hum8 (SEQ ID NO:73) DIVMTQSPDSLAVSLGERATMNC KSSQSLLNSRTRKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFLTISSLQAEDVAVYYC KQSFILRT FGGGTKLEIK > mAb007VH_hum5 (SEQ ID NO: 45) QVQLVQSGAEVKKPGASVKVSCKASGSTFT DNYIH WVRQAPGQRLEWMG WIYPGSVNIKYNEKFKD RVTITRDTSASTAYMELSSLRSEDTAVYYCAR DISRYYFDY WGQGTTVTVSS > mAb007VH_hum6 (SEQ ID NO: 46) QVQLVQSGAEVKKPGASVKVSCKASGSTFT DNYIH WVRQAPGQRLEWIG WIYPGSVNIKYNEKFKD RVTITADTSASTAYMELSSLRSEDTAVYYCAR DISRYYFDY WGQGTTVTVSS > mAb007VH_hum7 (SEQ ID NO: 47) QVQLVQSGAEVKKPGASVKVSCKASGSTFT DNYIH WVRQAPGQRLEWIG WIYPGSVNIKYNEKFKD RATITADTSASTAYMELSSLRSEDTAVYYCAR DISRYYFDY WGQGTTVTVSS > mAb007VH_hum8 (SEQ ID NO:48) QVQLVQSGAEVKKPGASVKVSCKASGSTFT DNYIH WVKQAPGQRLEWIG WIYPGSVNIKYNEKFKD RATITADTSATTAYMELSSLRSEDTAVYYCAR DISRYYFDY WGQGTTVTVSS

[0173] 3.3 Humanization of mouse anti-mAb008

[0174] 3.3.1 Humanization Scheme 1 of Mouse Anti-mAb008

[0175] (1) Selection of mAb008 humanized framework The humanized light chain template of mouse-derived antibody mAb008 is IIGKV2-30 IGKV2-30*01 X63403 V-KAPPA F, the humanized heavy chain template is IGHV1-8 IGHV1-8*01 M99637 VH F, and after humanization, the humanized antibody hAb008 is obtained, and the sequences of the humanized variable regions are as follows:

[0176] >hAb008 VH-CDR graft (SEQ ID NO:49) QVQLVQSGAEVKKPGSSVKVSCKASGYTFT DYTVN WVRQAPGQGLEWMG YINPFNSYTKYNQKFKD RVTITADESTSTAYMELSSLRSEDTAVYYCAN SVSTY WGQGTLVTVSS >hAb008 VL-CDR graft (SEQ ID NO:74) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WFQQRPGQSPRRLIY LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC WQGTHFPRT FGGGTKVEIK

[0177] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where underlined are CDR sequences and non-underlined are FR sequences.

[0178] (2) The design of the hAb008 back mutations is as shown in the table below.

[0179] TIFF2025510266000014.tif74166

[0180] NOTE: For example, F41L represents a mutation, numbered according to the natural order of the amino acid sequence, of the F at position 41 back to an L. "Grafting" refers to the transplantation of a murine antibody CDR into a human species-based FR region sequence.

[0181] (3) The combination of hAb008 humanized sequences is as follows:

[0182] TIFF2025510266000015.tif55166

[0183] Note: This table shows the sequences obtained by combining various mutations. As shown in hAb008-5, the humanized antibody hAb008-5 has three back mutations in the light chain mAb008VL_hum2 and the heavy chain mAb007VH_hum2. The rest are inferred.

[0184] (4) The specific sequence of hAb008 humanization is as follows:

[0185] > mAb008VL_hum1 (SEQ ID NO:74) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WFQQRPGQSPRRLIY LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC WQGTHFPRT FGGGTKVEIK > mAb008VL_hum2 (SEQ ID NO:75) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WLQQRPGQSPRRLIY LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC WQGTHFPRT FGGGTKVEIK > mAb008VL_hum3 (SEQ ID NO:76) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WLQQRPGQSPRRLIQ LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC WQGTHFPRT FGGGTKVEIK > mAb008VH_hum1 (SEQ ID NO:49) QVQLVQSGAEVKKPGSSVKVSCKASGYTFT DYTVN WVRQAPGQGLEWMG YINPFNSYTKYNQKFKD RVTITADESTSTAYMELSSLRSEDTAVYYCAN SVSTY WGQGTLVTVSS > mAb008VH_hum2 (SEQ ID NO:50) QVQLVQSGAEVAKPGSSVKVSCKASGYTFT DYTVN WVRQRPGQGLEWMG YINPFNSYTKYNQKFKD RVTITADESTSTAYMELSSLRSEDTAVYYCAN SVSTY WGQGTLVTVSS > mAb008VH_hum3 (SEQ ID NO:51) QVQLVQSGAEVAKPGSSVKVSCKASGYTFT DYTVN WVRQRPGQGLEWMG YINPFNSYTKYNQKFKD RVTITADESTSTAYMELSSLTSEDTAVYYCAN SVSTY WGQGTLVTVSS > mAb008VH_hum4 (SEQ ID NO:52) QVQLVQSGAEVAKPGSSVKVSCKTSGYTFT DYTVN WVRQRPGQGLEWMG YINPFNSYTKYNQKFKD RVTLTADESTSTAYMELSSLTSEDTAVYYCAN SVSTY WGQGTLVTVSS > mAb008VH_hum5 (SEQ ID NO:53) QVQLVQSGAEVAKPGSSVKVSCKTSGYTFT DYTVN WVRQRPGQGLEWMG YINPFNSYTKYNQKFKD RVTLTADKSTSTAYMQLSSLTSEDTAVYYCAN SVSTY WGQGTLVTVSS

[0186] 3.3.2 Humanization method 2 of hybridoma mouse anti-mAb008

[0187] (1) Selection of mAb008 humanized framework The humanized light chain template of mouse-derived antibody mAb008 is IIGKV2-30 IGKV2-30*01 X63403 V-KAPPA F, and the humanized heavy chain template is IGHV1-3 IGHV1-3*01 X62109 VH, and after humanization, the humanized antibody hAb008 is obtained, and the sequences of the humanized variable regions are as follows:

[0188] >hAb008 VH2-CDR graft (SEQ ID NO:54) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYTVN WVRQAPGQRLEWMG YINPFNSYTKYNQKFKD RVTITRDTSASTAYMELSSLRSEDTAVYYCAR SVSTY WGQGTTVTVSS >hAb008 VL2-CDR graft (SEQ ID NO:77) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WFQQRPGQSPRRLIY LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC WQGTHFPRT FGGGTKLEIK

[0189] NOTE: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where underlined are CDR sequences and non-underlined are FR sequences.

[0190] (2) The design of the hAb008 back mutations is as shown in the table below.

[0191] TIFF2025510266000016.tif61168

[0192] NOTE: For example, Y54Q represents a mutation, numbered according to the natural order of the amino acid sequence, of the Y at position 54 back to a Q. "Grafting" refers to the transplantation of a murine antibody CDR into a human species-based FR region sequence.

[0193] (3) The combination of hAb008 humanized sequences is as follows:

[0194] TIFF2025510266000017.tif38128

[0195] Note: This table shows the sequences obtained by combining various mutations. As shown in hAb008-22, the humanized antibody hAb008-22 has six back mutations in the light chain mAb008VL_hum6 and the heavy chain mAb007VH_hum7. The rest are inferred.

[0196] (4) The specific sequence of hAb008 humanization is as follows:

[0197] > mAb008VL_hum4 (SEQ ID NO:77) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WFQQRPGQSPRRLIY LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC WQGTHFPRT FGGGTKLEIK > mAb008VL_hum5 (SEQ ID NO:78) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WFQQRPGQSPRRLIQ LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVFYC WQGTHFPRT FGGGTKLEIK > mAb008VL_hum6 (SEQ ID NO:79) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WLQQRPGQSPRRLIQ LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVFYC WQGTHFPRT FGGGTKLEIK > mAb008VL_hum7 (SEQ ID NO:80) DVVMTQSPLSLPVTLGQPASISC KSSQSLLDSDGKTYLN WLLQRPGQSPRRLIQ LVSKLNS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVFYC WQGTHFPRT FGGGTKLEIK > mAb008VH_hum6 (SEQ ID NO:54) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYTVN WVRQAPGQRLEWMG YINPFNSYTKYNQKFKD RVTITRDTSASTAYMELSSLRSEDTAVYYCAR SVSTY WGQGTTVTVSS > mAb008VH_hum7 (SEQ ID NO:55) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYTVN WVRQAPGQRLEWI GYINPFNSYTKYNQKFKD RVTITADTSASTAYMELSSLRSEDTAVYYCAN SVSTY WGQGTTVTVSS > mAb008VH_hum8 (SEQ ID NO:56) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYTVN WVRQAPGQRLEWI GYINPFNSYTKYNQKFKD RATITADTSASTAYMELSSLRSEDTAVYFCAN SVSTY WGQGTTVTVSS > mAb008VH_hum9 (SEQ ID NO:57) QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYTVN WVKQAPGQRLEWI GYINPFNSYTKYNQKFKD RATITADTSASTAYMELSSLRSEDTAVYFCAN SVSTY WGQGTTVTVSS

[0198] 3.3.3 Hot spot mutations in the hybridoma mouse anti-mAb008 chimeric antibody Point mutations are made to the antibody mAb008 hotspot. The light chain mAb008 antibody has one potential mutation site and is mutated from G at position 34 of the light chain back to A to generate the hotspot mutation chimeric antibody CAb008DA.

[0199] >mAb008VL DA (SEQ ID NO:81) DVVMTQTPLTLSVTIGQPASISC KSSQSLLDSDAKTYLN WLLQRPGQSPKRLIQ LVSKLNS GVPDRFTGSGSGTDFTLKISRVEAEDLGIFYC WQGTHFPRT FGGGTKLEIK >mAb008VH or CAb008VH (SEQ ID NO: 89) QVQLQQSGAELARPGASVKMSCKTSGYTFT DYTVN WVKQRPGQGLEWIG YINPFNSYTKYNQKFKD KATLTADKSSSTAYMQLSSLTSEDSAVYFCAN SVSTY WGQGTTLTVSS >mAb008VL or CAb008VL (SEQ ID NO: 90) DVVMTQTPLTLSVTIGQPASISC KSSQSLLDSDGKTYLN WLLQRPGQSPKRLIQ LVSKLNS GVPDRFTGSGSGTDFTLKISRVEAEDLGIFYC WQGTHFPRT FGGGTKLEIK

[0200] 3.4 Hot spot mutations in the hybridoma mouse anti-mAb009 chimeric antibody Point mutations are made to the antibody mAb009 hotspot. The light chain mAb009 antibody has two potential mutation sites and is mutated from G back to A at position 32 of the light chain to generate the hotspot mutation chimeric antibody CAb009DADG.

[0201] >mAb009VL DADG (SEQ ID NO:82) DVVMTQTPLTLSVTIGQPASISC KSSQSLLDADGKTYLN WLLQRPGQSPKRLIQ LVSKLNS GVPDRFTGSGSGTDFTLKISRVEAEDLGIYYC WQGTHFPRT FGGGTKLEIK >mAb009VH or CAb009VH (SEQ ID NO: 85) QVQLQQSGAELTRPGASVKMSCKASGYTFT DYTIH WVKQRPGQGLEWIG YINPFSDYTKYNQKFKD KATLTADKSSSTAYIQLNSLTSEDSAVYYCAN SVSIY WGQGTTLTVSS >mAb009VL or CAb009VL (SEQ ID NO: 86) DVVMTQTPLTLSVTIGQPASISC KSSQSLLDGDGKTYLN WLLQRPGQSPKRLIQ LVSKLNS GVPDRFTGSGSGTDFTLKISRVEAEDLGIYYC WQGTHFPRT FGGGTKLEIK

[0202] A summary of the antibody sequences tested in this disclosure is provided in the table below.

[0203] [Table 6] TIFF2025510266000019.tif209166TIFF2025510266000020.tif108166

[0204] >CAb001 VH (SEQ ID NO:87) QVQLQQSGPELVKPGASVKISKASGYSFTNYYMHWVKQRPGQGLEWIGWTYPGNNNIKYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYYCARDGYGYYFFDYWGQGIILTVSS > CAb001 VL (SEQ ID NO:88) DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNNRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCKQSYTLRTFGGGTKLAIK >CAb007 VH (SEQ ID NO:91) QVQLQQSGPELVKPGASVKISCKASGSTFTDNYIHWVKQRPGQGLEWIGWIYPGSVNIKYNEKFKDKATLTADTSSTTAYMQLSSLSSEDSAIYYCARDISRYYFDYWGQGTTLTVSS >CAb007 VL (SEQ ID NO:92) DIVMSQSPSSLAVSAGERVTMNCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSFILRTFGGGTKLAIK

[0205] Example 5 Binding of humanized or hotspot mutation variants of CD3 antibodies to Jurkat cells

[0206] Jurkat cells were cultured at 3 × 10 in a T-175 cell culture flask. 6 The cells were expanded to 2 × 10 cells / ml, centrifuged to remove the medium, washed twice with PBS buffer (purchased from Invitrogen), counted, and then diluted with PBS buffer to 2 × 10 cells / ml. 6 The cells were diluted and 1% BSA blocking solution was added, where the percentages are mass %, incubated on ice for 30 minutes, and then washed twice with PBS buffer by centrifugation. The collected cells were diluted with FACS buffer (PBS + 1% BSA, where the percentages are mass %) at 2 × 10 6The cells were suspended in 100 μl per well of a 96-well FACS reaction plate, and the purified CD3 antibody sample obtained in Example 4 was added at 100 μl per well and incubated at 4° C. for 2 hours. After centrifugation and washing twice with FACS buffer, 100 μl of fluorescent (Alexa 488)-labeled secondary antibody (purchased from Jackson) was added at 100 μl per well and incubated at 4° C. for 1 hour. After centrifugation and washing three times with FACS buffer, the cells were suspended in 100 μl of FACS buffer and detected using a BD FACS LSRFortessa (purchased from BD), and the results were analyzed. The results are shown in FIG. 5, where the IgG control was human IgG1 (hIgG).

[0207] Example 6. Effect of antibodies on activation of human total T cells

[0208] Human Total T Cell Isolation Kit (EasySep TM Total human T cells were extracted using a Direct Human T Cell Isolation Kit (purchased from Stemcell, catalog number 19661). They were washed three times with complete medium. T cells and diluted CD3 antibodies were mixed uniformly in a 96-well plate and then cultured at 37°C for 3 days. After 3 days, the culture supernatant was collected and the human IFN-γ level in the culture supernatant was detected using an ELISA kit (IFN gamma Human Uncoated ELISA Kit, purchased from Invitrogen, catalog number 88-7316-88). The cultured cells were washed once with PBS, then stained with a labeled anti-human CD25 flow cytometry antibody (PE-Cy7 Mouse Anti-Human CD25, purchased from BD Pharmingen, catalog number 557741) for 30 minutes in the dark, and then washed twice with PBS. The expression of CD25 on human T cells was analyzed using FACS (BD FACS LSRFortessa, purchased from BD).

[0209] As a result, as shown in Figures 6 and 7, both the humanized variants and hot spot mutation variants of the CD3 antibody can significantly activate human total T cells, specifically, by up-regulating the expression of CD25 and the release of IFN-γ.

[0210] Example 7 Measurement of antibody affinity for human CD3e / g antigen

[0211] The equilibrium dissociation constant of CD3 antibody binding to human CD3e / g antigen (purchased from ACRO, catalog number CDG-H52W5) was determined by kinetic binding method using Biacore 8K (GE) system. By the capture method, first, NHS (N-hydroxysuccinimide) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were used to activate the carboxylated dextran surface of the CM5 chip, and then anti-human IgG (Fc) antibody was added, whose free amino group could form an amide bond with the activated carboxyl group to immobilize it on the chip surface. The anti-human IgG (Fc) antibody could bind to the Fc end of the CD3 antibody to capture the antibody, and then a series of concentrations of antigen were added to obtain the binding and dissociation curves of the antibody and antigen, which were calculated by Biacore Evaluation software to obtain the corresponding kinetic constants, specifically shown in the table below.

[0212] [Table 7] TIFF2025510266000022.tif45170

[0213] It should be understood that, after reading the above content of the present disclosure, one skilled in the art may make various changes or modifications to the present invention, and that equivalent forms thereof are also included within the scope defined by the claims appended to this application.

Claims

1. 1. An antibody or antigen-binding fragment thereof that specifically binds to CD3, wherein the heavy chain comprises CDR1, CDR2 and CDR3, (a) the heavy chain CDR1 comprises a sequence having at least 80% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; (b) the heavy chain CDR2 comprises a sequence having at least 80% identity to SEQ ID NO:18, SEQ ID NO:6, or SEQ ID NO:22; (c) an antibody or antigen-binding fragment thereof, wherein the heavy chain CDR3 comprises a sequence having at least 80% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:

11.

2. (a) a heavy chain CDR1 comprising a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a sequence having one amino acid addition, substitution, and / or deletion compared to any of SEQ ID NOs:1-4; (b) WTYPGX １ X ２ X ３ IKYNEKFKG (SEQ ID NO:5), SEQ ID NO:6, or a sequence having a total of 3 or less different amino acids added, substituted, and / or deleted from SEQ ID NO:6, or YINPFX ４ X ５ YTKYNQKFKD (SEQ ID NO: 7), wherein X １ , X ２ and X ３ are each independently N or Q; X ４ is N or S, and X ５ a heavy chain CDR2 which is S or D; (c) a heavy chain CDR3 comprising a sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or a sequence having a total of no more than two different amino acids added, substituted, and / or deleted from any of SEQ ID NOs:8-11; (d) KSSQSLLNX ６ RTRKNYLA (SEQ ID NO: 12) or KSSQSLLDX ７ D.X. ８ KTYLN (SEQ ID NO: 13), wherein X ６ is N, Q or S; X ７ is S, A or G, and X ８ a light chain CDR1 which is G or A; (e) a light chain CDR2 comprising a sequence selected from SEQ ID NO: 14 or SEQ ID NO: 15; and (f) KQSX ９ X １０ LRT (SEQ ID NO: 16) or SEQ ID NO: 17, wherein X ９ is Y or F, and X １０ and a light chain CDR3 that is T or I.

3. (1) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 1 and WTYPGX １ X ２ X ３ IKYNEKFKG (SEQ ID NO:5), １ , X ２ and X ３ a heavy chain CDR2, each independently N or Q; a heavy chain CDR3 comprising or consisting of SEQ ID NO:8; and KSSQSLLNX. ６ RTRKNYLA (SEQ ID NO: 12), ６ or a light chain CDR1 which is N or Q, a light chain CDR2 which comprises or consists of SEQ ID NO: 14, and a light chain CDR3 which comprises or consists of SEQ ID NO: 31; or (2) A heavy chain CDR1 comprising or consisting of SEQ ID NO:2, a heavy chain CDR2 comprising or consisting of SEQ ID NO:6, a heavy chain CDR3 comprising or consisting of SEQ ID NO:9, a light chain CDR1 comprising or consisting of SEQ ID NO:26, a light chain CDR2 comprising or consisting of SEQ ID NO:14, and a light chain CDR3 comprising or consisting of SEQ ID NO:32, or (3) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 3, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 22, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 10, and KSSQSLLDX. ７ D.X. ８ KTYLN (SEQ ID NO: 13), ７ is S and X ８ a light chain CDR1 which is G or A, a light chain CDR2 which comprises or consists of SEQ ID NO: 15, and a light chain CDR3 which comprises or consists of SEQ ID NO: 17; or (4) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 4, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 23, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 11, and KSSQSLLDX. ７ D.X. ８ KTYLN (SEQ ID NO: 13), ７ is G or A, and X ８ or G; a light chain CDR2 comprising or consisting of SEQ ID NO: 15; and a light chain CDR3 comprising or consisting of SEQ ID NO:

17. The antibody or antigen-binding fragment thereof of claim 1 ,

4. and / or the heavy chain CDR2 comprises a sequence selected from SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:23; and / or and / or the light chain CDR1 comprises a sequence selected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30; and / or The antibody or antigen-binding fragment thereof of claim 2, wherein the light chain CDR3 comprises an sequence selected from SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:

17.

5. A CDR according to any one of (1) to (12) below, namely: (1) A heavy chain CDR1 comprising or consisting of SEQ ID NO:1, a heavy chain CDR2 comprising or consisting of SEQ ID NO:18, a heavy chain CDR3 comprising or consisting of SEQ ID NO:8, a light chain CDR1 comprising or consisting of SEQ ID NO:24, a light chain CDR2 comprising or consisting of SEQ ID NO:14, and a light chain CDR3 comprising or consisting of SEQ ID NO:31, or (2) A heavy chain CDR1 comprising or consisting of SEQ ID NO:1, a heavy chain CDR2 comprising or consisting of SEQ ID NO:18, a heavy chain CDR3 comprising or consisting of SEQ ID NO:8, a light chain CDR1 comprising or consisting of SEQ ID NO:25, a light chain CDR2 comprising or consisting of SEQ ID NO:14, and a light chain CDR3 comprising or consisting of SEQ ID NO:31, or (3) A heavy chain CDR1 comprising or consisting of SEQ ID NO:1, a heavy chain CDR2 comprising or consisting of SEQ ID NO:19, a heavy chain CDR3 comprising or consisting of SEQ ID NO:8, a light chain CDR1 comprising or consisting of SEQ ID NO:24, a light chain CDR2 comprising or consisting of SEQ ID NO:14, and a light chain CDR3 comprising or consisting of SEQ ID NO:31, or (4) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 1, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 19, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 8, a light chain CDR1 comprising or consisting of SEQ ID NO: 25, a light chain CDR2 comprising or consisting of SEQ ID NO: 14, and a light chain CDR3 comprising or consisting of SEQ ID NO: 31, or (5) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 1, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 20, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 8, a light chain CDR1 comprising or consisting of SEQ ID NO: 24, a light chain CDR2 comprising or consisting of SEQ ID NO: 14, and a light chain CDR3 comprising or consisting of SEQ ID NO: 31, or (6) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 1, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 20, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 8, a light chain CDR1 comprising or consisting of SEQ ID NO: 25, a light chain CDR2 comprising or consisting of SEQ ID NO: 14, and a light chain CDR3 comprising or consisting of SEQ ID NO: 31, or (7) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 1, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 21, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 8, a light chain CDR1 comprising or consisting of SEQ ID NO: 24, a light chain CDR2 comprising or consisting of SEQ ID NO: 14, and a light chain CDR3 comprising or consisting of SEQ ID NO: 31, or (8) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 1, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 21, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 8, a light chain CDR1 comprising or consisting of SEQ ID NO: 25, a light chain CDR2 comprising or consisting of SEQ ID NO: 14, and a light chain CDR3 comprising or consisting of SEQ ID NO: 31, or (9) A heavy chain CDR1 comprising or consisting of SEQ ID NO:3, a heavy chain CDR2 comprising or consisting of SEQ ID NO:22, a heavy chain CDR3 comprising or consisting of SEQ ID NO:10, a light chain CDR1 comprising or consisting of SEQ ID NO:27, a light chain CDR2 comprising or consisting of SEQ ID NO:15, and a light chain CDR3 comprising or consisting of SEQ ID NO:17, or (10) A heavy chain CDR1 comprising or consisting of SEQ ID NO:3, a heavy chain CDR2 comprising or consisting of SEQ ID NO:22, a heavy chain CDR3 comprising or consisting of SEQ ID NO:10, a light chain CDR1 comprising or consisting of SEQ ID NO:28, a light chain CDR2 comprising or consisting of SEQ ID NO:15, and a light chain CDR3 comprising or consisting of SEQ ID NO:17, or (11) A heavy chain CDR1 comprising or consisting of SEQ ID NO: 4, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 23, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 11, a light chain CDR1 comprising or consisting of SEQ ID NO: 30, a light chain CDR2 comprising or consisting of SEQ ID NO: 15, and a light chain CDR3 comprising or consisting of SEQ ID NO: 17, or (12) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a heavy chain CDR1 comprising or consisting of SEQ ID NO: 4, a heavy chain CDR2 comprising or consisting of SEQ ID NO: 23, a heavy chain CDR3 comprising or consisting of SEQ ID NO: 11, a light chain CDR1 comprising or consisting of SEQ ID NO: 29, a light chain CDR2 comprising or consisting of SEQ ID NO: 15, and a light chain CDR3 comprising or consisting of SEQ ID NO:

17.

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 33 to 57, or an amino acid sequence having at least 85% identity to the aforementioned sequences.

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 58 to 82, or an amino acid sequence having at least 85% identity to the aforementioned sequences.

8. The antibody or antigen-binding fragment thereof may comprise the following group of heavy and light chain variable regions: (1) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61; or (2) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65; or (3) a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69; or (4) A heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73; or (5) A heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, and a light chain variable region having an amino acid sequence selected from SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76; or (6) The antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that it comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57, and a light chain variable region having an amino acid sequence selected from SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:

80.

9. The antibody or antigen-binding fragment thereof is selected from the group consisting of a heavy chain variable region and a light chain variable region of any one of the following (1) to (18): (1) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 38 and a light chain variable region whose amino acid sequence is SEQ ID NO: 62; or (2) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 38 and a light chain variable region whose amino acid sequence is SEQ ID NO: 63; or (3) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 38 and a light chain variable region whose amino acid sequence is SEQ ID NO: 64; or (4) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 39, and a light chain variable region whose amino acid sequence is SEQ ID NO: 63; or (5) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 39, and a light chain variable region whose amino acid sequence is SEQ ID NO: 64; or (6) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 39, and a light chain variable region whose amino acid sequence is SEQ ID NO: 65; or (7) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 46, and a light chain variable region whose amino acid sequence is SEQ ID NO: 71; or (8) a heavy chain variable region whose amino acid sequence is SEQ ID NO: 46, and a light chain variable region whose amino acid sequence is SEQ ID NO: 72; or (9) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 47, and a light chain variable region whose amino acid sequence is SEQ ID NO: 70; or (10) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 47, and a light chain variable region whose amino acid sequence is SEQ ID NO: 72; or (11) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 48, and a light chain variable region whose amino acid sequence is SEQ ID NO: 70; or (12) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 48, and a light chain variable region whose amino acid sequence is SEQ ID NO: 71; or (13) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 50, and a light chain variable region whose amino acid sequence is SEQ ID NO: 76; or (14) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 55, and a light chain variable region whose amino acid sequence is SEQ ID NO: 79, or (15) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 55, and a light chain variable region whose amino acid sequence is SEQ ID NO: 80; or (16) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 56, and a light chain variable region whose amino acid sequence is SEQ ID NO: 79; or (17) A heavy chain variable region whose amino acid sequence is SEQ ID NO: 56, and a light chain variable region whose amino acid sequence is SEQ ID NO: 80; or (18) The antibody or antigen-binding fragment thereof described in claim 8, characterized in that it comprises a heavy chain variable region whose amino acid sequence is SEQ ID NO:57, and a light chain variable region whose amino acid sequence is SEQ ID NO:

79.

10. The antibody or antigen-binding fragment thereof may be a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a single-chain antibody, a double-chain antibody, a triple-chain antibody, a quadruple-chain antibody, a Fab fragment, a F(ab') ２ 10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which is a Fv fragment, an scFv fragment, an Fv fragment, an Fab' fragment, or a domain antibody.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or antigen-binding fragment thereof binds to human CD3 and / or cynomolgus monkey CD3.

12. The antibody or antigen-binding fragment thereof of claim 11, which binds to CD3e / d and / or CD3e / g.

13. The antibody or antigen-binding fragment thereof (i) significantly activates total T cells, upregulates CD25 expression, and / or induces IFN-γ release, and / or (ii) inhibits 4×10 －８ K less than M Ｄ The antibody or antigen-binding fragment thereof of claim 11, which binds to human CD3 at a value of 1:

1.

14. An antibody or antigen-binding fragment thereof that competes with or cross-blocks binding to CD3, in particular CD3e / d or CD3e / g, with the antibody or antigen-binding fragment of any one of claims 1 to 13.

15. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

16. A nucleic acid vector comprising the isolated nucleic acid of claim 15.

17. 17. A host cell comprising the isolated nucleic acid of claim 15 or the nucleic acid vector of claim 16.

18. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, an isolated nucleic acid according to claim 15 or a nucleic acid vector according to claim 16, and a pharma- ceutically acceptable carrier.

19. 15. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, comprising incubating a host cell according to claim 17 under conditions permissive for expression of said antibody or antigen-binding fragment thereof.

20. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, an isolated nucleic acid according to claim 15, a nucleic acid vector according to claim 16 or a pharmaceutical composition according to claim 18 in the preparation of a medicament for inhibiting an immune response or for activating T cells.

21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the isolated nucleic acid of claim 15, the nucleic acid vector of claim 16 or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating cancer or an autoimmune disease.

22. 19. A method of inhibiting an immune response or activating T cells comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the isolated nucleic acid of claim 15, the nucleic acid vector of claim 16, or the pharmaceutical composition of claim 18.

23. A method for treating cancer or an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the isolated nucleic acid of claim 15, the nucleic acid vector of claim 16, or the pharmaceutical composition of claim 18.

24. 24. The use of claim 21 or the method of claim 23, wherein the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, large intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, bladder cancer, renal pelvis cancer, central nervous system tumor, glioma, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and T-cell lymphoma.

25. 24. The use according to claim 21 or the method according to claim 23, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, insulin-dependent diabetes mellitus, autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, Wegener's granulomatosis, Crohn's disease, ulcerative colitis, lupus such as systemic lupus erythematosus, atherosclerosis, chronic obstructive pulmonary disease, liver cirrhosis, renal transplant fibrosis, renal transplant nephropathy, and pulmonary fibrosis.

26. The method of any one of claims 22 to 25, wherein the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, the isolated nucleic acid of claim 15, the nucleic acid vector of claim 16 or the pharmaceutical composition of claim 18 is administered sequentially or simultaneously with another therapeutic agent.

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