CD3-targeting antibodies and uses thereof

Abstract

The present specification discloses antibodies that target CD3 and bispecific antibodies that target both CD3 and a tumor-associated antigen. The present specification also provides polynucleotides encoding these antibodies, pharmaceutical compositions containing these antibodies, and methods for producing the antibodies. Medical uses of the antibodies described herein are also disclosed.

Description

[Technical Field]

[0001] The present disclosure relates to molecular biology and immunology. The present disclosure provides an anti-CD3 antibody comprising a VHH fragment (a single variable domain VH of a heavy chain antibody), a bispecific antibody comprising the antibody, and the use of the antibody in disease treatment. [Background technology]

[0002] Bispecific antibodies targeting CD3 are a new therapeutic model in the field of cancer immunotherapy. In 2014, the FDA approved the anti-CD3 × CD19 bispecific T cell adapter blinatumomab for the treatment of patients with Philadelphia chromosome-negative B-cell acute lymphoblastic leukemia. More than 100 clinical trials have already been conducted using CD3 bispecific T cells to redirect cancer therapy, and some of these trials have shown favorable therapeutic effects in both hematological and solid tumors.

[0003] CD3 bispecific antibodies can simultaneously target CD3 on T cells and tumor-associated antigens (TAA) expressed on cancer cells. Upon cross-linking of these two cell types, CD3 bispecific antibodies can form an immune synapse similar to a natural TCR-MHC complex. This synapse secretes inflammatory cytokines and soluble cellular molecules that can activate T cells and, in the process, kill cancer cells. Unlike TCRs, CD3 bispecific antibodies can induce the redirected division of polyclonal T cells against cancer cells in a manner independent of MHC-peptide complexes. Therefore, CD3 bispecific antibodies can bind all available T cells (not just tumor-specific T cells) to target and divide tumors by redirecting other anti-TAA arms. Summary of the Invention [Problem to be solved by the invention]

[0004] With the accumulation of new knowledge in cancer biology and advances in antibody technology, many different forms of CD3 bispecific antibodies have been produced. However, to date, there have been no reports of bispecific antibodies using anti-CD3 single-domain antibodies. VHH antibodies derived from llama or alpaca heavy-chain antibodies retain their binding activity in a monomeric form. This offers the following advantages for constructing multispecific antibodies. First, by using VHH instead of VH and VL, the total number of chains can be reduced, avoiding mismatches due to unpaired chains. Second, due to their strict monomeric nature, VHH can be easily grafted onto different parts of a multivalent antibody without compromising their biophysical and biochemical properties. Since increasing evidence shows that redirecting the first or second signal of TAA alone with bispecific antibodies is not sufficient to treat solid tumors, there is a great need for trispecific antibodies, which include one arm targeting CD3 to activate T cells and participate in tumor division, one arm targeting the second signal to promote immune cell proliferation and anti-apoptosis, and a third arm targeting tumors. In such cases, anti-CD3 VHH antibodies can be easily grafted onto existing costimulatory or immune checkpoint inhibitor bispecific antibodies, which is of immense importance and great urgency. [Means for solving the problem]

[0005] The present specification provides antibodies that specifically bind to CD3, bispecific antibodies that further bind to tumor-associated antigens, related pharmaceutical compositions, polynucleotides, vectors, and recombinant cells. The present specification also provides methods for producing these antibodies and their uses.

[0006] Here, we demonstrate the discovery of anti-CD3 VHH antibodies. These anti-CD3 VHHs can bind to human CD3 protein and specifically bind to the human T lymphocyte line Jurkat, but not to a Jurkat mutant cell line in which CD3 / TCR is knocked out, and can activate Jurkat-NFAT-luciferase cells. Most importantly, when used in tumor-targeting bispecific antibodies constructed with anti-BCMA, anti-B7H4, or anti-ROR1 antibodies, the anti-CD3 VHHs can bind to T cells and disrupt tumor cells. Such functional anti-CD3 VHHs may play an essential role in the future involvement of T cells in cancer therapy.

[0007] A first aspect of the present disclosure provides an antibody that targets CD3, the antibody comprising a heavy chain variable region including HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17-18 and 20-22, HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 41-43, and HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 62-79.

[0008] In some embodiments, HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41 and 62, respectively.

[0009] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 18, 41, and 63, respectively.

[0010] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 64, respectively.

[0011] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 65, respectively.

[0012] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 66, respectively.

[0013] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 21, 42, and 64, respectively.

[0014] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 67, respectively.

[0015] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 68, respectively.

[0016] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 69, respectively.

[0017] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 70, respectively.

[0018] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 71, respectively.

[0019] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 72, respectively.

[0020] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 73, respectively.

[0021] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 74, respectively.

[0022] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 75, respectively.

[0023] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 76, respectively.

[0024] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 77, respectively.

[0025] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 22, 42, and 64, respectively.

[0026] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 43, and 64, respectively.

[0027] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 78, respectively.

[0028] In some other embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 79, respectively.

[0029] In this disclosure, these CDR regions are defined based on the Chothia numbering system.

[0030] In some embodiments, the heavy chain variable region (VH) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 107-108 and SEQ ID NOs: 110-132.

[0031] In some other embodiments, the VH fragment has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:107-108 and SEQ ID NOs:110-132.

[0032] In some embodiments, the antibody further comprises a heavy chain constant region.

[0033] In some preferred embodiments, the heavy chain constant region is derived from hIgG1, hIgG2, hIgG3 and hIgG4 and variants thereof, more preferably the heavy chain constant region is derived from hIgG1.

[0034] In some embodiments, the antibody further comprises one or more mutations in the heavy chain constant region.

[0035] In some preferred embodiments, the mutations are selected from L234A, L235A, G237A, P329G (Eu numbering), or any combination.

[0036] In some embodiments, the antibody is a heavy chain-only antibody, wherein the heavy chain-only antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs: 141-142 and SEQ ID NOs: 144-166.

[0037] In some embodiments, antibodies targeting CD3 may be in the form of HCAbs, VHHs, nanobodies, Fabs, Fab's, F(ab')2s, Fds, Fd's and dAbs.

[0038] A second aspect of the present disclosure provides a bispecific antibody, the bispecific antibody comprising a first antigen-binding fragment that targets CD3 and a second antigen-binding fragment that targets a tumor-associated antigen (TAA), wherein the first antigen-binding fragment that targets CD3 is as described in the first aspect.

[0039] In some embodiments, the TAA is CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, sLe, GM3, TGS5, HMWMAA, GD2, FOLR 1, FOLR2, TEM1 / CD248, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6-AML, SPA17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY- TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL 1, CD20, CD30, HER2, ROR1, FLT3, TAAG72, CD22, CD33, GD2, gp100Tn, FAP, TYR, EPCAM, CEA, IGF-1R, EphB2, MSLN, CDH17, CD32b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C, and TACSTD2.

[0040] In some preferred embodiments, the TAA is BCMA, B7H4, or ROR1.

[0041] In some specific embodiments, the second antigen-binding fragment targets B7H4.

[0042] In some preferred embodiments, the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively, and these CDR regions are defined according to the Chothia numbering system.

[0043] In some specific embodiments, the second antigen-binding fragment targets BCMA.

[0044] In some embodiments, the second antigen-binding fragment comprises one heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 12, 36, and 57, respectively, and these CDR regions are defined according to the Chothia numbering system.

[0045] In some other specific embodiments, the second antigen-binding fragment targets ROR1.

[0046] In some embodiments, the second antigen-binding fragment comprises one heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 15, 39, and 60, respectively, and these CDR regions are defined according to the Chothia numbering system.

[0047] In some exemplary embodiments, the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 62, respectively; and the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 12, 36, and 57, respectively.

[0048] In some other exemplary embodiments, the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 18, 41, and 63, respectively; and the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 12, 36, and 57, respectively.

[0049] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 62, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0050] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 18, 41, and 63, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0051] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0052] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 65, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0053] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 66, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0054] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 21, 42, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0055] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 67, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0056] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 68, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0057] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 69, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0058] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 70, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0059] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 71, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0060] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 72, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0061] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 73, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0062] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 74, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0063] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 75, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0064] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 76, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0065] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 77, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0066] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 22, 42, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0067] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 43, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0068] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 78, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0069] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 79, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively.

[0070] In some other exemplary embodiments, the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 62, respectively; and the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 15, 39, and 60, respectively.

[0071] In this disclosure, the CDRs are defined by any numbering system known in the art, such as the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, the AHO numbering system, the ANARCI numbering system, and the CONTACT numbering system, etc. In some preferred embodiments, the CDRs are defined by the Chothia numbering system.

[0072] In the above embodiment, the CDR regions are defined according to the Chothia numbering system.

[0073] In some exemplary embodiments, the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:107, and the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:101.

[0074] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:108, and the second antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:101.

[0075] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:107, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0076] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:108, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0077] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:110, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0078] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:111, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0079] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:112, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0080] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:113, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0081] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:114, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0082] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:115, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0083] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:116, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0084] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:117, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0085] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:118, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0086] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:119, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0087] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:120, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0088] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:121, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0089] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:122, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0090] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:123, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0091] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:124, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0092] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:125, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0093] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:126, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0094] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:127, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0095] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:128, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0096] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:129, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0097] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:130, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0098] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:131, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0099] In some other exemplary embodiments, the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:132, and the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO:134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO:103.

[0100] In some other exemplary embodiments, the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:107, and the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:105.

[0101] In some embodiments, the bispecific antibody comprises one polypeptide having the amino acid sequence shown in SEQ ID NO:172.

[0102] In some other embodiments, the bispecific antibody comprises one polypeptide having the amino acid sequence shown in SEQ ID NO:173.

[0103] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:175 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:170.

[0104] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:176 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:170.

[0105] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:177 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0106] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:179 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0107] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:180 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0108] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:181 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0109] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:182 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0110] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:183 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0111] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:185 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0112] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:186 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0113] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:187 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0114] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:188 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0115] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:189 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0116] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:190 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0117] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:191 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0118] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:192 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0119] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:193 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0120] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:194 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0121] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:195 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0122] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:196 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0123] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:197 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0124] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:198 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0125] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:199 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0126] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:200 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0127] In some other embodiments, the bispecific antibody comprises two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:201 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:178.

[0128] In some other embodiments, the bispecific antibody comprises one polypeptide having the amino acid sequence shown in SEQ ID NO:184.

[0129] In some embodiments, the antibody that targets CD3 is a chimeric antibody, a humanized antibody, or a human antibody.

[0130] In the present disclosure, the antibody or antigen-binding fragment that specifically binds to BCMA, or the antibody or antigen-binding fragment that specifically binds to B7H4, or the antibody or antigen-binding fragment that specifically binds to ROR1 is preferably selected from Fab, Fv, VHH, and scFv.

[0131] In the present disclosure, an antibody or antigen-binding fragment that specifically binds to BCMA is preferably an anti-BCMA VHH, an antibody or antigen-binding fragment that specifically binds to B7H4 is an anti-B7H4 scFv, and an antibody or antigen-binding fragment that specifically binds to ROR1 is preferably an anti-ROR1 VHH.

[0132] In some embodiments, the bispecific antibody comprises: a) a polypeptide comprising an anti-CD3 VHH-anti-BCMA VHH-mono Fc, or b) a first polypeptide comprising an anti-CD3 VHH-Fc and a second polypeptide comprising an anti-B7H4 scFv-Fc, wherein the two Fc moieties form a dimeric Fc region (in the form of a VH-Fc-scFv); or c) comprises a polypeptide comprising two copies of an anti-ROR1 VHH and one copy of an anti-CD3 VHH.

[0133] In some embodiments, one Fc interacts with another Fc via common technical means known in the art, such as chemical bonding or knob-into-hole structures.

[0134] In some specific embodiments, the bispecific antibody comprises a heavy chain constant region, which may have a mutation combination comprising L234A and L235A (LALA), or a mutation combination comprising L234A, L235A and G237A (AAA), or a mutation combination comprising L234A, L235A and P329G (AAG) to eliminate ADCC effects.

[0135] A third aspect of the present disclosure provides a polynucleotide, which encodes the CD3-targeting antibody according to the first aspect or the bispecific antibody according to the second aspect.

[0136] In some embodiments, the polynucleotide is selected from DNA or RNA.

[0137] In some embodiments, the polynucleotide is mRNA.

[0138] A fourth aspect of the present disclosure provides a vector, which comprises a polynucleotide according to the third aspect.

[0139] A fifth aspect of the present disclosure provides a cell, which comprises an antibody targeting CD3 according to the first aspect, a bispecific antibody according to the second aspect, a polynucleotide according to the third aspect or a vector according to the fourth aspect.

[0140] A sixth aspect of the present disclosure provides a method for preparing an antibody that targets CD3 according to the first aspect or a bispecific antibody according to the second aspect, wherein the method comprises culturing cells comprising the polynucleotide or vector according to the fifth aspect and obtaining the antibody that targets CD3 or the bispecific antibody from the culture.

[0141] A seventh aspect of the present disclosure provides a pharmaceutical composition, comprising an antibody targeting CD3 according to the first aspect, or a bispecific antibody according to the second aspect, or a polynucleotide according to the third aspect, or a vector according to the fourth aspect, or a cell according to the fifth aspect, and optionally a pharmaceutically acceptable carrier.

[0142] An eighth aspect of the present disclosure provides a use of an antibody targeting CD3 according to the first aspect, or a bispecific antibody according to the second aspect, or a polynucleotide according to the third aspect, or a vector according to the fourth aspect, or a cell according to the fifth aspect, or a pharmaceutical composition according to the seventh aspect, in the manufacture of a medicament for treating a disease.

[0143] A ninth aspect of the present disclosure provides use of the antibody targeting CD3 according to the first aspect, or the bispecific antibody according to the second aspect, or the polynucleotide according to the third aspect, or the vector according to the fourth aspect, or the cell according to the fifth aspect, or the pharmaceutical composition according to the seventh aspect, as a medicament, preferably the medicament being used for preventing and / or treating a disease.

[0144] A tenth aspect of the present disclosure provides a method for preventing and / or treating a disease in a subject in need thereof, the method comprising administering an effective amount of an antibody targeting CD3 according to the first aspect, or a bispecific antibody according to the second aspect, or a polynucleotide according to the third aspect, or a vector according to the fourth aspect, or a cell according to the fifth aspect, or a pharmaceutical composition according to the seventh aspect. In some preferred embodiments, the effective amount is effective for treatment.

[0145] An eleventh aspect of the present disclosure provides a composition for preventing and / or treating a disease, wherein the composition comprises an antibody targeting CD3 according to the first aspect, or a bispecific antibody according to the second aspect, or a polynucleotide according to the third aspect, or a vector according to the fourth aspect, or a cell according to the fifth aspect, or a pharmaceutical composition according to the seventh aspect.

[0146] A twelfth aspect of the present disclosure provides a composition, which is used in the manufacture of a medicament for preventing and / or treating a disease, wherein the composition comprises an antibody targeting CD3 according to the first aspect, or a bispecific antibody according to the second aspect, or a polynucleotide according to the third aspect, or a vector according to the fourth aspect, or a cell according to the fifth aspect, or a pharmaceutical composition according to the seventh aspect.

[0147] In the present disclosure, the disease is selected from myeloma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, nasopharyngeal cancer, oral cancer, esophageal cancer, gastric cancer, bladder cancer and lymphoma. [Brief explanation of the drawings]

[0148] [Figure 1] 1 shows the binding activity of antibodies to Jurkat cells or J.RT3-T3.5 cells measured by FACS. [Figure 2] 1 shows the binding activity of antibodies to CHOK1 / TCR / CD3 or CHO-K1 cells measured by FACS. [Figure 3] Luciferase luminescence signals produced after Jurkat-NFAT-Luc reporter cells were activated by binding with anti-CD3 antibody are shown. [Figure 4] The diagram shows the structures for producing various forms of recombinant antibodies by fusing the VHH region with monoFc or general human IgG1 Fc. [Figure 5] SDS-PAGE results after transient expression and affinity capture of recombinant A1-monoFc (PR005860) and E2-monoFc (PR005861) are shown. [Figure 6] Figure 1 shows that A1-monoFc (PR005860) has a dose-dependent effect on T cell-mediated cytotoxicity of NCI-H929 cells, which highly express BCMA. [Figure 7] The HCAb forms of PR006364 (A1-HCAb) and PR010991 (3G11-HCAb) are shown. [Figure 8] The results of antibody binding activity to Jurkat cells (A), J.RT3-T3.5 cells (B), or Jurkat-NFAT-Luc cells (C) measured by FACS are shown. [Figure 9-1] The results of ELISA binding to multiple CD3 recombinant proteins (A to C) are shown. [Figure 9-2] The results of ELISA binding to multiple CD3 recombinant proteins (D to F) are shown. [Figure 10] The bispecific antibody forms of VH-Fc-scFv and Fab-Fc-scFv are shown, and the LALA mutation refers to a mutation combination including L234A and L235A. [Figure 11] 1 shows the cytotoxicity (A to B) of the B7H4×CD3 bispecific antibody against MDA-MB-468 cells that highly express B7H4, as measured by the RTCA method. [Figure 12] 1 shows the binding activity of humanized variants derived from anti-CD3 A1 to the human CD3e&d heterodimeric protein. [Figure 13] Binding activity of anti-CD3 A1-derived humanized variants to Jurkat-NFAT-Luc reporter cells is shown. [Figure 14] Figure 1 shows the cytotoxicity of the B7H4xCD3 bispecific antibody derived from the anti-CD3 humanized A1 variant against MDA-MB-468 cells, which highly express B7H4, as measured by the RTCA method. [Figure 15] Figure 1 shows the cytotoxicity of the B7H4xCD3 bispecific antibody derived from the anti-CD3 humanized A1 variant against B7H4-negative MDA-MB-231 cells as measured by the RTCA method. [Figure 16] Binding activity (A to B) to cynomolgus monkey PBMCs measured by FACS is shown. [Figure 17] Figure 1 shows the cytotoxicity of the anti-CD3 3G11-derived B7H4 x CD3 bispecific antibody PR008551 against MDA-MB-468 cells in the presence of cynomolgus monkey PBMCs (A-B). [Figure 18]1 shows an alignment of the VH sequences of 3G11 VH and its mutants. [Figure 19] 1 shows the cytotoxicity of B7H4×CD3 bispecific antibodies produced by 3G11-derived variants against MDA-MB-468 cells (A-C). [Figure 20] 1 shows the cytotoxicity of anti-CD3 A1-derived ROR1xCD3 bispecific antibodies against PANC-1 cells. [Figure 21] The graph shows the change in tumor volume in each treatment group in the PANC-1 PBMC model. DETAILED DESCRIPTION OF THE INVENTION

[0149] [Definition] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Also, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those known and commonly used in the art.

[0150] It should be noted that the term "one" or "one" entity refers to one or more of that entity, e.g., "an antibody" should be understood to represent one or more antibodies. As such, the terms "one" (or "one"), "one or more," and "at least one" can be used interchangeably herein.

[0151] As used herein, the term "and / or" should be considered to mean that each of the two named features or components is either specifically disclosed or not with the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following aspects: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0152] The terms "polypeptide," "peptide," "protein," "polypeptide chain," "peptide chain," and grammatical equivalents thereof can be used interchangeably herein and refer to a polymer of amino acids of any length, which may be linear or branched. It may contain non-natural or modified amino acids or may be interrupted by non-amino acids. A polypeptide, peptide, polypeptide chain, peptide chain, or protein may be further modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

[0153] The terms "polynucleotide," "nucleic acid," and grammatical equivalents can be used interchangeably herein to refer to polymers of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

[0154] As used herein, the term "specifically binds" refers to more frequent, more rapid, longer lasting, or higher affinity interactions of a polypeptide or molecule with an epitope, protein, or target molecule, or more frequent, more rapid, longer lasting, or higher affinity interactions with some combination of the above, compared to alternative substances (including related and unrelated proteins).

[0155] The terms "same," "percent identity," and grammatical equivalents thereof, as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence (introducing gaps, if necessary), have the same or a specified percentage of the same nucleotide or amino acid residues, without considering any conservative amino acid substitutions as part of the sequence identity.

[0156] The term "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody, any antigen-binding fragment thereof, or a single chain thereof. Thus, the term "antibody" includes any protein or peptide containing the following molecules, including at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen: a heavy or light chain complementarity-determining region (CDR) or ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region (FWR), or any portion thereof, or at least a portion of a binding protein. The CDRs of the light and heavy chains are primarily responsible for the antibody's interaction with the antigen. Each VH or VL generally consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4 (see further Chothia and Lesk, J. Mol. Biol., 1987, vol. 196, pp. 901-17).

[0157] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the β-sheet framework of an immunoglobulin (Ig or antibody) VH, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the β-sheet framework of an antibody VL. CDR regions (HCDR1, HCDR2, and HCDR3 refer to the three CDRs of the VH, and LCDR1, LCDR2, and LCDR3 refer to the three CDRs of the VL) are well known to those skilled in the art and have been defined by several methods / systems. These systems and / or definitions have been developed and refined over the years and include Kabat, Chothia, IMGT, AbM, and CONTACT. Software programs (e.g., abYsis) for analyzing antibody sequences and determining CDRs are available and known to those skilled in the art. Unless otherwise specified, amino acid sequences are written using the Chothia numbering system.

[0158] The term "antibody" covers a wide variety of biochemically distinguishable polypeptides. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and several subclasses thereof (e.g., gamma to gamma). The nature of the chain determines the "class" of the antibody, which is IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, are well characterized and are known to have specialized functional capabilities. According to the present disclosure, modifications of each of these classes and isotypes are readily discernible by those skilled in the art and, therefore, are within the scope of the present disclosure. While all immunoglobulin classes are expressly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. The structure of immunoglobulins has been well characterized (see, e.g., Fundamental Immunology, Chapter 7, edited by Paul, W., 2nd ed., Raven Press, NY (1989)). Generally, immunoglobulins comprise two pairs of polypeptide chains, one pair of light chains and one pair of heavy chains, with all four pairs of chains linked to each other via disulfide bonds.

[0159] Each light chain of an immunoglobulin generally comprises a light chain variable region ("VL region") and a light chain constant region ("CL region"). Based on the amino acid sequence of the CL region, two different types of light chains exist, called lambda (λ) and kappa (κ). The amino acid sequence of the CL region is known in the art. Each heavy chain generally comprises a heavy chain variable region ("VH region") and a heavy chain constant region ("CH region").

[0160] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of immunoglobulin heavy (VH) and light (VL) chains. In some embodiments, these regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility, serine- or threonine-rich for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant regions and the introduction of the linker, the protein still retains the specificity of the original immunoglobulin. ScFv molecules are known and described in the art.

[0161] As used herein, the term "Fc region" includes native sequence Fc regions and variant Fc regions. In some embodiments, the Fc domains of the two heavy chains of a bispecific antibody according to the present invention may contain paired modifications that promote their association with each other instead of forming homodimers.

[0162] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, or single-chain fragment variable region (scFv). Regardless of structure, an antibody fragment binds with the same antigen recognized by the whole antibody. The term "antibody fragment" includes aptamers, enantiomers, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy-chain antibodies (HCAbs), light-chain antibodies (LCAbs), disulfide-linked scFvs (dsscFv), diabodies, triabodies, tetrabodies, microbodies, dual variable domain antibodies (DVDs), single variable domain antibodies (sdAbs, e.g., camelid antibodies, alpaca antibodies), and single variable domains of heavy-chain antibodies (VHHs), as well as bispecific or multispecific antibodies formed by antibody fragments. The term "antibody fragment" further includes any synthetic or genetically engineered protein, which acts like an antibody by binding to a specific antigen to form a complex.

[0163] The terms "humanized," "humanized," and "human-like" can be used interchangeably and refer to an antibody in which at least one binding domain contains at least one complementarity-determining region ("CDR") derived from a non-human antibody or a fragment thereof. Humanization methods are described, for example, in WO 91 / 09968 and U.S. Pat. No. 6,407,213. As a non-limiting example, the term covers cases in which the variable region of at least one binding domain contains a single CDR region (e.g., the third CDR region of a VH) derived from another non-human animal (e.g., a rodent), and one or two variable regions contain CDRs derived from the non-human animal in each of their corresponding first, second, and third CDRs. When all CDRs of an antibody binding domain are replaced with their corresponding counterparts, for example, derived from a rodent (commonly referred to as "CDR-grafting"), the term should be understood to be included in the term "humanized" or its grammatically related variants as used herein. The term "humanized" or its grammatically related variants further covers the case where, in addition to the replacement of one or more CDR regions within the VH and / or VL of the first and / or second binding domain, a further mutation (e.g., substitution) of at least one single amino acid residue in the framework region ("FR") between the CDRs is further carried out so that the amino acid at that / those positions corresponds to the amino acid at that / those positions in the animal from which the CDR regions for replacement are derived. As is known in the art, such a single mutation in the framework region is generally carried out after CDR grafting in order to restore the initial binding affinity of the non-human antibody used as a CDR donor to its target molecule. In addition to the amino acid substitution in the framework region as described above, the term "humanization" may further cover the replacement of amino acids in the CDR regions derived from a non-human animal with amino acids in the corresponding CDR regions derived from a human antibody. Polynucleotides

[0164] The term "polynucleotide" covers polynucleotides that contain only the coding sequence for a polypeptide as well as polynucleotides that contain additional coding and / or non-coding sequences. Polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA may be cDNA, genomic DNA, or synthetic DNA, and may be double-stranded or single-stranded. Single-stranded DNA may be the coding (sense) strand or the non-coding (antisense) strand. Polynucleotides of the present disclosure may be mRNA.

[0165] In some embodiments, the present specification provides polynucleotides encoding the anti-CD3 antibodies and bispecific antibodies. As used herein, the term "code" and its grammatical equivalents refer to the inherent property of a particular nucleotide sequence in a polynucleotide or nucleic acid (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, these polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties produced thereby. Thus, a gene encodes a protein when transcription and translation of mRNA corresponding to the gene produces the protein. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may contain introns.

[0166] In some embodiments, a polynucleotide comprises a coding sequence for a polypeptide (e.g., an antibody) fused in the same reading frame to a polynucleotide responsible for the expression and secretion of the polypeptide from a host cell (e.g., a leader sequence used as a secretory sequence to control polypeptide transport). The polypeptide may have a leader sequence in its "mature" form that is cleaved by the host cell to form the polypeptide.

[0167] In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure.

[0168] [vector] As used herein, the term "vector" and its grammatical equivalents refer to a vehicle for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell and replicated and / or expressed in the host cell. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes. These vectors may contain operable selection sequences or markers that can be stably integrated into a host cell chromosome. These vectors may also contain one or more selection marker genes and appropriate expression control sequences. The included selection marker genes may, for example, provide resistance to antibiotics or toxins, complement nutrient deficiencies, or provide important nutrients missing from the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as known in the art. When two or more polynucleotides are coexpressed, the two polynucleotides can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoded polynucleotides can be operably linked to a common expression control sequence, or can be linked to different expression control sequences, for example, one inducible promoter and one constitutive promoter. Introduction of the polynucleotide into the host cell can be confirmed using methods known in the art. Those skilled in the art will understand that the polynucleotide will be expressed in an amount sufficient to produce the desired product, and will further understand that expression levels can be optimized to obtain sufficient expression using methods known in the art.

[0169] [cell] Multiple expression vectors can be used to co-transfect a host cell, with each expression vector encoding a polypeptide chain of an antibody described herein. These vectors may contain the same selection marker, which allows for equal expression of all polypeptides. Optionally, a single vector can be used to encode two or more polypeptides. The coding sequences for the polypeptides of the compounds described herein may comprise cDNA or genomic DNA.

[0170] [Production method] Antibodies or polypeptides described herein can be produced and isolated using methods known in the art. Polypeptides can be synthesized in whole or in part using chemical methods. Peptide synthesis can be performed using a variety of solid-phase techniques, and automated synthesis is feasible. Peptides can also be synthesized by combinatorial methods. Synthetic residues and polypeptides can be synthesized using a variety of programs and methods known in the art.

[0171] A variety of host-expression vector systems can be used for recombinant expression of the antibodies described herein or one or more polypeptide chains thereof. Suitable host cells for expression include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells controlled by a suitable promoter. Suitable clones and expression vectors and methods for protein production for bacterial, fungal, yeast, and mammalian cell hosts, including antibody production, are known in the art. Such host-expression systems represent vehicles in which the coding sequences of the antibodies described herein can be produced and subsequently purified, and further represent cells which, when transformed or transfected with an appropriate polynucleotide coding sequence, can express the antibodies described herein in situ.

[0172] Once recombinantly expressed, the antibodies described herein or the polypeptides described herein can be purified by any method known in the art for purifying polypeptides, polyproteins, or antibodies (e.g., similar to antibody purification schemes based on antigen selectivity), such as chromatography (e.g., ion exchange, affinity, particularly affinity for a particular antigen (optionally after Protein A selection, where the compound comprises an Fc domain (or portion thereof)), and classifier column chromatography), centrifugation, differential solubility, or any other standard technique for purifying polypeptides or antibodies.

[0173] [Pharmaceutical composition] The term "pharmaceutically acceptable carrier" or "drug excipient" refers to a substance that is suitable for administration to an individual, together with an active agent, without causing undesired biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition.

[0174] In some embodiments, the pharmaceutical compositions disclosed herein may include one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, and / or a surfactant, and various combinations thereof. The use of preservatives, tonicity agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those of skill in the art.

[0175] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous glucose solutions, and glycerin solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat powdered milk, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methyl p-hydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for adjusting tonicity such as sodium chloride or glucose may also be considered. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be prepared into suppositories with conventional adhesives and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. The formulation should be adapted to the mode of administration. The parent formulation can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0176] Standard clinical techniques can be used to determine the amount of an antibody, fragment, or antibody-drug conjugate of the disclosure that effectively treats, inhibits, and prevents inflammatory, immune, or malignant diseases, disorders, or conditions. In vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will further depend on the route of administration and the severity of the disease, disorder, or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0177] [Treatment method] As used herein, the terms "administration" or "drug administration" and their grammatical equivalents refer to the act of delivering or causing a therapeutic agent or pharmaceutical composition to be delivered to the body of a subject using methods described herein or other methods known in the art. Administration may be systemic or local. A therapeutic agent may be a compound, polypeptide, antibody, cell, or cell population. Administering a therapeutic agent or pharmaceutical composition includes formulating the therapeutic agent or pharmaceutical composition for delivery into the body of a subject. Exemplary dosage forms include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms including creams, gels, powders, or patches; buccal dosage forms; inhalation powders; sprays; suspensions; and rectal suppositories.

[0178] In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions used for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may further include a solubilizing agent and a local anesthetic, such as lignocaine, to reduce pain at the injection site. Typically, these ingredients are provided singly or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container (e.g., an ampoule or sachet) indicating the quantity of active agent. When administered by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical-grade water or saline. When administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to use.

[0179] As used herein, the terms "effective amount," "therapeutically effective amount," and grammatical equivalents refer to the administration of an agent, alone or as part of a pharmaceutical composition, and in a single dose or as part of a series of doses, to a subject in an amount that, when administered to a subject, is capable of having any detectable positive effect on any symptom, aspect, or feature of a disease, disorder, or condition. A therapeutically effective amount can be determined by measuring the relevant physiological effect. The precise amount required will depend on the subject, and will depend on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate "effective amount" in any individual case can be ascertained by one of ordinary skill in the art using routine experimentation.

[0180] The terms "treatment" or "treatment" refer to therapeutic treatment and prophylactic or preventative measures, where the objective is to prevent or alleviate (reduce) the progression of an undesirable physiological change or disorder, such as cancer. Beneficial or desired clinical results include alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delaying or slowing of disease progression, improvement or reduction in the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder and those prone to the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0181] The terms "subject" or "individual" or "animal" or "patient" or "mammal" refer to any subject in need of diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, and the like.

[0182] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects (e.g., mammalian subjects) who would benefit from the administration of an antibody or composition of the disclosure, wherein the antibody or composition is used, for example, in a detection, diagnostic program, and / or treatment.

[0183] The following examples are for illustrative purposes only, and unless otherwise specified, these examples are not intended to be limiting. As such, the present disclosure is not limited in any way to the following examples, but should be construed to cover any and all variations that become apparent from the teachings herein.

[0184] In order to describe the technical solution of the present disclosure in detail, preferred embodiments of the present disclosure will be described below based on the drawings. [Example]

[0185] Example 1: Immunization of alpacas and screening of anti-CD3 VHH antibodies Alpacas were immunized with human pan T cells at 3-week intervals. After immunization for over 3 months, 50 mL of alpaca blood was extracted and used for PBMC isolation. A phage library encoding VHH gene fragments was constructed using a standard scheme (DOI: 10.1038 / nprot.2014.039).

[0186] In the first panning run, the phage library was panned against cynomolgus monkey CD3 epsilon (cyno CD3e) protein (ACRO Biosystems, CDE-C5226) and Jurkat (ATCC, TIB-152) cells. VHH genes from the panning export pool were cloned into the pTT5-VHH-anti-BCMA-his vector, which linked the VHH gene fragment to the N-terminus of the VH region (SEQ ID NO: 101) from the previously identified anti-BCMA HCAb PR001046 (disclosed in patent WO 2021063349A1). Single plasmids carrying the tandem anti-CD3 VHH and anti-BCMA VH were then prepared before transfection. HEK293T cells were transfected with each single plasmid carrying the tandem anti-CD3 VHH and anti-BCMA VH, and the supernatants were harvested 96 hours after transfection. The supernatants were then screened for binding to Jurkat cells by FACS. Two clones (A1 and E2) were identified as positive hits that bound to Jurkat cells. Their VHH genes were cloned into the pTT5-VHH-anti-BCMA-monoFc vector for protein expression, designated A1-monoFc (PR005860) and E2-monoFc (PR005861), respectively, as shown in Figure 4. The amino acid sequences of A1 and E2 and their CDR regions are listed in Table 1.

[0187] In the second panning run, the phage library was panned against cynomolgus monkey CD3 epsilon and delta heterodimer (cyno CD3e / d) protein (ACRO Biosystems, CDD-C52W9) and cynomolgus monkey PBMC cells. VHH genes from the panning export pool were cloned into the pTT5-VHH-monoFc vector, which carries human IgG Fc variants and allows them to be accurately expressed in a monomeric form, rather than the normal Fc homodimeric form. HEK293T cells were transfected with each of the prepared single plasmids, and the supernatants were harvested 96 hours after transfection. The supernatants were then screened for binding to Jurkat cells by FACS. One clone (3G11) was identified as a positive hit that bound to Jurkat, and its VHH gene was cloned into the pTT5-VHH-hFc vector for protein expression, designated as HCAb PR010991, as shown in Figure 7. The amino acid sequence of 3G11 and its CDR regions are listed in Table 1.

[0188] [Table 1]

[0189] As shown in Figure 4, the A1 and E2 VHH genes were cloned into the pTT5-VHH-anti-BCMA-monoFc vector, which carried the mutated human Fc sequence monoFc67 (SEQ ID NO:203) to produce the recombinant antibodies A1-monoFc (PR005860) and E2-monoFc (PR005861), respectively. PR005860 and PR005861 share the same anti-BCMA VH fragment, where the amino acid sequence of the anti-BCMA VH fragment is as shown in SEQ ID NO:101. HCDR1, HCDR2, and HCDR3 of the anti-BCMA VH fragment are as shown in SEQ ID NOs:12, 36, and 57, respectively, and are defined, for example, according to the Chothia numbering system.

[0190] The A1 and E2 VHH genes were also cloned into the pTT5-VHH-hFc vector to produce the recombinant antibodies A1-HCAb (PR006364) and E2-HCAb (PR006365), respectively. A C220S mutation was introduced into the hinge region (located between the VH and CH2 of the Fc) to prevent disulfide bond formation between the Cys residue at position 220 (Eu numbering system) or other free thiol groups. To eliminate Fc effector functions, such as ADCC, a triple mutation (L234A, L235A, P329G, designated "AAG") was introduced into positions 234, 235, and 329 (Eu numbering system) of the CH2 domain, respectively.

[0191] The 3G11 VHH gene was cloned into the pTT5-VHH-hFc vector to produce the recombinant antibody 3G11-HCAb (PR010991), as shown in Figure 7. A C220S mutation was introduced into the hinge region, and a triple mutation L234A, L235A, G237A (represented as "AAA") was introduced into CH2 to eliminate Fc effector functions.

[0192] The amino acid sequences of A1-monoFc (PR005860), E2-monoFc (PR005861), A1-HCAb (PR006364), E2-HCAb (PR006365), and 3G11-HCAb (PR010991) are shown in Table 2.

[0193] [Table 2-1] [Table 2-2] [Table 2-3]

[0194] Example 2: Evaluation of the binding activity of the supernatant of recombinant anti-CD3 antibodies to CD3-expressing cells The A1-monoFc (PR005860) and E2-monoFc (PR005861) plasmids were transfected into 293F host cells (ThermoFisher, R79007), respectively. After culturing the cells for 3 to 5 days, the supernatant was harvested and then exchanged into PBS buffer by ultracentrifugation at 10 KDa. The PBS solution containing the antibody was defined as the stock solution for binding and reporter gene assays.

[0195] Binding activity to Jurkat or J.RT-T3.5 cell lines

[0196] J.RT3-T3.5 cells (ATCC, TIB-153) are a derivative mutant of the Jurkat leukemia cell line (ATCC, TIB-152) that lacks the β chain of the T cell antigen receptor and does not express CD3 or the T cell receptor α / β heterodimer on the cell surface.

[0197] The binding activity of the supernatants of A1-monoFc (PR005860) and E2-monoFc (PR005861) to target cells was assessed by FACS. Supernatants of recombinant antibodies (i.e., A1-monoFc, E2-monoFc) were diluted from the original solution to eight different concentrations using three-fold serial dilutions. The diluted samples were incubated with target cells (Jurkat cells (ATCC, TIB-152) or J.RT-T3.5 cells (ATCC, TIB-153)) for 1 hour, followed by the addition of a secondary antibody (Alexa Fluor™ 488 AffinityPure goat anti-human IgG (Fcγ fragment specific), Jackson ImmunoResearch, 109-545-098) and incubation for 1 hour. The cells were washed twice with staining buffer and subjected to flow cytometry. Data were processed by FlowJo v10 (FlowJo, LLC) and analytical nonlinear curve fitting was analyzed by GraphPad Prism 8.

[0198] As shown in Figure 1, the recombinant antibodies A1-monoFc (PR005860) and E2-monoFc (PR005861) could strongly bind to CD3 / TCR-positive Jurkat cells in a dose-dependent manner, but showed little interaction with CD3 / TCR-negative J.RT-T3.5 cells, indicating that they could specifically bind to the CD3 / TCR complex.

[0199] [Binding activity to CHOK1 / TCR / CD3 recombinant cell lines]

[0200] CHOK1 / TCR / CD3 is a recombinant stable cell line produced on CHO-K1 (ATCC, CCL-61) that overexpresses the human CD3 / TCR complex, which has multiple polypeptide subunits, including the human TCR α chain, TCR β chain, and human CD3 ζ chain, CD3 ε chain, CD3 δ chain, and CD3 γ chain. Native CHO-K1 cells do not express CD3 or TCR.

[0201] Recombinant antibody (i.e., A1-monoFc, E2-monoFc) supernatants were diluted from the original solution to six different concentrations using a three-fold serial dilution. The diluted samples were incubated with CHO-K1 or CHOK1 / TCR / CD3 cells for 1 hour, followed by the addition of a secondary antibody (Alexa Fluor™ 488 AffinityPure goat anti-human IgG (Fcγ fragment specific), Jackson ImmunoResearch, 109-545-098) and incubation for 1 hour. Cells were washed twice with staining buffer and subjected to flow cytometry. Data were processed using FlowJo v10 (FlowJo, LLC), and nonlinear curve fitting analysis was performed using GraphPad Prism 8.

[0202] As shown in Figure 2, A1-monoFc and E2-monoFc could strongly bind to CHOK1 / TCR / CD3 cells but could not bind to CHO-K1 cells, indicating that they could specifically bind to the CD3 / TCR complex.

[0203] Example 3: T cell activation bioassay performed with Jurkat-NFAT-Luc reporter cells Jurkat-NFAT-Luc is a genetically engineered Jurkat cell line that expresses endogenous TCR, CD3, and CD28 receptors and a luciferase reporter driven by an NFAT-response element. When the TCR / CD3 complex on Jurkat-NFAT-Luc is bound by an anti-TCR / CD3 antibody, the TCR / CD3 transduces the transcription of the downstream NFAT gene within the cell, and the expressed NFAT transcription factor binds to the luciferase gene promoter and regulates luciferase gene expression. In the presence of a substrate, the amount of luciferase can be quantified and reflects TCR / CD3 activation.

[0204] The Jurkat-NFAT-Luc reporter cell assay was used to evaluate the T cell activation activity of anti-CD3 antibodies. 1 × 10 cells were used. 5 Cells were seeded at 50 μL per well in complete medium. Supernatants of recombinant antibodies (i.e., A1-monoFc, E2-monoFc) were diluted from the stock solution to nine different concentrations using a three-fold serial dilution. Diluted samples were added at 25 μL per well in duplicate. For each diluted sample, 25 μL of a bridging antibody (goat F(ab')2 anti-human IgG-Fc (Abcam, ab98587)) at a final concentration of 100 nM was added to duplicate wells, or 25 μL of complete medium was added as a non-bridging control. The plates and cells with the diluted samples were incubated at 37°C for 6 hours, and the ONE-Glo™ Luciferase Assay System (Promega, E6110) was used to quantify the luminescent signal. Data collection and processing followed the scheme provided by the supplier (Promega, J1621). GraphPad Prism 8 software was used to analyze data including relative light units (RLU) and antibody concentration Log10 or stock dilution Log10, nonlinear curve fitting, and EC50 assay.

[0205] As shown in Figure 3, A1-monoFc and E2-monoFc were able to activate Jurkat-NFAT-Luc reporter cells in a dose-dependent manner in the presence of cross-linking antibodies. However, in the absence of cross-linking antibodies in the antibody and cell mixture, A1-monoFc and E2-monoFc were significantly less able to activate reporter cells than their cross-linked counterparts. These results demonstrate that A1-monoFc and E2-monoFc are cross-linking-dependent TCR / CD3 agonist antibodies.

[0206] Example 4: Antibody production and purification This example describes a method for purifying the recombinant antibodies listed in Table 2 or other antibodies of the invention.

[0207] Plasmids encoding specific recombinant antibodies were transiently transfected into 293-F cells (ThermoFisher, R79007) using PEI (Polyscience, 24885). After transfection, the cells were incubated at 37°C with 5% CO2 and shaken at 120 rpm. Six to seven days after transfection, the cell culture supernatant containing the target antibody was harvested, and the target antibody was purified using a protein A resin by affinity capture. The purity of the target antibody was tested by SEC-HPLC (Agilent 1260 Infinity II HPLC system, Welch Xtimate SEC-300 chromatography column, 1x PBS, pH 7.4, as the mobile phase) and SDS-PAGE (SurePAGE, Bis-Tris, 4%-12%, Genscript, M00653).

[0208] The recombinant antibodies were successfully expressed and purified for further characterization, and the percentage of the major components of each antibody was greater than 90%, indicating that the desired high purity was achieved.

[0209] A1-monoFc (PR005860) and E2-monoFc (PR005861) were tested on an SDS-PAGE gel under non-reducing and reducing conditions, as shown in Figure 5. The results showed that purified A1-monoFc and E2-monoFc retained their monomeric nature under both non-reducing and reducing conditions, indicating that their CD3 agonist activity was due to cross-linking with the anti-human Fc antibody (cross-linking antibody in Example 3) rather than cross-linking with dimeric Fc.

[0210] Example 5: Bioassay of T cell-mediated cytotoxicity performed against BCMA+ cells by the LDH method The T cell-mediated cytotoxicity of the BCMA x CD3 bispecific antibody A1-monoFc (PR005860) was evaluated by the LDH method. Briefly, 3 × 10 BCMA+ cells of NCI-H929 (ATCC, CRL-3580) were cultured in a 200-well plate. 4 Seed 3 x 10 human PBMCs in 50 µL of RPMI 1640 medium in a U-shaped 96-well plate at 10 cells / well. 5 Cells were added to 50 μL of assay medium at 1000 cells / well. Purified A1-monoFc (PR005860) was diluted to a final concentration of 2 μg / mL by 6-fold serial dilution and then added to the plate at 50 μL / well. Each diluted sample was prepared in triplicate to correct for experimental variations. Cytotoxicity was measured by the LDH method using the CytoTox 96™ Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780).

[0211] As shown in Figure 6, PR005860 exhibited dose-dependent cytotoxicity against NCI-H929 cells, indicating that when BCMA bound to NCI-H929 cells, PR005860 was able to activate T cells in PBMCs.

[0212] Example 6: Binding activity of purified anti-CD3 antibodies to CD3-expressing cells The binding activity of purified A1-HCAb (PR006364) and E2-HCAb (PR006365) to Jurkat cells and J.RT3-T3.5 cells was measured by FACS according to the method described in Example 2. As shown in Figures 8(A) and (B), PR006364 and PR006365 could bind to Jurkat cells but not to J.RT3-T3.5 cells, which was consistent with the results shown in Figure 1.

[0213] The binding activity of purified 3G11-HCAb (PR010991) to Jurkat-NFAT-Luc reporter cells and J.RT3-T3.5 cells was measured by FACS using the method described in Example 2. Purified PR010991 was diluted to 11 different concentrations starting from 400 nM using two-fold serial dilutions. As shown in Figure 8(C), PR010991 was able to bind to Jurkat-NFAT-Luc cells in a dose-dependent manner, but not to J.RT3-T3.5 cells, indicating that PR010991 specifically recognizes the CD3 / TCR complex on Jurkat cells.

[0214] [Example 7: Measurement of binding activity to CD3 recombinant protein by ELISA] The binding activity of the recombinant anti-CD3 antibodies of the present invention to various CD3 proteins was measured by ELISA. Because the CD3 / TCR complex has multiple subunits, multiple monomeric or heterodimeric CD3 recombinant proteins purchased from Acro Biosystems were used in the ELISA assay, for example, as listed in Table 3. The test antibodies used in the ELISA assay are listed in Table 4.

[0215] The ELISA assay was performed according to the following brief description: CD3 protein was coated onto an ELISA plate at 1 μg / mL. Test antibodies OKT3 and PR006364 (A1-HCAb) were diluted in a 3-fold serial dilution from 2 μg / mL to eight different concentrations, or test antibodies PR006370 (SP34) and PR010991 (3G11-HCAb) were diluted in a 2-fold serial dilution from 100 nM to 11 different concentrations. PR006364, PR006370, and PR010991 were detected with a secondary antibody (anti-human IgG Fc-HRP), and OKT3 binding signals were detected with a secondary antibody (anti-mouse IgG Fc-HRP). Absorbance signals at 450 nm (OD450) were recorded using a reader (Molecular Devices, SpectraMax). Data were processed and analyzed by nonlinear curve fitting using GraphPad Prism 8.

[0216] [Table 3]

[0217] [Table 4]

[0218] As shown in Figure 9-1(A), OKT3 showed minimal binding to the human CD3E&D heterodimer protein only at high concentrations, whereas PR006364 (A1-HCAb) showed strong and dose-dependent binding activity to the human CD3E&D heterodimer protein, indicating that the binding epitope of A1 is on the CD3 subunit, not on the TCR α or β subunit.

[0219] As shown in Figures 9-1(B) and (C), both PR010991 (3G11-HCAb) and PR006370 (SP34) were able to bind to both human CD3E&D heterodimer protein (hCD3e&d-his) and cynomolgus monkey CD3E&D heterodimer protein (cynoCD3e&d-his). This indicates that 3G11 is an anti-CD3 VHH cross-reactive with both human and cynomolgus monkey CD3, as is SP34, a well-known CD3 agonist antibody cross-reactive with cynomolgus monkeys. However, as shown in Figure 9-2(D) and (E), PR006370 (SP34) could bind to both human CD3E&G heterodimer protein (hCD3e&g-his) and human CD3E monomer protein (hCD3e-his), which was consistent with the fact that the binding epitope of SP34 is located at the N-terminus of the CD3E chain. However, PR010991 (3G11-HCAb) could not bind to them, indicating that it is a different epitope from that of SP34.

[0220] 3G11 can bind to the human CD3E&D heterodimeric protein (Figure 9-1(B)), but not to monomeric CD3E (Figure 9-2(E)) or CD3D (Figure 9-2(F)), indicating that 3G11 may bind to a conformational epitope produced by both CD3E and CD3D.

[0221] Example 8: Production of B7H4xCD3 bispecific antibodies To evaluate whether the agonistic activity of anti-CD3 can induce T cell-mediated cytotoxicity, we combined one B7H4-binding moiety from anti-B7H4 PR003366 with one CD3-binding moiety from various anti-CD3 antibodies of the present invention or from other sources to generate multiple B7H4xCD3 bispecific antibodies (listed in Table 5) in two formats: "Fab-Fc-scFv" and "VH-Fc-scFv," as shown in Figure 10 . To form the B7H4xCD3 heterodimeric Fc, we employed the "knob-into-hole" technique. "knob" mutations (S354C, T366W) were introduced into the heavy chain encoding the anti-CD3 domain, and "hole" mutations (Y349C, T366S, L368A, Y407V) were introduced into the heavy chain encoding the anti-B7H4 domain. To eliminate Fc effector function, a double mutation, L234A and L235A (designated "LALA"), or a triple mutation, L234A, L235A, G237A (designated "AAA"), or a triple mutation, L234A, L235A, P329G (designated "AAG"), was introduced into CH2 of the two heavy chains. The sequences of each B7H4xCD3 polypeptide chain are listed in Table 6.

[0222] PR003366 is an anti-B7H4 antibody in the form of a homodimeric scFv-Fc, which was identified from Harbour Mice™ H2L2 transgenic mice, as described in patent WO 2022002012A1, and its sequence is listed in Table 7.

[0223] PR001848 and PR003886 are humanized variants derived from anti-CD3 clone SP34 (PR006370), which have different binding activities, as described in patent WO 2021063330A1, and their sequences are listed in Table 8. The mutations "LALA" or "AAG" were introduced to remove Fc effector functions.

[0224] PR006361 is an anti-CD3 HCAb with human IgG1 Fc by inserting the anti-CD3 VHH derived from patent WO 2016180982A1 into the pTT5-VHH-hFc vector, and its sequence is listed in Table 8. The mutations C220S and "AAG" were introduced into PR006361.

[0225] Recombinant B7H4xCD3 bispecific antibodies were produced by transfecting the plasmids encoding each antibody polypeptide chain into mammalian host cells, followed by affinity capture or other purification methods as described in Example 4. The expression yields and purities of B7H4xCD3 bispecific antibodies, as determined by SEC-HPLC, are listed in Table 9.

[0226] [Table 5]

[0227] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0228] [Table 7-1] [Table 7-2]

[0229] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

[0230] [Table 9]

[0231] Example 9: T cell-mediated cytotoxicity of B7H4xCD3 bispecific antibodies measured by RTCA method The T cell-mediated cytotoxicity of the B7H4xCD3 bispecific antibody against specific target cells was measured using the RTCA (Real Time Quantitative Cell Analysis) method. Briefly, target cells with high B7H4 expression, specifically tumor cells MDA-MB-468 (ATCC, HTB-132), were cultured at 2 × 10 4 1 x 10 cells / well in 50 µL of assay medium, and 2 x 10 effector cells, specifically human PBMCs or isolated pan T cells. 5 Cells / well were seeded in 50 μL of assay medium. Diluted test bispecific antibodies were added to the wells in 50 μL of assay medium. The assay plate was incubated at 37°C and 5% CO2 for 24 hours, followed by real-time monitoring on an Agilent xCELLigence RTCA instrument. Data were recorded and processed according to the scheme provided by the supplier, and results were analyzed and presented using GraphPad Prism 8.

[0232] The cytotoxicity of B7H4xCD3 bispecific antibodies (PR003733, PR003899, PR006002, PR006003, PR008551) against MDA-MB-468 was tested, and the results are shown in Figure 11 and Table 10.

[0233] [Table 10]

[0234] As shown in Figure 11(A), the B7H4xCD3 bispecific antibodies PR006002 and PR006003 derived from A1 and E2 were able to produce strong, dose-dependent cytotoxicity against MDA-MB-468 cells, with efficacy comparable to that of PR003733 derived from humanized SP34. As shown in Figure 11(B), the B7H4xCD3 bispecific antibody PR008551 derived from 3G11 was also able to produce dose-dependent cytotoxicity against MDA-MB-468 cells, with the highest cytotoxicity reaching >90%. The results demonstrated the effective biological activity of the anti-CD3 VHH sequences of the present invention (A1, E2, 3G11).

[0235] Example 10: Humanization of PR006364 (A1-HCAb) The variable regions of the alpaca-derived anti-CD3 PR006364 (A1-HCAb) were further humanized using established techniques, such as "CDR grafting." Briefly, the three CDR regions of PR006364 were grafted into four framework regions provided by the human germline gene segments IGHV3-23 and IGHJ4 to generate a humanized variant sequence in the order FWR1-HCDR1-FWR2-HCDR2-FWR3-HCDR3-FWR4, where the sequences of framework regions FWR1, FWR2, and FWR3 are derived from IGHV3-23, and the sequence of framework region FWR4 is derived from IGHJ4, as defined, for example, by the Chothia numbering system. Additionally, one or more key residues in the framework regions were modified back to the corresponding residues in the parent VH of PR006364 to retain biological activity.

[0236] A total of five humanized variants of PR006364 were produced: PR011628, PR011629, PR011630, PR011631, and PR011632. The amino acid sequences and their CDR regions of the humanized variants derived from PR006364 are listed in Table 12. The "humanness H-score" represents the degree of similarity of a specific antibody sequence to real-world human antibodies, and this score was used to sort these humanized sequences together with the alpaca-derived parent VH sequence. The "humanness H-score" of each VH sequence was calculated using the online tool Hscore (URL: http: / / www.bioinf.org.uk / abs / shab / ) and listed in Table 11. The H-score values ​​of the humanized variants were higher than that of PR006364, indicating that the humanized variants had high homology to human antibodies.

[0237] Humanized variants were produced by the method described in Example 4. The purified antibody samples were then characterized for their binding activity to human CD3E&D heterodimer protein by the method described in Example 7, and for their binding activity to Jurkat-NFAT-Luc reporter cells by the method described in Example 6.

[0238] [Table 11]

[0239] As shown in Figures 12 and 13, all humanized variants of PR006364, despite their different affinities, were able to bind to human CD3E&D heterodimer protein and Jurkat-NFAT-Luc reporter cells in a dose-dependent manner, demonstrating the successful humanization of A1-HCAb.

[0240] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4]

[0241] Example 11: Humanized PR006364-derived B7H4xCD3 bispecific antibody To assess whether the humanized variants of anti-CD3 PR006364 (A1-HCAb) could induce T cell-mediated cytotoxicity, multiple B7H4xCD3 bispecific antibodies (listed in Table 13) were generated in the "VH-Fc-scFv" format shown in Figure 10 by combining one B7H4-binding moiety from anti-B7H4 PR003366 with one CD3-binding moiety from the humanized variant. As described in Example 8 above, the "knob-into-hole" technique was used to enforce heterodimeric heavy chain formation, and the "AAA" mutation was used to remove Fc effector function. The sequences of each B7H4xCD3 polypeptide chain are listed in Table 14.

[0242] Recombinant B7H4xCD3 bispecific antibodies were produced by the method described in Example 8. Expression yields and purity as determined by SEC-HPLC are listed in Table 13. All B7H4xCD3 samples showed good production rates.

[0243] [Table 13]

[0244] [Table 14-1] [Table 14-2] [Table 14-3]

[0245] The T cell-mediated cytotoxicity of the B7H4xCD3 bispecific antibody against specific target cells was measured by the RTCA method as described in Example 9. In this example, the tumor cells MDA-MB-468 (ATCC, HTB-132), which highly express B7H4, and the B7H4-negative tumor cells MDA-MB-231 (ATCC, HTB-26) were used as target cells, and isolated human T cells were used as effector cells at an E:T ratio of 10:1. The cells were incubated with the test antibody sample for 24 hours before analysis.

[0246] As shown in Figure 14 and Table 15, all B7H4xCD3 bispecific antibodies produced by PR006364 and the humanized variants were able to produce strong, dose-dependent cytotoxicity against MDA-MB-468 cells. The maximum cytotoxicity reached nearly 100%, indicating complete disruption of B7H4 target cells. The EC50 values ​​were different, consistent with their different binding activities to CD3, as previously described (Figure 13). In another aspect, as shown in Figure 15, they failed to produce significant cytotoxicity against MDA-MB-231 cells, demonstrating the specificity of the cytotoxicity of B7H4xCD3 to B7H4-positive cells. The different cytotoxic efficacies of these humanized variants, with nearly identical maximum cytotoxicity but different EC50 values, provide multiple options for the production of T cell adapter bispecific antibodies in different therapeutic schemes.

[0247] [Table 15]

[0248] [Example 12: Evaluation of cross-reactivity with cynomolgus monkeys] The cross-reactivity of 3G11-HCAb (PR010991) and its derivative B7H4xCD3 PR008551 to cynomolgus monkeys was assessed by measuring their binding activity to cynomolgus monkey PBMCs using the FACS method, and T cell-mediated cytotoxicity against cynomolgus monkey PBMCs was measured using the RTCA method. In this example, the anti-CD3 / TCR HCAb PR006361 derived from WO 2016 / 180982 and its derivative B7H4xCD3 PR006001 were used as control antibodies. These antibodies were produced as described in Example 8.

[0249] Cynomolgus monkey PBMCs were isolated from 10 mL of fresh cynomolgus monkey blood using Ficoll Paque Plus (Cytiva 17-1440-02) according to the supplier's manual. Recombinant antibodies were diluted from 200 nM to 11 different concentrations using two-fold serial dilutions. The diluted samples were incubated with cynomolgus monkey PBMCs for 1 hour, followed by the addition of secondary antibody (Alexa Fluor™ 488 AffinityPure Goat Anti-Human IgG (Fcγ Fragment Specific), Jackson ImmunoResearch, 109-545-098) and incubation for 1 hour. Cells were washed twice with staining buffer and subjected to flow cytometry. Data were processed using FlowJo v10 (FlowJo, LLC), and nonlinear curve fitting analysis was performed using GraphPad Prism 8.

[0250] As shown in Figure 16, 3G11-HCAb (PR010991) and the derived B7H4xCD3 PR008551 could bind to cynomolgus monkey PBMCs in a dose-dependent manner, whereas the control antibody PR006361 and the derived B7H4xCD3 PR006001 did not bind to the cynomolgus monkey PBMCs, indicating that 3G11 has cross-reactivity with cynomolgus monkeys, which is superior to the prior art PR006361.

[0251] The T cell-mediated cytotoxicity of B7H4xCD3 PR008551 against cynomolgus monkey PBMCs was measured by RTCA as described in Example 9. In this example, MDA-MB-468 cells, which highly express B7H4, were used as target cells, and cynomolgus monkey PBMCs were used as effector cells, with an E:T ratio of 10:1. Test antibodies were diluted from 100 nM to eight concentrations using a five-fold serial dilution. Cells were incubated with test antibody samples for 24 or 48 hours before analysis.

[0252] As shown in Figure 17, PR008551 induced dose-dependent cynomolgus monkey PBMC-mediated cytotoxicity against MDA-MB-468 cells, with the highest cytotoxicity reaching nearly 80% of the maximum after 24 hours of incubation and increasing to more than 95% after 48 hours of incubation, indicating that the target cells were nearly completely disrupted.

[0253] Combined, the results in Figures 16 and 17 demonstrate that 3G11-HCAb (PR010991) is cross-reactive to cynomolgus monkeys; it can bind to and activate CD3 on T cells in cynomolgus monkey PBMCs, and produce cytotoxicity against tumor cells in the context of a CD3-adapter bispecific antibody.

[0254] Example 13: Mutants derived from 3G11-HCAb To identify which residues in the VH region of 3G11-HCAb (PR010991) contribute to its binding activity to CD3, multiple VH variants were derived from 3G11 by introducing mutations into the CDRs by saturation mutagenesis or other techniques.

[0255] A total of 17 VH variants were obtained. These VH variants were assembled into the vector pTT5-VHH-hFc to produce recombinant HCAb derivatives of PR010991, and further assembled into a plasmid encoding a polypeptide chain of PR008551 to produce recombinant B7H4xCD3 derivatives of PR008551. The newly produced HCAb antibodies and B7H4xCD3 bispecific antibodies are listed in Table 16, and their amino acid sequences are listed in Tables 17 and 18. An alignment of the VH sequences of 3G11 VH (as part of PR008551) and variants from derivatives of PR008551 is shown in Figure 18.

[0256] [Table 16]

[0257] [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6] [Table 17-7] [Table 17-8] [Table 17-9] [Table 17-10]

Table 17-11

[0258]

Table 18-1

Table 18-2

Table 18-3

Table 18-4

Table 18-5

Table 18-6

Table 18-7

Table 18-8

Table 18-9

[0259] Using the RTCA method described in Example 9, the T cell-mediated cytotoxicity of the newly produced B7H4xCD3 bispecific antibodies listed in Table 16 was further evaluated. In this example, B7H4 cells, which highly express MDA-MB-468, were used as target cells, and human T cells isolated from PBMCs were used as effector cells, with an E:T ratio of 10:1. Test antibodies were diluted from 100 nM to eight concentrations using a five-fold serial dilution. Cells were incubated with the test antibody samples for 24 or 48 hours before analysis. The results are shown in Figure 19, indicating that all B7H4xCD3 bispecific antibodies derived from the 3G11 variants retained most of their activity in inducing cytotoxicity against MDA-MB-468. Some variants (e.g., PR012683, PR012685) even showed enhanced cytotoxicity at lower concentrations.

[0260] Example 14: Binding affinity to CD3 protein measured by BLI method To measure the binding affinity of anti-CD3 antibodies to human or cynomolgus CD3e&d heterodimeric proteins, a biofilm interference technology (BLI) assay was performed using Octet™ RED96e.

[0261] Biotinylated human CD3e&d protein (Acro Biosystems, CDD-H82W6) or biotinylated cynomolgus monkey CD3e&d protein (Acro Biosystems, CDD-C82W6) was first loaded onto the surface of an SA Octet biosensor (Sartorius, 18-5019) to achieve a capture level between 0.5 nm and 0.8 nm. The loaded biosensor was then immersed in wells containing two-fold serial dilutions of the test antibody to detect the association signal, followed by a dissociation step in a well containing kinetic buffer. The association phase lasted 180 s, and the dissociation phase lasted 900 s. Sensing maps were recorded, and the reference signal was subtracted before curve fitting using ForteBio Data Analysis 11.0 software. The association rate (k) and dissociation rate (k) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (K) was calculated as the ratio of k / k.

[0262] As shown in Table 19, PR010991 (3G11-HCAb) showed strong binding affinity to both human CD3e&d protein and cynomolgus monkey CD3e&d protein, which was consistent with the ELISA results (Figures 9-1 and 9-2). However, the prior art PR006361 could only bind to human CD3e&d protein, but was unable to bind to cynomolgus monkey CD3e&d protein. Furthermore, PR006364 (A1-HCAb) and its humanized variant PR011629 also showed strong binding affinity to human CD3 protein.

[0263] [Table 19]

[0264] Example 15: ROR1 x CD3 bispecific antibody produced by A1-HCAb The ROR1xCD3 bispecific antibody PR011870 was generated by combining two ROR1-binding VH domains from a previously discovered anti-ROR1 HCAb (PR005340) with one CD3-binding VH domain from the A1-HCAb (PR006364) in a single-chain VH tandem format, with a peptide linker between the domains. A poly-His tag was added to the C-terminus of PR011870 to facilitate purification. The amino acid sequences of ROR1xCD3 PR011870 and anti-ROR1 PR005340 are listed in Table 20.

[0265] [PR011870-Protein]

[0266] PR011870 protein was produced by affinity capture using a Ni-excel column (Cytiva) as described in Example 4.

[0267] [PR011870-mRNA]

[0268] In this example, PR011870-mRNA encoding PR011870 was prepared and packaged into lipid nanoparticles (LNPs).

[0269] Briefly, the coding sequence of PR011870 (listed in Table 21) was cloned into a pUC plasmid vector containing a T7 promoter, human α-globulin 5'UTR, mtRNR1 and AES 3'UTR motifs, and a poly-A tail. The plasmid was then linearized and used as a transcription template. mRNA was produced using in vitro T7 RNA polymerase-mediated transcription. N1-Me-PseudoUTP was used instead of UTP, followed by lithium chloride precipitation purification.

[0270] Lipid nanoparticle (LNP) formulations were prepared in a microfluidic device by mixing ethanol and an aqueous phase at a volume ratio of 1:3. The ethanol phase was prepared by dissolving a mixture of ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and PEG-lipid in a molar ratio of 50:10:38.5:1.5. The aqueous phase was prepared in citrate buffer containing PR011870-mRNA. The LNPs were dialyzed against PBS and concentrated to the desired concentration. Sucrose was added as a cryoprotectant. The formulated LNPs were stored at -70°C until further use. All formulations were tested for particle size, distribution, RNA concentration, and packaging.

[0271] [Fluc-mRNA]

[0272] mRNA encoding firefly luciferase (Fluc) (sequence in Table 21) was also prepared using the same program and subsequently packaged into LNPs. Fluc-mRNA was used as a negative control in other examples.

[0273] [Table 20]

[0274] [Table 21-1] [Table 21-2]

[0275] [Example 16: T cell-mediated cytotoxicity of ROR1×CD3 bispecific antibody] The T cell-mediated cytotoxicity of the ROR1 x CD3 bispecific antibody containing PR011870 protein and PR011870 mRNA against specific target cells was measured by the RTCA method as described in Example 9. In this example, tumor cells PANC-1 (ATCC, CRL-1469), which highly express ROR1, were used as target cells, and isolated human T cells were used as effector cells at an E:T ratio of 5:1. Before analysis, the cells were incubated with test samples (PR011870 protein, PR011870 mRNA, and Fluc mRNA prepared in Example 15) for 27 hours.

[0276] The results are shown in Figure 20 and Table 22. Both the recombinant protein and mRNA forms of the ROR1 x CD3 bispecific antibody PR011870 were able to produce strong and dose-dependent cytotoxicity against the target cells PANC-1, demonstrating that A1-HCAb can be used to generate multiple TAA x CD3 adaptor bispecific antibodies, regardless of whether they target B7H4 or ROR1 or other tumor-associated antigens.

[0277] [Table 22]

[0278] Example 17: In vivo pharmacological study of ROR1 x CD3 bispecific antibodies In vivo pharmacological studies were performed using NCG mice (GemPharmatech, product no. T001475) to establish a PANC-1 tumor model and human PBMC immune system. Briefly, on the day of cell inoculation, each NCG mouse was subcutaneously inoculated with PANC-1 (ATCC, CRL-1469) cells. The average tumor volume of each group of mice was 88 mm. 3When the tumor volume reached 100 mg / kg, the mice were divided into 6 groups and administered three doses of each test sample by intravenous tail vein injection once a week (on days 39, 46, and 53 after tumor cell inoculation), as listed in Table 23. After the start of administration, body weight and tumor volume were measured twice a week. Tumor volume was calculated as follows: Tumor volume (mm 3 ) = 0.5 × tumor's long diameter × tumor's short diameter 2 Data were analyzed using t-tests.

[0279] The results of tumor volume change are shown in Figure 21, and the tumor growth inhibition rate (TGI) (%) on day 60 is summarized in Table 23. Specifically, the average tumor volume of the PBS vehicle group mice on day 60 after inoculation was 330 mm 3 In the Fluc-mRNA (2.5 mg / kg) treatment group, the mean tumor volume 60 days after inoculation was 283 mm 3 The mean tumor volume at day 60 in the PR011870-mRNA (2.5 mg / kg) treatment group was 7 mm 3 The TGI (%) was 97.92%, which was significantly different from the vehicle group (p<0.05). PR011870-mRNA showed effective antitumor activity at 1.0 mg / kg and 0.5 mg / kg, with TGI (%) of 97.73% and 95.53%, respectively (p<0.05). The mean tumor volume in the PR011870-protein treatment group was 83 mm 3 The TGI (%) was 74.72% (p value < 0.05). During the treatment period, all animals tolerated the treatment well and showed no obvious weight loss.

[0280] The study showed that both the recombinant protein and mRNA forms of the ROR1 x CD3 bispecific antibody PR011870 were highly effective in treating PANC-1 tumors. Surprisingly, the mRNA form showed stronger antitumor activity than the protein form at the same dose, resulting in a nearly complete response (TGI=95.53% vs. TGI=74.72%), and the efficacy of the mRNA form could be longer-lasting.

[0281] [Table 23]

[0282] This study further demonstrates the biological activity of the anti-CD3 antibodies of the present invention, which is of great value for the production of innovative biologics to treat diseases.

[0283] It should be understood that the above description of two preferred embodiments is only intended to illustrate the principles of the present disclosure and is in no way intended to be exhaustive, and that modifications and variations thereof will be apparent to those skilled in the art, and that the present disclosure is not intended to be limiting unless expressly set forth in the following claims.

Claims

1. an antibody that targets CD3, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3; wherein the HCDR1 comprises an amino acid sequence shown in any one of SEQ ID NOs: 17-18 and 20-22, the HCDR2 comprises an amino acid sequence shown in any one of SEQ ID NOs: 41-43, and the HCDR3 comprises an amino acid sequence shown in any one of SEQ ID NOs: 62-79.

2. The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41 and 62, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 18, 41 and 63, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 64, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41 and 65, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 66, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 21, 42 and 64, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 67, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 68, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 69, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 70, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 71, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 72, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 73, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 74, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 75, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 76, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 77, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 22, 42 and 64, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 43 and 64, respectively; or The HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42 and 78, respectively; or 2. The antibody of claim 1, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 79, respectively, and the CDR regions are defined according to the Chothia numbering system.

3. the heavy chain variable region (VH) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 107-108 and 110-132; Alternatively, the VH fragment has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with an amino acid sequence set forth in any one of SEQ ID NOs: 107-108 and SEQ ID NOs: 110-132.

4. The antibody of claim 1, further comprising a heavy chain constant region, preferably the heavy chain constant region is derived from hIgG1, hIgG2, hIgG3 and hIgG4 and variants thereof, more preferably the heavy chain constant region is derived from hIgG1.

5. 5. The antibody of claim 4, wherein the antibody further comprises one or more mutations in the heavy chain constant region, preferably the mutations may be selected from L234A, L235A, G237A, P329G (Eu numbering), or any combination.

6. 4. The antibody of claim 3, wherein the antibody is a heavy chain-only antibody, and the heavy chain-only antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 141-142 and SEQ ID NOs: 144-166.

7. A bispecific antibody comprising a first antigen-binding fragment that targets CD3 and a second antigen-binding fragment that targets a tumor-associated antigen (TAA); wherein the first antigen-binding fragment targeting CD3 is as defined in any one of claims 1 to 6, and preferably the TAA is selected from the group consisting of CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, sLe, GM3, TGS5, HMWMAA, GD2, FOLR 1, FOLR2, TEM1 / CD248, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6-AML, SPA17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD20, CD30, HER2, R The bispecific antibody may be any one of OR1, FLT3, TAAG72, CD22, CD33, GD2, gp100Tn, FAP, TYR, EPCAM, CEA, IGF-1R, EphB2, MSLN, CDH17, CD32b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C, and TACSTD2, and is more preferably BCMA, B7H4, or ROR1.

8. The second antigen-binding fragment targets B7H4 and preferably comprises:

8. The bispecific antibody of claim 7, wherein the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 14, 38, and 59, respectively; and the CDR regions are defined according to the Chothia numbering system.

9. The second antigen-binding fragment targets BCMA and preferably comprises:

8. The bispecific antibody of claim 7, wherein the second antigen-binding fragment comprises one heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 12, 36, and 57, respectively, and the CDR regions are defined according to the Chothia numbering system.

10. The second antigen-binding fragment targets ROR1 and preferably comprises:

8. The bispecific antibody of claim 7, wherein the second antigen-binding fragment comprises one heavy chain variable region (VH), the VH region comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 15, 39, and 60, respectively, and the CDR regions are defined according to the Chothia numbering system.

11. the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 62, respectively; the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 12, 36, and 57, respectively; or the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 18, 41, and 63, respectively; the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 12, 36, and 57, respectively; or The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 62, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 62, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 18, 41, and 63, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 18, 41, and 63, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 65, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 65, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 66, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 21, 42, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 11, 12, 13, and 14, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 67, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 68, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 69, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 70, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 71, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 72, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 73, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 74, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 75, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 76, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 77, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 22, 42, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 22, 42, and 64, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 43, and 64, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 78, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; The first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 42, and 79, respectively; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 88, 93, and 98, respectively; and the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 104, 105, and 106, respectively. or comprising the amino acid sequences set forth in SEQ ID NOs: 14, 38 and 59; the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 41, and 62, respectively; the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 15, 39, and 60, respectively; 11. The bispecific antibody of claim 7, wherein the CDR regions are defined according to the Chothia numbering system.

12. the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 107; and the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 101; or the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 108; the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 101; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 107; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 108; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 110; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 111; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 112; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 113; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 114; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 115; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 116; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 117; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 118; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 119; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 120; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 121; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 122; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 123; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 124; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 125; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 126; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 127; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 128; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 129; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 130; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 131; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or the first antigen-binding fragment comprises one heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 132; the second antigen-binding fragment comprises one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 134, and the VH region comprises the amino acid sequence set forth in SEQ ID NO: 103; or 12. The bispecific antibody of claim 7 , wherein the first antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO: 107, and the second antigen-binding fragment comprises a heavy chain variable region (VH), wherein the VH region comprises the amino acid sequence set forth in SEQ ID NO:

105.

13. the bispecific antibody comprises one polypeptide having the amino acid sequence shown in SEQ ID NO: 172; or comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 173, or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 175 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 170; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 176 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 170; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 177 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 179 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 180 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 181 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 182 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 183 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 185 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 186 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 187 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 188 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 189 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 190 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 191 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 192 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 193 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 194 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 195 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 196 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 197 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 198 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 199 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 200 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or two polypeptides, wherein a first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 201 and a second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 178; or 13. A bispecific antibody according to any one of claims 7 to 12, comprising one polypeptide having the amino acid sequence shown in SEQ ID NO:

184.

14. A polynucleotide encoding a CD3-targeting antibody according to any one of claims 1 to 6 or a bispecific antibody according to any one of claims 7 to 13, Preferably, said polynucleotide is selected from DNA or RNA, more preferably said polynucleotide is mRNA.

15. A vector comprising the polynucleotide of claim 14.

16. A cell comprising an antibody targeting CD3 according to any one of claims 1 to 6, a bispecific antibody according to any one of claims 7 to 13, a polynucleotide according to claim 14 or a vector according to claim 15.

17. 16. A method for preparing a CD3-targeting antibody according to any one of claims 1 to 6 or a bispecific antibody according to any one of claims 7 to 13, the method comprising culturing a cell comprising the polynucleotide according to claim 14 or the vector according to claim 15, and obtaining the CD3-targeting antibody or the bispecific antibody from the culture.

18. 17. A pharmaceutical composition comprising an antibody targeting CD3 according to any one of claims 1 to 6, or a bispecific antibody according to any one of claims 7 to 13, or a polynucleotide according to claim 14, or a vector according to claim 15, or a cell according to claim 16, and optionally a pharmaceutically acceptable carrier.

19. Use of an antibody targeting CD3 according to any one of claims 1 to 6, or a bispecific antibody according to any one of claims 7 to 13, or a polynucleotide according to claim 14, or a vector according to claim 15, or a cell according to claim 16, or a pharmaceutical composition according to claim 18 in the manufacture of a drug for the prevention and / or treatment of a disease.

20. 19. Use of an antibody targeting CD3 according to any one of claims 1 to 6, or a bispecific antibody according to any one of claims 7 to 13, or a polynucleotide according to claim 14, or a vector according to claim 15, or a cell according to claim 16, or a pharmaceutical composition according to claim 18, as a drug, preferably wherein the drug is used for preventing and / or treating a disease.

21. 19. A method for preventing and / or treating a disease in a subject in need thereof, said method comprising administering an effective amount of an antibody targeting CD3 according to any one of claims 1 to 6, or a bispecific antibody according to any one of claims 7 to 13, or a polynucleotide according to claim 14, or a vector according to claim 15, or a cell according to claim 16, or a pharmaceutical composition according to claim 18.

22. 22. The use of claim 19 or 20 or the method of claim 21, wherein the disease is selected from myeloma, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, nasopharyngeal cancer, oral cancer, esophageal cancer, gastric cancer, bladder cancer and lymphoma.

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