Antigen-binding molecule that specifically binds to PSMA and CD3 and its medical use

Abstract

The present invention provides an antigen-binding molecule that specifically binds to PSMA and CD3, and a pharmaceutical use thereof. The antigen-binding molecule can be used to treat tumor-related diseases.

Description

[Technical field]

[0001] This application claims priority to Chinese patent application No. 202210116684.6, filed on February 7, 2022.

[0002] The present disclosure is in the field of biotechnology, and more specifically, the present disclosure relates to antigen-binding molecules and uses thereof. [Background technology]

[0003] The statements herein are not necessarily intended to constitute prior art, but merely to provide background information relevant to the present disclosure.

[0004] PSMA belongs to glutamate carboxypeptidase II (GCPII) and consists of 750 amino acids, 19 intracellular, 24 transmembrane and 707 extracellular. The extracellular portion of PSMA consists of three domains, protease-like, apical and C-terminal. All three domains are involved in substrate binding, protease-like and apical directly bind substrates, and C-terminal functions in the formation of PSMA dimers. In prostate cancer patients, the expression level of PSMA in tumor tissue is 100-1000 times higher than that in normal prostate. PSMA is also expressed on the cell surface of endometrial cancer, neovasculature in gastric cancer (66.4%) and colorectal cancer (84.6%), neovasculature in non-small cell lung cancer and some tumor cells. Therefore, PSMA is not only a target for prostate cancer development, but also applicable to other tumor types.

[0005] CD3 is a homo- or heterodimeric antigen expressed on T cells. Functional CD3 is formed by dimerization of two of four different chains, ε, ζ, δ, and γ. CD3 dimer sequences include γ / ε, δ / ε, and ζ / ζ. CD3 binds to the T cell receptor complex (TCR) and is required for T cell activation. Therefore, anti-CD3 antibodies that activate T cells have been proposed to achieve therapeutic purposes. However, administration of anti-CD3 antibodies may trigger T cell activation and associated cytokine release. Excessive cytokine release can cause severe cytokine release syndrome (CRS), which is a major challenge in the clinical administration of anti-CD3 antibodies.

[0006] Therefore, there is an unmet need to provide PSMA / CD3 bispecific antibodies with high activity and low cytokine release. Summary of the Invention

[0007] The present disclosure provides antigen-binding molecules that specifically bind to PSMA and CD3, and antibodies that specifically bind to PSMA.

[0008] In one aspect, the present disclosure provides an antigen-binding molecule, which comprises at least one antigen-binding module that specifically binds to PSMA and at least one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to PSMA comprises a heavy chain variable region (PSMA-VH) and a light chain variable region (PSMA-VL), and the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region (CD3-VH) and a light chain variable region (CD3-VL).

[0009] In some embodiments, the antigen-binding molecule comprises: (i) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 77, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 78, respectively; or (ii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 73, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 74, respectively; or (iii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 75, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 76, respectively; or (iv) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 79, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 80, respectively; or (v) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 81, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 82, respectively; or (vi) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 83, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 84, respectively; or (vii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 85, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 86, respectively; or (viii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 87, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 88, respectively; or (ix) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 89, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 90, respectively; or (x) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 91, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 92 or 152, respectively; or (xi) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 93, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 94, respectively; or (xii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH respectively comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 95, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL respectively comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 96.

[0010] In some embodiments, the PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

[0011] In some embodiments, the antigen-binding molecule of any one of the above, (i) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 15, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, or (ii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:1, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:3, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:4, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:6; or (iii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:7, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:9, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:10, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:12, or (iv) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or (v) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:25, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:27, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:28, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:29, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:30; or (vi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 33, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 36, or (vii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 42, or (viii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 45, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 46, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 48, or (ix) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 49, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 52, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 54, or (x) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58 or 159, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 59, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 60; or (xi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 61, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 66, or (xii) The above PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 67, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 69, and the above PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 72.

[0012] In some embodiments, the antigen binding molecule of any one of the above, wherein the PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 are defined according to the Kabat numbering convention.

[0013] In some embodiments, the antigen-binding molecule according to any one of the above is at a concentration of 1×10 at 25° C. -7 M, 5×10 -8 M, 1×10 -8 It binds to human PSMA with a KD of less than M, as measured by surface plasmon resonance.

[0014] In some specific embodiments, the antigen-binding molecule according to any one of the above is capable of binding to at least 9×10 -9 M, 8×10 -9 M, 7×10 -9 M, 6×10 -9 M, 5×10 -9 M, 4×10 -9 M, 3×10 -9 M, 2.9×10 -9 M, 2.8×10 -9 M, 2.7×10 -9 M, 2.6×10 -9 M, 2.5×10 -9 M, 2.4×10 -9 M, 2.3×10 -9 M, 2.2×10 -9 M, 2.1×10 -9 M, 2.0×10 -9 M, 1.9×10 -9M, 1.8×10 -9 M, 1.7×10 -9 M, 1.6×10 -9 M, 1.5×10 -9 M, 1.4×10 -9 It binds to human PSMA with a KD of less than M, as measured by surface plasmon resonance.

[0015] In some embodiments, the antigen-binding molecule of any one of the above, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 77, 107, 108, 109, 110, 111, or 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 78, 113, 114, or 115; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73 or 99, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74 or 100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, 101, 102, or 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76, 104, 105, or 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 79, 116, or 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 80, 118, or 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81 or 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82, 121, 122, 123, 124, 125, or 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 83 or 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 84, 128, 129, or 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, 131, or 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86, 133, 134, or 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, 136, 137, or 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88, 139, or 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, 141, 142, 143, or 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90, 145, 146, or 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, 148, or 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92, 150, 151, or 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, 153, 154, 155, or 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94, 157, or 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, 277, 278, or 279, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96, 280, 281, 282, or 283.

[0016] In some embodiments, the antigen-binding molecule of any one of the above, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:77 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:78; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:114; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:99 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO:79, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:80; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 121; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 122; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 123; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 124; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 125; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 83, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 84; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 129; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 136 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 137 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96.

[0017] In some embodiments, the antigen-binding molecule of any one of the above, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128.

[0018] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises an Fc region (including an IgG Fc region or an IgG1 Fc region). In some embodiments, the Fc region comprises one or more amino acid substitutions compared to a wild-type Fc region, which can reduce binding to an Fc receptor, in particular binding to an Fcγ receptor. In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 175.

[0019] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule further comprises an Fc region, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2) capable of associating with each other, and the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region.

[0020] In this disclosure, ordinal numbers such as "first," "second," "third," "1," "2," "3" (e.g., "first subunit," "Fc2," "third chain," "linker 2") are used merely to distinguish different features, elements, components or steps and are not intended to limit the quantity, order, or level.

[0021] In some embodiments, the Fc1 and Fc2 each independently have one or more knobs-in-hole amino acid substitutions. In some embodiments, the Fc1 has a knobs structure from the knobs-in-hole technology and the Fc2 has a hole structure from the knobs-in-hole technology, or vice versa.

[0022] In some embodiments, the Fc1 has an amino acid residue at position 366 that is W, and the Fc2 has an amino acid residue at position 366 that is S, an amino acid residue at position 368 that is A, and an amino acid residue at position 407 that is V, numbered according to the EU index, or vice versa.

[0023] In some embodiments, the Fc1 has an amino acid residue at position 354 that is C and the Fc2 has an amino acid residue at position 349 that is C, numbered according to the EU index, or vice versa.

[0024] In some embodiments, the Fc1 has the amino acid sequence of SEQ ID NO:169, and the Fc2 has the amino acid sequence of SEQ ID NO:170.

[0025] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to PSMA and one antigen-binding module that specifically binds to CD3. In some embodiments, the antigen-binding module that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 are scFvs.

[0026] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises one first chain having a structure represented by formula (a) and one second chain having a structure represented by formula (b), Formula (a) [PSMA-VH]-[Linker 1]-[PSMA-VL]-[Linker 2]-[CD3-VH]-[Linker 3]-[CD3-VL]-[Linker 4]-[Fc2]; Formula (b) [Fc1], The structures shown in formula (a) and formula (b) are arranged from the N-terminus to the C-terminus, and the linkers 1, 2, 3 and 4 are the same or different peptide linkers (as a non-limiting example, the structure shown in FIG. 1E). It should be understood that Fc1 and Fc2 here function in the formation of a dimer and are therefore interchangeable.

[0027] In some embodiments, the antigen-binding molecule of any one of the above, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 191 and one second chain comprising the amino acid sequence of SEQ ID NO: 169; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 192 and one second chain comprising the amino acid sequence of SEQ ID NO: 169; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO:198 and one second chain comprising the amino acid sequence of SEQ ID NO:169.

[0028] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding module that specifically binds to PSMA or the antigen-binding module that specifically binds to CD3 comprises a Titin chain and an Obscurin chain (capable of forming a dimer).

[0029] In some embodiments, the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 210 to 228, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 229 to 269. In some embodiments, the Titin chain comprises the amino acid sequence of SEQ ID NO: 226, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 264.

[0030] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to PSMA and one antigen-binding module that specifically binds to CD3. In some embodiments, the antigen-binding module that specifically binds to PSMA is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab comprising a Titin chain and an Obscurin chain (capable of forming a dimer).

[0031] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises one first chain having a structure represented by formula (c), one second chain having a structure represented by formula (d), one third chain having a structure represented by formula (e), and one fourth chain having a structure represented by formula (f); Formula (c) [PSMA-VH]-[CH1]-[Fc1], Formula (d) [PSMA-VL]-[CL], Formula (e) [CD3-VH]-[Linker 5]-[Obscurin chain]-[Fc2], Formula (f) [CD3-VL]-[Linker 6]-[Titin chain], The structures represented by formulae (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus, and the linker 5 and the linker 6 are the same or different peptide linkers (as a non-limiting example, the structure shown in FIG. 1A). It should be understood that Fc1 and Fc2 here function in the formation of a dimer and are therefore interchangeable.

[0032] In some embodiments, the antigen-binding molecule of any one of the above, The antigen-binding molecule comprises one first chain comprising the amino acid sequence of SEQ ID NO: 193, one second chain comprising the amino acid sequence of SEQ ID NO: 194, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 172; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 201, one second chain comprising the amino acid sequence of SEQ ID NO: 202, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 172; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 206, one second chain comprising the amino acid sequence of SEQ ID NO: 207, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 172; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 284, one second chain comprising the amino acid sequence of SEQ ID NO: 285, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 172.

[0033] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises two antigen-binding modules that specifically bind to PSMA and one antigen-binding module that specifically binds to CD3. In some embodiments, the antigen-binding module that specifically binds to PSMA is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab comprising a Titin chain and an Obscurin chain (capable of forming a dimer).

[0034] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises one first chain having a structure represented by formula (c), two second chains having a structure represented by formula (d), one third chain having a structure represented by formula (g), and one fourth chain having a structure represented by formula (h); Formula (c) [PSMA-VH]-[CH1]-[Fc1], Formula (d) [PSMA-VL]-[CL], Formula (g) [PSMA-VH]-[CH1]-[CD3-VH]-[Linker 7]-[Titin chain]-[Fc2], Formula (h) [CD3-VL]-[Linker 8]-[Obscurin chain], The structures represented by formulae (c), (d), (g) and (h) are arranged from the N-terminus to the C-terminus, and the linker 7 and the linker 8 are the same or different peptide linkers. (As a non-limiting example, the structure shown in FIG. 1B.) It should be understood that Fc1 and Fc2 here function in the formation of a dimer and are therefore interchangeable.

[0035] In some embodiments, the antigen-binding molecule of any one of the above, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 180, two second chains comprising the amino acid sequence of SEQ ID NO: 181, one third chain comprising the amino acid sequence of SEQ ID NO: 182, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 187, two second chains comprising the amino acid sequence of SEQ ID NO: 188, one third chain comprising the amino acid sequence of SEQ ID NO: 189, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 193, two second chains comprising the amino acid sequence of SEQ ID NO: 194, one third chain comprising the amino acid sequence of SEQ ID NO: 195, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 206, two second chains comprising the amino acid sequence of SEQ ID NO: 207, one third chain comprising the amino acid sequence of SEQ ID NO: 208, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 174.

[0036] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises two antigen-binding modules that specifically bind to PSMA and two antigen-binding modules that specifically bind to CD3. In some embodiments, the antigen-binding module that specifically binds to PSMA is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab comprising a Titin chain and an Obscurin chain (capable of forming a dimer).

[0037] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises two first chains having a structure represented by formula (j), two second chains having a structure represented by formula (d), and two third chains having a structure represented by formula (h); Formula (j) [PSMA-VH]-[CH1]-[CD3-VH]-[Linker 9]-[Titin chain]-[one subunit of Fc]; Formula (d) [PSMA-VL]-[CL], Formula (h) [CD3-VL]-[Linker 8]-[Obscurin chain], The structures represented by formulae (j), (d) and (h) are arranged from the N-terminus to the C-terminus, and the linker 8 and the linker 9 are the same or different peptide linkers. (As a non-limiting example, the structure shown in FIG. 1C.) It should be understood that Fc1 and Fc2 here function in the formation of a dimer and are therefore interchangeable.

[0038] In some embodiments, the antigen-binding molecule of any one of the above, The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 183, two second chains comprising the amino acid sequence of SEQ ID NO: 181, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 185, two second chains comprising the amino acid sequence of SEQ ID NO: 186, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 196, two second chains comprising the amino acid sequence of SEQ ID NO: 194, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 203, two second chains comprising the amino acid sequence of SEQ ID NO: 204, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 286, two second chains comprising the amino acid sequence of SEQ ID NO: 285, and two third chains comprising the amino acid sequence of SEQ ID NO: 174.

[0039] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises two antigen-binding modules that specifically bind to PSMA and two antigen-binding modules that specifically bind to CD3. In some embodiments, the antigen-binding module that specifically binds to PSMA is a Fab, and the antigen-binding module that specifically binds to CD3 is an scFv.

[0040] In some embodiments, the antigen-binding molecule of any one of the above, wherein the antigen-binding molecule comprises two first chains having a structure represented by formula (k) and two second chains having a structure represented by formula (d), Formula (k) [PSMA-VH]-[CH1]-[CD3-VH]-[linker 9]-[CD3-VL]-[linker 10]-[one subunit of Fc]; Formula (d) [PSMA-VL]-[CL], The structures shown in formula (k) and formula (d) are arranged from the N-terminus to the C-terminus, and the linker 9 and the linker 10 are the same or different peptide linkers. (As a non-limiting example, the structure shown in FIG. 1D.) It should be understood that Fc1 and Fc2 here function in the formation of a dimer and are therefore interchangeable.

[0041] In some embodiments, the antigen-binding molecule of any one of the above, The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 184 and two second chains comprising the amino acid sequence of SEQ ID NO: 181; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 190 and two second chains comprising the amino acid sequence of SEQ ID NO: 188; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 197 and two second chains comprising the amino acid sequence of SEQ ID NO: 194; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 199 and two second chains comprising the amino acid sequence of SEQ ID NO: 200; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 205 and two second chains comprising the amino acid sequence of SEQ ID NO: 204; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO:209 and two second chains comprising the amino acid sequence of SEQ ID NO:207.

[0042] In another aspect, the disclosure further provides an isolated antibody, which is capable of specifically binding to PSMA, said antibody comprising a heavy chain variable region PSMA-VH and a light chain variable region PSMA-VL, wherein (i) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 77, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 78, respectively; or (ii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 73, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 74, respectively; or (iii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 75, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 76, respectively; or (iv) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 79, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 80, respectively; or (v) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 81, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 82, respectively; or (vi) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 83, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 84, respectively; or (vii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 85, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 86, respectively; or (viii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 87, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 88, respectively; or (ix) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 89, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 90, respectively; or (x) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 91, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 92 or 152, respectively; or (xi) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 93, respectively, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 94, respectively; or (xii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in the above PSMA-VH respectively comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in SEQ ID NO: 95, and PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in the above PSMA-VL respectively comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 96.

[0043] In some embodiments, the PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

[0044] In some embodiments, the isolated antibody is an antibody comprising: (i) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 15, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, or (ii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:1, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:3, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:4, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:6; or (iii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:7, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:9, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:10, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:12, or (iv) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or (v) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:25, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:27, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:28, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:29, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:30; or (vi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 33, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 36, or (vii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 42, or (viii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 45, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 46, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 48, or (ix) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 49, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 51, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 52, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 54, or (x) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58 or 159, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 59, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 60; or (xi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 61, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 66, or (xii) The above PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 67, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 69, and the above PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 72.

[0045] In some embodiments, the PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 are defined according to the Kabat numbering convention.

[0046] In some embodiments, the antibody has an EC of less than 10 μg / mL, 1.5 μg / mL, 1.3 μg / mL, 1.1 μg / mL, 1 μg / mL, 0.7 μg / mL, 0.6 μg / mL, 0.5 μg / mL, 0.4 μg / mL, 0.3 μg / mL, 0.2 μg / mL, 0.1 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL. 50 Binds to PSMA at EC 50 is measured by ELISA.

[0047] In some embodiments, the antibody of any one of the preceding claims, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 77, 107, 108, 109, 110, 111, or 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 78, 113, 114, or 115; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73 or 99, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74 or 100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, 101, 102, or 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76, 104, 105, or 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 79, 116, or 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 80, 118, or 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81 or 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82, 121, 122, 123, 124, 125, or 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 83 or 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 84, 128, 129, or 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, 131, or 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86, 133, 134, or 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, 136, 137, or 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88, 139, or 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, 141, 142, 143, or 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90, 145, 146, or 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, 148, or 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92, 150, 151, or 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, 153, 154, 155, or 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94, 157, or 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, 277, 278, or 279, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96, 280, 281, 282, or 283.

[0048] In some embodiments, the antibody of any one of the preceding claims, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:77 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:78; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:114; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:99 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO:79, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:80; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 121; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 122; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 123; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 124; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 125; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 83, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 84; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 129; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 136 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 137 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156 and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96.

[0049] In some embodiments, the antibody of any one of the preceding claims, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128.

[0050] In some embodiments, the antibody of any one of the preceding claims, wherein the antibody is a bispecific antibody. In some embodiments, the bispecific antibody specifically binds to PSMA and CD3.

[0051] In another aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the antigen-binding molecules described above or any one of the antibodies described above, and one or more pharma- ceutically acceptable vectors, diluents, buffers, or excipients. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent. In some embodiments, the second therapeutic agent is an antibody capable of specifically binding to CD28, or an antibody capable of specifically binding to PSMA and CD28.

[0052] In another aspect, the present disclosure further provides an isolated nucleic acid encoding any one of the antigen-binding molecules described above or any one of the antibodies described above.

[0053] In another aspect, the disclosure further provides a host cell comprising the above-described nucleic acid. The host cell is incapable of developing into a plant or an animal.

[0054] In another aspect, the present disclosure further provides a method for treating a disease, the method comprising administering to a subject a therapeutically effective amount of any one of the antigen-binding molecules described above or any one of the antibodies described above or a composition thereof.

[0055] In another aspect, the present disclosure further provides the use of any one of the antigen-binding molecules described above or any one of the antibodies described above or compositions thereof in the preparation of a medicament for treating or preventing a disease.

[0056] In another aspect, the present disclosure further provides the antigen-binding molecule or the antibody or composition thereof described in any one of the above for use as a medicament. In some embodiments, the medicament is used for the treatment of a disease.

[0057] In some embodiments, the disease described in any one of the above is a proliferative disease, a tumor, or an immune disease. In some embodiments, the disease described in any one of the above is any one selected from prostate cancer, lung cancer, endometrial cancer, renal cancer, bladder cancer, colorectal cancer (including colon cancer and rectal cancer), and gastric cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is castration-resistant prostate cancer.

[0058] In some embodiments, the disease is a PSMA-associated disease. In some embodiments, the disease is a disease in which expression of PSMA is abnormal.

[0059] The antigen-binding molecules provided in the present disclosure have good characteristics of therapeutic activity, safety, pharmacokinetic properties and druggability (e.g., stability). [Brief description of the drawings]

[0060] [Figure 1A] FIG. 1 is a schematic diagram of the structure of Format 1. [Figure 1B] FIG. 2 is a schematic diagram of the structure of Format 2. [Figure 1C] FIG. 1 is a schematic diagram of the structure of Format 3. [Figure 1D] FIG. 1 is a schematic diagram of the structure of Format 4. [Figure 1E] FIG. 1 is a schematic diagram of the structure of Format 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] term

[0062] The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0063] Unless otherwise indicated by the context, throughout the specification and claims, the words "comprise", "have", "contain" and the like should be understood to have an inclusive meaning, i.e., "including but not limited to", rather than an exclusive or exhaustive meaning. Unless otherwise stated, "comprise" includes "consists of". For example, in the case of PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, it expressly covers PSMA-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 1.

[0064] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0065] The term "and / or", e.g., "X and / or Y", should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both meanings or either meaning.

[0066] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α carbon attached to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that act in a similar manner to a naturally occurring amino acid.

[0067] The term "amino acid mutation" includes amino acid substitution, deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, provided that the final construct has the desired properties, such as reduced binding to Fc receptors. Deletions and insertions in the amino acid sequence include deletions and insertions at the amino and / or carboxy termini of the polypeptide chain. A specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods of modifying amino acid side groups other than genetic engineering, such as chemical modification, may also be available. As used herein, various names may be used to refer to the same amino acid mutation. As used herein, the amino acid residue at a particular site may be designated by the position + amino acid residue, e.g., 366W indicates that the amino acid residue at position 366 is a W. T366W indicates that the amino acid residue at position 366 has been mutated from the original T to a W.

[0068] The term "antigen-binding molecule" is used in the broadest sense as long as it exhibits the desired antigen-binding activity, and covers various molecules that specifically bind to antigens, including, but not limited to, antibodies, other polypeptides having antigen-binding activity, and antibody fusion proteins formed by fusing both. The antigen-binding molecule of the present specification includes a variable region (VH) and a variable region (VL) that together constitute an antigen-binding domain. Exemplarily, the antigen-binding molecule of the present specification is a bispecific antigen-binding molecule (e.g., a bispecific antibody).

[0069] The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), provided that they exhibit the desired antigen-binding activity. For example, a native IgG antibody is a heterotetrameric protein of about 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has one variable region (VH), also called the variable heavy chain domain, heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has one variable region (VL), also called the variable light chain domain or light chain variable domain, followed by one constant light chain domain (light chain constant region, CL).

[0070] The term "bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, for example, a single-chain antibody) capable of specifically binding to two different antigens or at least two different antigen epitopes of the same antigen. Bispecific antibodies of various structures have been disclosed in the prior art. Depending on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Depending on the number of antigen-binding regions, they can be divided into bispecific antibodies with bivalent, trivalent, tetravalent or more. Depending on whether the structure is symmetrical or not, they can be divided into bispecific antibodies with symmetrical structure and bispecific antibodies with asymmetrical structure. Here, antibody fragment-type bispecific antibodies, for example, Fab fragments with Fc fragment deletion, form bispecific antibodies with low immunogenicity, small molecular weight, and high tumor tissue permeability by binding two or more Fab fragments within a single molecule, and typical antibody structures of this type are, for example, F(ab)2, scFv-Fab, and (scFv)2-Fab. Antibodies such as IgG-Like bispecific antibodies (e.g., having Fc fragments) have a relatively large molecular weight, and the Fc fragment contributes to the purification of the antibody, improves its solubility and stability, and the Fc portion may bind to the receptor FcRn to increase the antibody serum half-life. Typical bispecific antibody structure models, such as KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb , IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585-608 (2019), Chen S1 et al., J Immunol Res.2019 Feb 11;2019:4516041).

[0071] The term "variable region" or "variable domain" refers to a domain that binds to an antigen in an antigen-binding molecule. In this specification, the heavy chain variable region of an antigen-binding module that specifically binds to PSMA is designated PSMA-VH, and the light chain variable region is designated PSMA-VL. The heavy chain variable region of an antigen-binding module that specifically binds to CD3 is designated CD3-VH, and the light chain variable region is designated CD3-VL. VH and VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDRs). Among them, the term "complementarity determining region" or "CDR" refers to the region that mainly promotes binding to an antigen in the variable domain, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. In this specification, the three CDR regions in PSMA-VH are designated as PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3, the three CDR regions in PSMA-VL are designated as PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3, the three CDR regions in CD3-VH are designated as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, and the three CDR regions in CD3-VL are designated as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3. Each VH and VL is, from the N-terminus to the C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A single VH or VL can be sufficient to confer antigen binding specificity.

[0072] The amino acid sequence boundaries of the CDRs can be determined by various known methods, such as, for example, the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), and the correspondence between various numbering systems is well known to those of skill in the art. The numbering conventions of the present disclosure are as shown in Table 1 below.

[0073] [Table 1]

[0074] Unless otherwise stated, both variable region and CDR sequences in the examples of the present disclosure are applied to the "Kabat" numbering convention. In a specific embodiment, one numbering system (e.g., Kabat) is used to define amino acid residues, but technical solutions corresponding to other numbering systems are considered as equivalent technical solutions.

[0075] The term "antibody fragment" refers to a molecule distinct from an intact antibody and includes a portion of the intact antibody that retains the intact antibody's antigen-binding ability. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies composed of antibody fragments.

[0076] The term "Fc region" or "fragment crystallizable region" is intended to define the C-terminal region of an antibody heavy chain, and includes native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be identical or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226, or from Pro230, to its carboxy terminus. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1. Unless otherwise stated, the numbering convention for the Fc region is the EU index.

[0077] The term "Titin chain" refers to a peptide fragment of Titin protein having a length of 78 to 118 amino acids and including Titin Ig-Like 152 domain, or a functional mutant thereof. The Titin chain can bind to the Obscurin chain to form a dimerized complex.

[0078] The term "Obscurin chain" refers to a peptide fragment of the Obscurin protein having a length of 87 to 117 amino acids and containing the Obscurin Ig-Like 1 domain, or a functional variant thereof, or a peptide fragment of the Obscurin-Like 1 protein having a length of 78 to 118 amino acids and containing the Obscurin-Like Ig-Like 1 domain, or a functional variant thereof. The Obscurin chain can bind to a Titin chain to form a dimerized complex.

[0079] The Titin chain and Obscurin chain of the present disclosure can be used to replace CH1 and CL in a Fab to form a substituted Fab (Fab-S), which substitution does not affect the binding of the antigen-binding molecule to an antigen or its epitope.

[0080] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0081] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while at the same time having relatively low immunogenicity in humans. Humanization may be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (i.e., the constant and variable region framework region portions).

[0082] The term "affinity" refers to the strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to internal binding affinity and reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can usually be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art (such as those described herein). The term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed in molar concentration (M). The KD value of an antibody can be measured by methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).

[0083] The term "monoclonal antibody" refers to a population of essentially homogeneous antibodies or members thereof, i.e., the amino acid sequences of the antibody molecules in the population are the same, except for minor natural mutations that may exist. In contrast, a "polyclonal antibody" typically contains a plurality of different antibodies having different amino acid sequences in their variable domains that are generally specific for different epitopes. "Monoclonal" describes the character of an antibody obtained from an essentially homogeneous antibody population, and should not be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0084] The term "antigen" refers to a molecule or portion of a molecule that can be selectively recognized or bound by an antigen-binding molecule (e.g., an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding molecules (e.g., antibodies).

[0085] The term "epitope" refers to an area or region on an antigen capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope may be formed from a contiguous string of amino acids (linear epitope) or may contain non-contiguous amino acids (conformational epitope), for example, where the non-contiguous amino acids are brought into spatial proximity to one another by folding of the antigen (i.e., tertiary folding of a proteinaceous antigen). Conformational and linear epitopes differ in that antibody binding to conformational epitopes is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by methods routine in the art, including, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide truncation analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0086] The terms "capable of specifically binding," "specifically binds," or "binding" refer to the ability of an antibody to bind to an antigen or epitope with greater affinity than to other antigens or epitopes. Generally, antibodies bind to antigens or epitopes with greater affinity than other antigens or epitopes with greater affinity than other antigens or epitopes. -7 M or less (for example, about 1×10 -8An antibody binds to an antigen or epitope with an equilibrium dissociation constant (KD) of 100 M or less. In some embodiments, the KD of an antibody for binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody for binding to a non-specific antigen (e.g., BSA, casein). KD can be measured by known methods, for example, FACS or surface plasmon resonance assays. However, an antibody that specifically binds to an antigen or its epitope may be cross-reactive with other related antigens, for example, with corresponding antigens from other species (homologs) (e.g., humans or monkeys such as Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) and Callithrix jacchus (common marmoset, marmoset)).

[0087] The term "does not bind" means that the antibody is unable to bind to an antigen or its epitope in the manner of specific binding described above. For example, the antibody may be capable of binding to an antigen having a specific binding activity of about 1×10 -6 It binds to an antigen or its epitope with an equilibrium dissociation constant (KD) of greater than or equal to M.

[0088] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to a target antigen or an epitope thereof. A specific antigen-binding module includes, for example, an antigen-binding domain of an antibody, including a heavy chain variable region and a light chain variable region. The term "antigen-binding module that specifically binds to PSMA" refers to a module that can bind to PSMA or an epitope thereof with sufficient affinity such that a molecule containing the module can be used as a diagnostic and / or therapeutic agent targeting PSMA. For example, an antigen-binding module that specifically binds to PSMA has an affinity of < about 1 x 10 -8 M or less, as measured by a surface plasmon resonance assay. Antigen-binding modules include antibody fragments, such as Fab, substituted Fab, or scFv, as defined herein.

[0089] The term "linker" refers to a linking unit that links two polypeptide fragments. In this specification, linkers appearing in the same structural formula may be the same or different. The linker may be a peptide linker and comprises one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. The linkers used in this specification may be the same or different. When "-" appears in a structural formula, it indicates that the units on both sides are directly linked by a covalent bond.

[0090] "Tm" is the melting denaturation temperature (intrinsic fluorescence). When a protein is denatured (by heating or by the action of a denaturing agent), the tertiary structure opens and the microenvironment of aromatic amino acids is changed, which results in a change in the emission fluorescence spectrum. In this disclosure, Tm1 refers to the temperature at which the fluorescence is changed to half of its maximum value.

[0091] "Tonset" is the onset temperature of denaturation. It means the temperature at which the protein starts to denature, i.e., the temperature at which the fluorescence value starts to change.

[0092] "Tagg" is the aggregation onset temperature. The aggregates were detected by static light scattering at two wavelengths, 266 nm and 473 nm, to monitor the temperature at the onset of sample aggregation. Tagg 266 refers to the onset of aggregation temperature monitored at 266 nm.

[0093] The term "nucleic acid" may be used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term covers nucleic acids containing known nucleoside analogs or modified backbone residues or linkers, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid and are metabolized in the same manner as the reference nucleoside. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid encoding an antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including one or more such nucleic acid molecules contained in a single vector or separate vectors, and one or more such nucleic acid molecules present in one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence also implicitly covers conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly stated. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced with a degenerate base and / or deoxyinosine residue.

[0094] The terms "polypeptide" and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, and apply to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0095] The term sequence "identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned. During alignment, gaps can be introduced as necessary to obtain the maximum percentage of sequence identity, and any conservative substitutions are not considered as part of the sequence identity. To measure the percentage of sequence identity, alignment can be achieved by techniques known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0096] The terms "fused" or "linked" refer to covalent linkage of elements (e.g., an antigen binding module and an Fc domain) either directly or via a linker.

[0097] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA circle that can be ligated to an additional DNA segment. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which another DNA segment can be ligated to the viral genome. Some vectors can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector that is useful for transformation of a host cell and contains (in conjunction with the host cell) nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, as well as RNA splicing of the coding region operably linked to introns, if any.

[0098] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its derived progeny, without regard to the number of passages. The progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. As used herein, the term includes mutant progeny that have the same function or biological activity as the cell screened or selected from the original transformed cell. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells, including, but not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, and the like. stiptis), Pichia methanolicamethanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum gramineum, Fusarium venenatum, Physcomitrella patens, Neurospora crassa, Aspergillus spp., Yarrowia lipolytica, Pichia spp., any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa. The host cells of this patent do not include those not permitted by patent law.

[0099] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but does not necessarily occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0100] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules or antibodies described herein and other chemical components, such as physiologically / pharmaceutical acceptable vectors and excipients.

[0101] The term "pharmaceutically acceptable vector, diluent, buffer, or excipient" refers to an ingredient other than the active ingredient in a pharmaceutical formulation that is non-toxic to a subject. Pharmaceutically acceptable vectors, diluents, buffers, or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0102] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the above terms "patient" or "subject" may be used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to cynomolgus monkeys (Macaca fascicularis). In some embodiments, the individual or subject is a human.

[0103] "Administration" or "giving" as applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid means contact of an exogenous agent, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid.

[0104] The term "sample" refers to a collection (e.g., fluid, cell, or tissue) isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymphatic fluid, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected from bronchial washings, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as media (including conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically resected tissues, organ cultures, or cell cultures.

[0105] "Treatment" and "treatment" (and grammatical variations thereof) refer to a clinical intervention intended to be given to the individual being treated, and can be performed prophylactically or in the clinical pathological process. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction / reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or amelioration of the disease state, and regression or improvement of prognosis. In some embodiments, the molecules of the present disclosure are used to delay the formation of disease or to slow the progression of disease.

[0106] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate those symptoms and / or their underlying causes, prevent the occurrence of the symptoms and / or their underlying causes, and / or ameliorate or ameliorate the damage caused by or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0107] A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a condition or symptom associated with the disease state, or to prevent, inhibit, slow or reverse the progression of the disease state or any other undesirable symptom associated in any way with the disease.

[0108] A "prophylactically effective amount" is an amount that, when administered to a subject, provides a desired prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or prophylactic effect does not necessarily occur when one dose is administered, but may occur after a series of doses are administered. Thus, a therapeutically or prophylactically effective amount can be administered in a single or multiple dose regime. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on various factors, such as, for example, the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to induce a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient. target molecule

[0109] "PSMA" should be understood broadly and is intended to cover various molecular forms of PSMA at various stages in the mammalian body, including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification processes of the PSMA gene (e.g., precursor PSMA, mature PSMA, membrane-expressed PSMA, PSMA splice variants, modified PSMA, or fragments thereof), and the term also covers artificially prepared or in vitro expressed PSMA.

[0110] "CD3" should be understood broadly and is intended to cover various forms of CD3 molecules at various stages in the mammalian body, including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification processes of the BCMA gene (e.g., precursor CD3, mature CD3, membrane-expressed CD3, CD3 splice variants, modified CD3, or fragments thereof), and the term also covers artificially prepared or in vitro expressed CD3. Antigen-binding molecules of the present disclosure

[0111] The present disclosure provides antigen-binding molecules that have many advantageous properties, such as high affinity for PSMA and PSMA-expressing cells, in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties and druggability (e.g., yield, purity and stability, etc.). Exemplary antigen-binding molecules

[0112] Antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (e.g., bispecific antibodies) that specifically bind to PSMA and CD3, and anti-PSMA antibodies. In particular, antigen-binding molecules of the present disclosure have one or more of the following: The antibody has a high affinity for PSMA. In some embodiments, the antibody has an EC of less than 10 μg / mL, 1.5 μg / mL, 1.3 μg / mL, 1.1 μg / mL, 1 μg / mL, 0.7 μg / mL, 0.6 μg / mL, 0.5 μg / mL, 0.4 μg / mL, 0.3 μg / mL, 0.2 μg / mL, 0.1 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL. 50 Binds to PSMA at EC 50 is measured by ELISA.

[0113] b. High affinity of the antigen-binding molecule (e.g., bispecific antibody) to PSMA. In some embodiments, the antigen-binding molecule has a binding affinity of 1×10 -7 M, 1×10 -8 M, 9×10 -9 M, 8×10 -9 M, 7×10 -9 M, 6×10 -9 M, 5×10 -9 M, 4×10 -9 M, 3×10 -9 M, 2.9×10 -9 M, 2.8×10 -9 M, 2.7×10 -9 M, 2.6×10 -9 M, 2.5×10 -9 M, 2.4×10 -9 M, 2.3×10 -9 M, 2.2×10 -9 M, 2.1×10-9 M, 2.0×10 -9 M, 1.9×10 -9 M, 1.8×10 -9 M, 1.7×10 -9 M, 1.6×10 -9 M, 1.5×10 -9 M, 1.4×10 -9 It binds to human PSMA with a KD of less than M, as measured by surface plasmon resonance.

[0114] c. High affinity for cell surface PSMA. The antibody has an EC of less than 10 μg / mL, 2 μg / mL, 1.5 μg / mL, 1.3 μg / mL, 1.2 μg / mL, 1.1 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL. 50 Binds to cell surface PSMA and inhibits EC 50 is measured by ELISA. The cells are CHO overexpressing human PSMA.

[0115] d. In vitro specific killing activity against PSMA-expressing cells.

[0116] e. Induction of low level cytokine (IL6 and IFNγ) release.

[0117] f. Greater in vivo therapeutic activity.

[0118] The present disclosure provides an antigen-binding molecule, which comprises at least one antigen-binding module that specifically binds to PSMA and at least one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to PSMA comprises PSMA-VH and PSMA-VL, and the antigen-binding module that specifically binds to CD3 comprises CD3-VH and CD3-VL.

[0119] The present disclosure further provides isolated antibodies, which can specifically bind to PSMA, including PSMA-VH and PSMA-VL. Specifically, the examples herein disclose antibody series 2, 13, 15, 19, 27, 31, 41, 43, 46, 50, 71, and 73. Hereinafter, the antibodies of the present disclosure will be described using antibody 15 as an example.

[0120] The PSMA-VH of the antigen-binding molecule or anti-PSMA antibody that specifically binds to PSMA and CD3 has a PSMA-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 13, a PSMA-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 14, and a PSMA-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 15, and the PSMA-VL has a PSMA-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 16, a PSMA-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 17, and a PSMA-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 18.

[0121] In some embodiments, the antigen-binding molecule or antibody, the PSMA-VH and / or the PSMA-VL are murine or humanized. In some embodiments, the PSMA-VH and / or the PSMA-VL are humanized. In some embodiments, FR1, FR2 and FR3 of the humanized PSMA-VH have at least 60%, 70% or 80% sequence identity to FR1, FR2 and FR3 of SEQ ID NO: 77, FR4 of the humanized PSMA-VH have at least 80% or 90% sequence identity to FR4 of SEQ ID NO: 77, FR1, FR2 and FR3 of the humanized PSMA-VL have at least 60%, 70% or 80% sequence identity to FR1, FR2 and FR3 of SEQ ID NO: 78, and / or FR4 of the humanized PSMA-VL have at least 80% or 90% sequence identity to FR4 of SEQ ID NO: 78. In some embodiments, the PSMA-VH has FR1, FR2, FR3 from IGHV1-3*01 and FR4 from IGHJ1*01, and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 2F, 28S, 44C, 48I, 67A, 69L, 71V, 73Q, and 76T, and / or the PSMA-VL has FR1, FR2, FR3 from IGKV1-27*01 and FR4 from IGKJ2*01, and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S, 60D, and 100C. In some embodiments, 44C and 100C are present simultaneously. In some embodiments, the variable regions and CDRs are defined according to the Kabat numbering convention.

[0122] In some embodiments, the antigen-binding molecule or antibody of any one of the above, wherein the amino acid sequence of the PSMA-VH has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 77, 107, 108, 109, 110, 111 or 112, and the amino acid sequence of the PSMA-VL has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 78, 113, 114 or 115. In some embodiments, the amino acid sequence of the PSMA-VH is set forth in SEQ ID NO: 77, 107, 108, 109, 110, 111 or 112, and the amino acid sequence of the PSMA-VL is set forth in SEQ ID NO: 78, 113, 114 or 115. In some embodiments, the amino acid sequence of the PSMA-VH is set forth in SEQ ID NO: 107, 108, 109, 110, 111, or 112, and the amino acid sequence of the PSMA-VL is set forth in SEQ ID NO: 113, 114, or 115.

[0123] In some embodiments, the antigen-binding molecule or antibody according to any one of the above, the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 112 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 115; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 110, and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 114; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 77 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 78; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 107 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 113; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 108 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 113; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 109 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 113; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 110, and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 113; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 111 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 113; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 107 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 114; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 108 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 114; or the amino acid sequence of said PSMA-VH is set forth in SEQ ID NO: 109 and the amino acid sequence of said PSMA-VL is set forth in SEQ ID NO: 114; or The amino acid sequence of the PSMA-VH is shown in SEQ ID NO:111, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO:114.

[0124] In some embodiments, the antigen-binding molecule or antibody of any one of the above, wherein the amino acid sequence of the PSMA-VH is set forth in SEQ ID NO: 112 and the amino acid sequence of the PSMA-VL is set forth in SEQ ID NO: 115; or The amino acid sequence of the PSMA-VH is shown in SEQ ID NO:110, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO:114.

[0125] In some embodiments, in the above-mentioned antigen binding molecule, the CD3-VH has a CD3-HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 160, a CD3-HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 161, and a CD3-HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 162, and the CD3-VL has a CD3-LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 163, a CD3-LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 164, and a CD3-LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 165.

[0126] In some embodiments, the antigen binding molecule of any one of the above, wherein the CD3-VH and / or the CD3-VL are murine or humanized. In some embodiments, the CD3-VH and / or the CD3-VL are humanized. In some embodiments, the amino acid sequence of the CD3-VH is set forth in SEQ ID NO: 166, and the amino acid sequence of the CD3-VL is set forth in SEQ ID NO: 167. In some embodiments, the variable regions and CDRs are defined according to the Kabat numbering convention.

[0127] According to the technical solutions of antibody series 2, 13, 19, 27, 31, 41, 43, 46, 50, 71 and 73 disclosed in the Examples of this specification, these antibodies have a similar technical solution scope as antibody 15 described above. Structure of antigen-binding molecules

[0128] The bispecific antigen-binding molecule of the present disclosure is not limited to a specific molecular structure as long as it has the desired antigen-binding function. For example, the bispecific antigen-binding molecule of the present disclosure may be bivalent (1+1), trivalent (2+1) or tetravalent (2+2). The antigen-binding module in the antigen-binding molecule may be any antibody fragment having antigen-binding activity fused via a peptide linker. The peptide linker of the present disclosure (e.g., linkers 1-11) may be any suitable peptide chain as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide of 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of L1-(GGGGS)n-L2, where L1 is a bond, A, GS, GGS, GGGS (SEQ ID NO: 276), SGGGGS (SEQ ID NO: 178), GGGTKLTVLGGG (SEQ ID NO: 177), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG (SEQ ID NO: 179), and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of (GGGGS)n, where n is 1, 2, or 3. In some aspects, the peptide linker is GGGGS (SEQ ID NO: 173), GGGGSGGGGSGGGGS (SEQ ID NO: 176), GGGTKLTVLGGG (SEQ ID NO: 177), SGGGGS (SEQ ID NO: 178), or GGG. In some embodiments, the amino acid sequences of linker 1, linker 3, and linker 10 are set forth in SEQ ID NO: 176, the amino acid sequence of linker 2 is set forth in SEQ ID NO: 178, the amino acid sequences of linker 4 and linker 11 are GGG, and the amino acid sequences of linker 5, linker 6, linker 7, linker 8, and linker 9 are set forth in SEQ ID NO: 173.

[0129] Exemplarily, the antigen-binding molecule of the present disclosure comprises one first chain having a structure represented by formula (ai) and one second chain having a structure represented by formula (b): Formula (ai) [PSMA-VH]-[GGGGSGGGSGGGGS]-[PSMA-VL]-[SGGGGS]-[CD3-VH]-[GGGGSGGGGSGGGS]-[CD3-VL]-[GGG]-[Fc2], Formula (b) [Fc1], The structures represented by formulae (ai) and (b) are arranged from the N-terminus to the C-terminus.

[0130] Exemplary bivalent antigen-binding molecules include: The antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO: 192 and one second chain whose amino acid sequence is set forth in SEQ ID NO: 169; or The antigen-binding molecule comprises one first chain whose amino acid sequence is set forth in SEQ ID NO:191 and one second chain whose amino acid sequence is set forth in SEQ ID NO:169. Mutants of antigen-binding molecules

[0131] In some embodiments, amino acid sequence variants of the antigen-binding molecules provided herein are covered. For example, it is desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antigen-binding molecule. The final construct can be obtained by making any combination of deletion, insertion and substitution, provided that the final construct has the desired properties, such as antigen-binding properties. Mutant substitutions, insertions and deletions

[0132] In one embodiment, variants of antigen-binding molecules having one or more amino acid substitutions are provided. Substitutions can be made in the CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". More substantial changes are provided in Table 2 under the heading of "exemplary substitutions" and as further described below with reference to amino acid side chain classifications. Amino acid substitutions can be introduced into an antibody of interest and the product screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0133] [Table 2]

[0134] According to common side chain properties, amino acids can be grouped as follows: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0135] Non-conservative substitutions refer to the replacement of a member of one class with a member of another class.

[0136] One type of substitution variant involves the substitution of one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study has an altered (e.g., improved) biological property (e.g., improved affinity, reduced immunogenicity) relative to the parent antibody and / or essentially retains some biological property of the parent antibody. One exemplary substitution variant is an affinity matured antibody, which can be readily produced, for example, by phage display-based affinity maturation techniques (e.g., techniques as described herein). Briefly, one or more CDR residues are mutated, the mutated antibodies are displayed on phage, and screened for a particular biological activity (e.g., binding affinity). For example, changes (e.g., substitutions) can be made in the CDRs to improve antibody affinity. Such changes can be made to CDR "hot spots", i.e., residues encoded by codons frequently mutated during the somatic maturation process and / or residues that contact the antigen, and the resulting variant VH or VL can be tested for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves CDR-targeting, where a few CDR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify CDR residues involved in antigen binding.

[0137] In certain embodiments, substitutions, insertions or deletions may be made in one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) can be made to the CDRs that do not substantially reduce binding affinity. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two or three amino acid substitutions.

[0138] A method that can be used to identify residues or regions that can be target sites for mutagenesis in an antibody is called "alanine scanning mutagenesis". In this method, a residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to see if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Also, contact points between the antibody and the antigen can be identified by examining crystal structures of antigen-antibody complexes. These contact and adjacent residues can be targeted or removed as substitution candidates. Mutants can be screened to see if they contain the desired properties.

[0139] Amino acid sequence insertions include the fusion of polypeptides of one residue or 100 or more residues in length at the amino and / or carboxyl terminus, and the insertion of one or more amino acid residues into the sequence. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions to the N- or C-terminus of the antibody with an enzyme (or a polypeptide which extends the serum half-life of the antibody). Fab modification

[0140] In one embodiment, in the antigen-binding molecule of the present disclosure, one of the antigen-binding module that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 is a replacement Fab containing a heavy chain variable region, a light chain variable region, a Titin chain, and an Obscurin chain. In the replacement Fab, the original CH1 and CL in the Fab are replaced with the Titin chain and the Obscurin chain. Exemplarily, the sequences of the Titin chain and the Obscurin chain are as shown in Table 3-1 and Table 3-2.

[0141] [Table 3-1-1] [Table 3-1-2] [Table 3-2-1] [Table 3-2-2] [Table 3-2-3] [Table 3-2-4] [Table 3-2-5] Fc Region Modification

[0142] In one aspect, the Fc region of the antigen binding molecule of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an Fcγ receptor, and reduce or eliminate effector function. A native IgG Fc region is specifically an IgG1 Fc region or an IgG4 Fc region, which may allow the antigen binding molecule of the present disclosure to target cells expressing the Fc receptor rather than cells expressing the antigen. The modified Fc region of the present disclosure exhibits reduced binding affinity to an Fc receptor and / or reduced effector function. In some embodiments, the modified Fc region has a reduced binding affinity to an Fc receptor of 50%, 80%, 90%, or 95% or more compared to the native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the altered Fc region also has reduced binding affinity to complement (e.g., C1q) relative to the native Fc region. In some embodiments, the altered Fc region does not have reduced binding affinity to the neonatal Fc receptor (FcRn) relative to the native Fc region. In some examples, the altered Fc region has reduced effector function, which may include, but is not limited to, one or more of reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen presenting cell uptake, reduced binding to NK cells, reduced binding to macrophage cells, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced cell apoptosis, reduced dendritic cell maturation, or reduced triggering by T cells.

[0143] For an IgG1 Fc region, effector function can be reduced by amino acid residue substitutions at positions such as 238, 265, 269, 270, 297, 327 and 329. In some embodiments, the Fc region is a human IgG1 Fc region and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For an IgG4 Fc region, effector function can be reduced by amino acid residue substitutions at positions such as 228.

[0144] The antigen-binding molecule may further include disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit. To increase the serum half-life of the antigen-binding molecule, the following mutations can be introduced: 252Y, 254T, and 256E.

[0145] When an antigen-binding molecule contains different antigen-binding modules fused to two subunits in the Fc region, this may lead to undesired homodimerization. To improve yield and purity, it is advantageous to introduce heterodimerization-promoting modifications to the Fc region of the antigen-binding molecule of the present disclosure. In some embodiments, the Fc region of the present disclosure includes a knob-into-hole (KIH) technique modification, which involves introducing a knob structure at the interface of the first subunit and a hole structure at the interface of the second subunit. This allows the knob structure to be positioned at the hole structure, promoting the formation of heterodimers and inhibiting the production of homodimers. The knob structure is constructed by replacing a small amino acid side chain from the interface of the first subunit with a relatively large side chain (e.g., tyrosine or tryptophan). Meanwhile, the hole structure is constructed at the interface of the second subunit by replacing a large amino acid side chain with a relatively small amino acid side chain (e.g., alanine or threonine). The knob and hole structures are prepared by altering the nucleic acid encoding the polypeptide, with selectable amino acid substitutions as shown in the table below.

[0146] [Table 4]

[0147] Except for the knobs-in-holes technique, other techniques for modifying the CH3 domain of the heavy chain so as to achieve heterodimerization are also known in the art, e.g. WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 and WO013 / 096291.

[0148] The C-terminus of the Fc region may be a complete C-terminus terminating in amino acid residue PGK, or may be a truncated C-terminus, e.g., one or two C-terminal amino acid residues removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus terminating in PG. Thus, in some embodiments, a composition of complete antibodies may comprise antibodies in which any K447 residue and / or G446+K447 residues have been removed. In some embodiments, a composition of complete antibodies may comprise antibodies in which the K447 residue and / or G446+K447 residues have not been removed. In some embodiments, a composition of complete antibodies comprises antibodies of an antibody mixture with and without the K447 residue and / or the G446+K447 residues. Recombinant methods

[0149] Antigen-binding molecules can be produced by recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecule are provided.

[0150] In the case of a natural antibody, a natural antibody fragment, or a bispecific antibody with a homodimeric heavy chain, two nucleic acids are required: one for the light chain or a fragment thereof, and one for the heavy chain or a fragment thereof. Such nucleic acids encode an amino acid sequence comprising the antibody VL and / or an amino acid sequence comprising the antibody VH (e.g., the light and / or heavy chains of the antibody). These nucleic acids can be present on the same expression vector or on different expression vectors.

[0151] In the case of a bispecific antibody having a heterodimeric heavy chain, for example, four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising a first heterologous monomeric Fc region polypeptide, one for the second light chain and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors, typically these nucleic acids are located in two or three expression vectors, i.e. one vector may contain one or more of these nucleic acids.

[0152] In one embodiment, the disclosure provides an isolated nucleic acid encoding such an antibody. Each such nucleic acid can independently encode any one of the polypeptide chains. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In another aspect, the disclosure provides a host cell comprising such a nucleic acid. In one embodiment, a method of preparing an antigen-binding molecule is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0153] To produce a recombinant antigen-binding molecule, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be easily isolated and sequenced by routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an antibody), or produced by recombinant methods, or obtained by chemical synthesis.

[0154] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, antibodies can be isolated from bacterial cell paste in a soluble fraction and can be further purified.

[0155] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns. Suitable host cells for the expression (glycosylation) of antibodies may be derived from multicellular organisms (invertebrates and vertebrates), examples of invertebrate cells include plants and insect cells. Many baculovirus strains have already been identified that may be used in conjunction with insect cells, in particular for transfection of Spodoptera frugiperda cells, and plant cell cultures such as US5959177, US6040498, US6420548, US7125978 and US6417429 may also be used as hosts, as well as vertebrate cells, e.g. mammalian cell lines suitable for growth in suspension. Other examples of suitable mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, MRC 5 cells and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of several mammalian host cell lines suitable for the production of antibodies, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pages 255-268. Diagnostic and Therapeutic Compositions

[0156] In some embodiments, the antigen binding molecules provided herein can be used to detect the presence of PSMA and / or CD3 in a biological sample. As used herein, the term "detection" covers quantitative or qualitative detection. In some embodiments, the biological sample includes cells or tissues such as tumor tissue.

[0157] In one embodiment, the antigen-binding molecule is used in a diagnostic or detection method. In another aspect, a method for detecting the presence of PSMA and / or CD3 in a biological sample is provided. In one embodiment, the method comprises contacting a biological sample with an antigen-binding molecule under suitable conditions, and detecting whether a complex is formed between the detection agent and the antigen. Such a method may be an in vitro method or an in vivo method. In one embodiment, the antigen-binding molecule is used to select subjects suitable for treatment, for example, PSMA or CD3 is a biomarker for selecting patients.

[0158] Exemplary disease states that can be diagnosed using the antigen binding molecules of the present disclosure are, for example, tumors and cancers.

[0159] In one embodiment, a labeled antigen-binding molecule is provided. Labels include, but are not limited to, directly detectable labels or modules (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent and radioactive labels) and indirectly detectable modules (e.g., modules that are indirectly detected by enzymatic reactions or molecular interactions, such as enzymes and ligands).

[0160] In another embodiment, a pharmaceutical composition comprising the antigen-binding molecule is provided, for example, for use in any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and a pharma- ceutical acceptable vector. In another embodiment, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and at least one other therapeutic agent.

[0161] The pharmaceutical composition of the antigen-binding molecule described in the present disclosure is prepared by mixing such an antigen-binding molecule having the required purity with one or more pharma- ceutically acceptable vectors, and the pharmaceutical composition is in the form of a lyophilized composition or an aqueous solution. Preparations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. Treatment Methods and Routes of Administration

[0162] Any of the antigen binding molecules provided herein can be applied in therapeutic methods.

[0163] In a further aspect, the disclosure provides a use of an antigen-binding molecule in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a tumor or cancer. Also, the medicament is present in an amount effective for the disease. In some embodiments, the effective amount is a daily dose or a weekly dose. In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents). The "subject" according to any of the above embodiments may be a human.

[0164] In a further aspect, there is provided a pharmaceutical composition comprising the antigen-binding molecule, for example for use in any of the pharmaceutical uses or methods of treatment described above. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0165] The antigen-binding molecules of the present disclosure can be used alone or in combination with other reagents for treatment. For example, the antigen-binding molecules of the present disclosure can be co-administered with at least one additional therapeutic agent.

[0166] The antigen-binding molecules of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, pulmonary and nasal administration, and, where localized treatment is required, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0167] The antigen-binding molecules of the present disclosure are prepared, administered and administered in accordance with GOOD MEDICAL PRACTICE. Factors to consider in this context include the specific condition being treated, the specific mammal being treated, the clinical status of the individual patient, the cause of the condition, the delivery site of the reagent, the method of administration, the administration schedule and other factors known to medical practitioners. The antigen-binding molecules are optionally prepared in combination with one or more reagents currently used to prevent or treat the above conditions. The effective amount of such other reagents depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of condition or treatment and other factors discussed above. These are generally used in the same doses and administration routes as described herein, or from about 1% to about 99% of the doses described herein, or in any dose and by any route empirically / clinically confirmed to be appropriate.

[0168] The appropriate dose of the antigen-binding molecule according to the present disclosure (used alone or in combination with one or more additional therapeutic agents) for preventing or treating a disease is determined by the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether the administration is for prevention or treatment, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is administered to the patient in one or a series of treatments, as appropriate. Depending on the type and severity of the disease, about 1 μg / kg to about 15 mg / kg of the antigen-binding molecule may be an initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dose may range from about 1 μg / kg to about 100 mg / kg or more, depending on the factors mentioned above. Thus, for a 50 kg body weight, an exemplary daily dose is 50 μg to 5 g. product

[0169] In another aspect of the disclosure, an article of manufacture is provided that includes materials applicable for treating, preventing, and / or diagnosing the above-mentioned medical conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as, for example, glass or plastic. The container may contain a composition that, alone or in combination with another composition, effectively treats, prevents, and / or diagnoses a disease, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a plug that can be pierced by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule according to the present disclosure. The label or package insert indicates that the composition is used to treat a selected medical condition. The article of manufacture may also include (a) a first container that contains a composition that includes an antigen-binding molecule according to the present disclosure, and (b) a second container that contains a composition that includes another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the disclosure may further include a package insert, the package insert indicating that the composition is applicable for treating a particular medical condition. Optionally, or in addition, the article of manufacture may further include a second (or third) container containing a pharma-ceutically acceptable buffer. From a business and user standpoint, it may further include other materials required, including other buffers, diluents, filters, needles and syringes.

[0170] In one example, the article is prepared in the form of a kit. Examples and Tests

[0171] The present disclosure will be further described below in conjunction with examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples and test examples of the present disclosure generally follow standard conditions, such as those in the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual, or conditions recommended by manufacturers of raw materials or products. Reagents for which a specific source is not specified are standard commercially available reagents.

[0172] Example 1 Antigen-binding molecule containing Titin chain / Obscurin chain The Titin chain / Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including those derived from WO2021139758A1 (incorporated herein in its entirety by reference) and CN202110527339.7, and patents which claim priority thereto (incorporated herein in their entirety by reference). A bispecific antibody was constructed, in which CL is the kappa light chain constant region in WO2021139758A1, the amino acid sequences of the Titin chain and Obscurin chain are shown in Tables 3-1 and 3-2, the linker sequence comprises GGGGS (SEQ ID NO: 173), ASTKG (SEQ ID NO: 270) or RTVAS (SEQ ID NO: 271), and the amino acid sequences of Fc1, Fc2, and CH1 in this example are as follows:

[0173] >Fc1 (knob, SEQ ID NO: 272) [ka] >Fc2 (hole, SEQ ID NO: 273) [ka] >CH1 (sequence number 168).

[0174] 1.1 DI bispecific antibodies With reference to Example 5 of WO2021139758A1, anti-hNGF and hRANKL DI bispecific antibodies: DI-2 to DI-20 were constructed, which comprise the following first heavy chain, second heavy chain, first light chain and second light chain: First heavy chain: [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2] from the N-terminus to the C-terminus; First light chain: from the N-terminus to the C-terminus, it is [VL1-I]-[Linker 2]-[Titin chain]; A second heavy chain: from N-terminus to C-terminus, [VH2-D]-[CH1]-[Fc1]; and The second light chain is, from the N-terminus to the C-terminus, [VL2-D]-[CL]; Among them, VH1-I and VL1-I are the heavy chain variable region and light chain variable region of I0 in WO2021139758A1, respectively, and VH2-D and VL2-D are the heavy chain variable region and light chain variable region of D0 in WO2021139758A1, respectively. In this embodiment, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the DI bispecific antibody are shown in the table below.

[0175] [Table 5] Note: The numbers of the Titin and Obscurin chains in the table are shown in Tables 3-1 and 3-2.

[0176] The binding activity of the DI-2 to DI-20 bispecific antibodies and their antigens was detected by the method in Test Example 4 of WO2021139758A1. The thermal stability of the antibodies was studied. Research method: The antibody was diluted to a concentration of 5 mg / mL with PBS, and its thermal stability was measured using a high-throughput differential scanning fluorometer (UNCHAINED, model number: Unit). The experimental results showed that the modified bispecific antibodies had no significant change in binding activity to the antigen, and compared with DI-2, DI-4 to DI-8, DI-10 to DI-16, and DI-20 had significantly improved Tm1 (℃) and Tonset (℃), indicating that the thermal stability of the bispecific antibodies was superior.

[0177] [Table 6] [Table 7]

[0178] A solution containing DI bispecific antibodies was prepared using a buffer solution of 10 mM acetic acid, pH 5.5, and 9% sucrose, and the solution was incubated in a 40°C incubator for 4 weeks. After incubation, the antibody concentration was concentrated to the same concentration as at the start of incubation, and the precipitation state of the solution was observed. The experimental results showed that precipitation appeared in the solution of the DI-2 bispecific antibody group, and DI-2 has better stability than DI-3 to DI-7.

[0179] [Table 8]

[0180] 1.2 PL bispecific antibodies Anti-hPDL1 and hCTLA4 PL bispecific antibodies: PL-1 to PL-19 were constructed, which contain the first heavy chain, second heavy chain, first light chain and second light chain described above: First heavy chain: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1] from the N-terminus to the C-terminus; First light chain: [VL1-P]-[Linker 2]-[Titin chain] from the N-terminus to the C-terminus. A second heavy chain: from N-terminus to C-terminus, [VH2-L]-[CH1]-[Fc2]; and The second light chain is, from the N-terminus to the C-terminus, [VL2-L]-[CL]; Of these, VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1, respectively, and the amino acid sequences of VH2-L and VL2-L are as follows.

[0181] >VH2-L (SEQ ID NO: 274) [ka] >VL2-L (SEQ ID NO: 275) [ka] In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the PL bispecific antibody are shown in the table below.

[0182] [Table 9-1] [Table 9-2]

[0183] Note: The numbers of the Titin and Obscurin chains in the table are shown in Tables 3-1 and 3-2.

[0184] The binding activity of the PL bispecific antibody was detected by referring to the ELISA method in Test Example 4 in WO2021139758A1, among which the hPDL1 and hCTLA4 antigens were purchased from Sino biology. The thermal stability of the antibody was studied. Method: The antibody was diluted with PBS to a concentration of 1.4-3mg / mL, and its thermal stability was measured by a high-throughput differential scanning fluorometer (UNCHAINED, model number: Unit). The experimental results showed that the PL bispecific antibody still had good binding activity to the antigen, and compared with PL-1, PL-2-PL-19 had obviously improved Tm1 (℃), Tagg 266 (℃), and Tonset (℃), and the thermal stability of the bispecific antibody was better.

[0185] [Table 10] [Table 11-1] [Table 11-2]

[0186] 1.3 HJ bispecific antibody Anti-hIL5 and hTSLP HJ bispecific antibodies: HJ-3 to HJ11 were constructed, which contain the following first heavy chain, second heavy chain, first light chain and second light chain: First heavy chain: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1] from the N-terminus to the C-terminus; First light chain: from the N-terminus to the C-terminus, it is [VL1-H]-[Linker 2]-[Obscurin chain]; A second heavy chain: from N-terminus to C-terminus, [VH2-J]-[CH1]-[Fc2]; and The second light chain is, from the N-terminus to the C-terminus, [VL2-J]-[CL]; Here, VH1-H and VL1-H are the heavy chain variable region and light chain variable region of H0 in WO2021139758A1, respectively, and VH2-J and VL2-J are the heavy chain variable region and light chain variable region of J1 in WO2021139758A1, respectively. In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the HJ bispecific antibody are shown in the table below.

[0187] [Table 12]

[0188] The antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 of WO2021139758A1. The thermal stability of the antibody was studied as follows: a diluted solution of HJ bispecific antibody was prepared with a buffer solution of 10 mM acetate pH 5.5, 9% sucrose, and the bispecific antibody was concentrated by the method of ultrafiltration concentration to obtain HJ bispecific antibody solutions of different concentrations (the concentrations of HJ bispecific antibody are shown in Table 13-2). The concentrated solutions were then placed in an incubator at 40°C for incubation, and the SEC purity of the samples was detected on day 0 (i.e., before the start of incubation at 40°C, D0), day 7 (day 7 of incubation at 40°C, D7), day 14 (day 14 of incubation at 40°C, D14), day 21 (day 21 of incubation at 40°C, D21) and day 28 (day 28 of incubation at 40°C, D28). After 28 days of incubation at 40°C, samples were immediately sampled to detect the CE-SDS purity of the samples. Experimental results show that the HJ bispecific antibodies constructed in the present disclosure have no significant change in antigen-binding activity, and compared with HJ-3, the HJ-5 to HJ-11 bispecific antibodies have better thermal stability.

[0189] [Table 13-1] [Table 13-2]

[0190] Example 2. Screening and identification of anti-human PSMA hybridoma antibodies The present disclosure uses hybridoma technology to prepare a monoclonal antibody against human PSMA. The obtained antibody can specifically bind to human PSMA with high affinity and cross-react with cynomolgus PSMA, and has good binding activity to human PSMA and cynomolgus PSMA on the cell surface.

[0191] Mice were cross-immunized with human PSMA-ECD-his, LnCap cells, or ChoK1-human PSMA cells as immunoreagents, or with human PSMA-ECD-his and LnCap cells as cross-immune reagents, and with TiterMax® Gold Adjuvant (Sigma Cat No. T2684) and Thermo Imject® Alum (Thermo Cat No. 77161) as adjuvants. After the first immunization and seven booster immunizations, mice 1-1, 1-2, 2-1, 2-5, 3-3, 3-4, 3-5, and 4-2# (titer > 307.2K) with high antibody titers in the serum were selected and subjected to splenocyte fusion. After fusion, the hybridoma culture supernatant was detected according to the growth density of the hybridoma cells, and antibodies that specifically bind to cell surface PSMA were screened. Monoclonal hybridoma cell lines with good activity were obtained by screening. Hybridoma cells in logarithmic growth phase were harvested, and RNA was extracted with NucleoZol (MN) (following the steps in the reagent kit's instruction manual) and reverse transcribed (PrimeScript (商標) The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced. The amino acid sequences of the CDRs and variable regions of the screened monoclonal hybridoma cell lines are as follows: [Table 14-1] [Table 14-2]

[0192] >2 Mouse heavy chain variable region (SEQ ID NO:73) [ka] >2 Mouse Light Chain Variable Region (SEQ ID NO:74) [ka] >13 Mouse heavy chain variable region (SEQ ID NO: 75) [ka] >13 Mouse light chain variable region (SEQ ID NO:76) [ka] >15 Mouse heavy chain variable region (SEQ ID NO:77) [ka] >15 Mouse Light Chain Variable Region (SEQ ID NO:78) [ka] >19 Mouse heavy chain variable region (SEQ ID NO: 79) [ka] >19 Mouse light chain variable region (SEQ ID NO: 80) [ka] >27 Mouse heavy chain variable region (SEQ ID NO:81) [ka] >27 Mouse light chain variable region (SEQ ID NO:82) [ka] >31 Mouse heavy chain variable region (SEQ ID NO:83) [ka] >31 Mouse light chain variable region (SEQ ID NO:84) [ka] >41 Mouse heavy chain variable region (SEQ ID NO: 85) [ka] >41 Mouse light chain variable region (SEQ ID NO: 86) [ka] >43 Mouse heavy chain variable region (SEQ ID NO:87) [ka] >43 Mouse light chain variable region (SEQ ID NO: 88) [ka] >46 Mouse heavy chain variable region (SEQ ID NO:89) [ka] >46 Mouse light chain variable region (SEQ ID NO: 90) [ka] >50 Mouse heavy chain variable region (SEQ ID NO:91) [ka] >50 Mouse Light Chain Variable Region (SEQ ID NO:92) [ka] >71 Mouse heavy chain variable region (SEQ ID NO: 93) [ka] >71 Mouse light chain variable region (SEQ ID NO: 94) [ka] >73 Mouse heavy chain variable region (SEQ ID NO:95) [ka] >73 Mouse light chain variable region (SEQ ID NO:96) [ka]

[0193] Note: The underlined regions are the CDR regions obtained according to the Kabat numbering convention.

[0194] The variable region sequence of a mouse anti-PSMA antibody was combined with the constant regions shown in SEQ ID NO: 97 and SEQ ID NO: 98 to obtain a chimeric antibody. Exemplarily, Chi13 is shown to contain a chimeric antibody of 13 mouse heavy chain variable regions, light chain variable regions, and the above constant regions. >hIgG1:CH1-Fc (SEQ ID NO:97) [ka] >CL (SEQ ID NO: 98) [ka]

[0195] Example 3. Humanized design of anti-human PSMA monoclonal antibody The humanization of mouse monoclonal antibodies was carried out according to the methods disclosed in many documents in this field. Briefly, based on the typical structure of the VH / VL CDR of the obtained mouse antibody, the homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) were searched for in the human germline database, the CDR region of the mouse antibody was grafted onto a human template, some of the residues of VL and VH were mutated, and the constant region of the mouse antibody was replaced with a human constant region to obtain the final humanized molecule.

[0196] 3-1. Humanization of Antibody 2 For the humanized antibody of mouse antibody 2, FR1, FR2, FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV6-21*02 and FR4 of IGKJ2*01 were selected as templates for the light chain framework region. Optionally, the amino acid residue at position 49 in the light chain variable region of the humanized antibody was substituted, specifically, the above mutation is K49Y (K at position 49 was mutated to Y according to the Kabat numbering system). The sequence of the antibody variable region is as follows: >hu2H1 (SEQ ID NO: 99) [ka] >hu2L1 (SEQ ID NO: 100) [ka]

[0197] 3-2. Humanization of Antibody 13 For the humanized antibody of mouse antibody 13, FR1, FR2, FR3 of IGHV2-26*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-39*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 1, 30, 44, 49, 73 and / or 89 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 41, 42, 43, 44 and / or 71 in the light chain variable region of the humanized antibody were substituted.

[0198] [Table 15-1] The sequences of the antibody variable regions are as follows: >hu13H1 (SEQ ID NO: 101) [ka] >hu13H2 (SEQ ID NO: 102) [ka] >hu13H3 (SEQ ID NO: 103) [ka] >hu13L1 (SEQ ID NO: 104) [ka] >hu13L2 (SEQ ID NO: 105) [ka] >hu13L3 (SEQ ID NO: 106) [ka]

[0199] 3-3. Humanization of Antibody 15 For the humanized antibody of mouse antibody 15, FR1, FR2, FR3 of IGHV1-3*01 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-27*01 and FR4 of IGKJ2*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 1, 2, 28, 44, 48, 67, 69, 71, 73 and / or 76 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 43, 60 and / or 100 in the light chain variable region of the humanized antibody were substituted.

[0200] [Table 15-2] The sequences of the antibody variable regions are as follows: >hu15H1 (SEQ ID NO: 107) [ka] >hu15H2 (SEQ ID NO: 108) [ka] >hu15H3 (SEQ ID NO: 109) [ka] >hu15H4 (SEQ ID NO: 110) [ka] >hu15H5 (SEQ ID NO: 111) [ka] >hu15H6 (SEQ ID NO: 112) [ka] >hu15L1 (SEQ ID NO: 113) [ka] >hu15L2 (SEQ ID NO: 114) [ka] >hu15L3 (SEQ ID NO: 115) [ka]

[0201] 3-4. Humanization of Antibody 19 For the humanized antibody of mouse antibody 19, FR1, FR2, FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-39*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, the amino acid residue at position 3 in the heavy chain variable region of the humanized antibody was substituted, and / or the amino acid residue at position 71 in the light chain variable region of the humanized antibody was substituted.

[0202] [Table 15-3] The sequences of the antibody variable regions are as follows: >hu19H1 (SEQ ID NO: 116) [ka] >hu19H2 (SEQ ID NO: 117) [ka] >hu19L1 (SEQ ID NO: 118) [ka] >hu19L2 (SEQ ID NO: 119) [ka]

[0203] 3-5. Humanization of Antibody 27 For the humanized antibody of mouse antibody 27, FR1, FR2, FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV6-21*02 and FR4 of IGKJ2*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 1, 22, 46, 47, 49, and / or 71 in the light chain variable region of the humanized antibody were substituted.

[0204] [Table 15-4] The sequences of the antibody variable regions are as follows: >hu27H1 (SEQ ID NO: 120) [ka] >hu27L1 (SEQ ID NO: 121) [ka] >hu27L2 (SEQ ID NO: 122) [ka] >hu27L3 (SEQ ID NO: 123) [ka] >hu27L4 (SEQ ID NO: 124) [ka] >hu27L5 (SEQ ID NO: 125) [ka] >hu27L6 (SEQ ID NO: 126) [ka]

[0205] 3-6. Humanization of Antibody 31 For the humanized antibody of mouse antibody 31, FR1, FR2, FR3 of IGHV3-7*01 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-39*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, the amino acid residue at position 94 in the heavy chain variable region of the humanized antibody was substituted, and / or the amino acid residues at positions 43, 45, 48, and / or 70 in the light chain variable region of the humanized antibody were substituted.

[0206] [Table 15-5] The sequences of the antibody variable regions are as follows: >hu31H1 (SEQ ID NO: 127) [ka] >hu31L1 (SEQ ID NO: 128) [ka] >hu31L2 (SEQ ID NO: 129) [ka] >hu31L3 (SEQ ID NO: 130) [ka]

[0207] 3-7. Humanization of Antibody 41 For the humanized antibody of mouse antibody 41, FR1, FR2, FR3 of IGHV1-18*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-39*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 1, 69, 71 and / or 73 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 36, 42, 44, 46, 66, 69 and / or 71 in the light chain variable region of the humanized antibody were substituted.

[0208] [Table 15-6] The sequences of the antibody variable regions are as follows: >hu41H1 (SEQ ID NO: 131) [ka] >hu41H2 (SEQ ID NO: 132) [ka] >hu41L1 (SEQ ID NO: 133) [ka] >hu41L2 (SEQ ID NO: 134) [ka] >hu41L3 (SEQ ID NO: 135) [ka]

[0209] 3-8. Humanization of Antibody 43 For the humanized antibody of mouse antibody 43, FR1, FR2, FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-12*01 or IGKV1-27*01 and FR4 of IGKJ2*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 4, 37, 72, 73, 78, 93 and / or 94 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residue at position 43 in the light chain variable region of the humanized antibody was substituted.

[0210] [Table 15-7] The sequences of the antibody variable regions are as follows: >hu43H1 (SEQ ID NO: 136) [ka] >hu43H2 (SEQ ID NO: 137) [ka] >hu43H3 (SEQ ID NO: 138) [ka] >hu43L1 (SEQ ID NO: 139) [ka] >hu43L2 (SEQ ID NO: 140) [ka]

[0211] 3-9. Humanization of Antibody 46 For the humanized antibody of mouse antibody 46, FR1, FR2, FR3 of IGHV3-21*01 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-39*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 45, 46, 49 and / or 93 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 60, 85 and / or 87 in the light chain variable region of the humanized antibody were substituted.

[0212] [Table 15-8] >hu46H1 (SEQ ID NO: 141) [ka] >hu46H2 (SEQ ID NO: 142) [ka] >hu46H3 (SEQ ID NO: 143) [ka] >hu46H4 (SEQ ID NO: 144) [ka] >hu46L1 (SEQ ID NO: 145) [ka] >hu46L2 (SEQ ID NO: 146) [ka] >hu46L3 (SEQ ID NO: 147) [ka]

[0213] 3-10. Humanization of Antibody 50 For the humanized antibody of mouse antibody 50, FR1, FR2, FR3 of IGHV1-18*01 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV2-28*01 and FR4 of IGKJ2*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 1, 27, 69, 71, 73 and / or 85 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 2, 3 and / or 28 in the light chain variable region of the humanized antibody were substituted. [Table 15-9] >hu50H1 (SEQ ID NO: 148) [ka] >hu50H2 (SEQ ID NO: 149) [ka] >hu50L1 (SEQ ID NO: 150) [ka] >hu50L2 (SEQ ID NO: 151) [ka] >hu50L3 (SEQ ID NO: 152) [ka] >hu50L3 LCDR1 RSSQSILHSYGNTYLE (sequence number 159).

[0214] 3-11. Humanization of Antibody 71 For the humanized antibody of mouse antibody 71, FR1, FR2, FR3 of IGHV3-30*09 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV2-40*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 1, 24, 37, 44, 72, 73, 76 and / or 93 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 2 and / or 4 in the light chain variable region of the humanized antibody were substituted.

[0215] [Table 15-10] >hu71H1 (SEQ ID NO: 153) [ka] >hu71H2 (SEQ ID NO: 154) [ka] >hu71H3 (SEQ ID NO: 155) [ka] >hu71H4 (SEQ ID NO: 156) [ka] >hu71L1 (SEQ ID NO: 157) [ka] >hu71L2 (SEQ ID NO: 158) [ka]

[0216] A fully humanized antibody was obtained by combining the heavy chain variable region and light chain variable region of each group described above with the constant regions shown in SEQ ID NO: 97 and SEQ ID NO: 98. hu2H1L1 represents a humanized antibody constructed by combining the humanized heavy chain variable region H1 (hu2H1) of antibody 2 with the constant region SEQ ID NO: 97 and the humanized light chain variable region L1 (hu2L1) with the constant region SEQ ID NO: 98, and the others are similarly inferred.

[0217] 3-12. Humanization of Antibody 73 For the humanized antibody of mouse antibody 73, FR1, FR2, FR3 of IGHV1-69*02 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region, and FR1, FR2, FR3 of IGKV1-27*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, amino acid residues at positions 27, 28, 39, 43, 69 and / or 93 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acid residues at positions 36, 41, 42, 43, 44, 45 and / or 71 in the light chain variable region of the humanized antibody were substituted.

[0218] [Table 15-11] >hu73H1 (SEQ ID NO: 277) [ka] >hu73H2 (SEQ ID NO: 278) [ka] >hu73H3 (SEQ ID NO: 279) [ka] >hu73L1 (SEQ ID NO: 280) [ka] >hu73L2 (SEQ ID NO: 281) [ka] >hu73L3 (SEQ ID NO: 282) [ka] >hu73L4 (SEQ ID NO: 283) [ka]

[0219] Example 4. Preparation of anti-PSMA-CD3 bispecific antibodies The CD3 binding molecules of the present disclosure may be derived from any suitable antibody. A specific embodiment of the present disclosure employs S107E.

[0220] [Table 16] The variable region sequence of S107E is as follows: >S107E-VH (SEQ ID NO: 166) [ka] >S107E-VL (SEQ ID NO: 167) [ka]

[0221] The present disclosure includes the following molecular structures:

[0222] Format 1 is a molecule with an asymmetric structure and contains: Chain 1: VH (anti-PSMA)-IgG1 (CH1)-IgG1Fc (Knob, L234A / L235A / S354C / T366W), Chain 2: VL(anti-PSMA)-CL, Chain 3 (Ob-hole): VH(S107E)-Linker a-Obscurin-IgG1Fc (Hole, L234A / L235A / Y349C / T366S / L368A / Y407V), and Strand 4 (VL-Titin): VL(S107E)-linker a-Titin.

[0223] A schematic diagram of this is shown in FIG. 1A (Ob stands for Obscurin, and so on below).

[0224] >IgG1(CH1) (SEQ ID NO: 168) [ka] >CL (SEQ ID NO: 98) >IgG1Fc (Knob, L234A / L235A / S354C / T366W) (SEQ ID NO: 169) [ka] > IgG1Fc (Hole, L234A / L235A / Y349C / T366S / L368A / Y407V) (SEQ ID NO: 170) [ka] >Ob-hole (sequence number 171) [ka] >VL-Titin: VL(S107E)-linker a-Titin (SEQ ID NO: 172) [ka] >Linker a (SEQ ID NO: 173) GGGGS Note: The single underlined regions are the CDR regions of the CD3 binding domain obtained according to the Kabat numbering convention, the double underlined regions are the Titin or Obscurin sequences, and the italics are the constant regions.

[0225] Format 2 is a molecule with an asymmetric structure and includes: Chain 1: VH (anti-PSMA)-IgG1 (CH1)-IgG1Fc (Knob, L234A / L235A / S354C / T366W), Chain 2 (two): VL(anti-PSMA)-CL, Chain 3: VH(anti-PSMA)-IgG1(CH1)-VH(S107E)-linker a-Titin-IgG1Fc(Hole, L234A / L235A / Y349C / T366S / L368A / Y407V), Strand 4 (VL-Ob): VL(S107E)-linker a-Ob, the schematic of which is shown in FIG. 1B.

[0226] >VL-Ob: VL(S107E)-linker a-Ob (SEQ ID NO: 174) [ka]

[0227] Format 3 is a molecule with a symmetric structure and includes: Chain 1 (2): VH(anti-PSMA)-IgG1(CH1)-VH(S107E)-Linker a-Titin-IgG1(AA)Fc; Chain 2 (two): VL(anti-PSMA)-CL, and Strand 4 (2 strands): VL-Ob: VL(S107E)-linker a-Ob, the schematic of which is shown in FIG. 1C.

[0228] >IgG1(AA)Fc (SEQ ID NO: 175) [ka]

[0229] Format 4 is a symmetric molecule that contains two identical heavy chains (chain 1) and two identical light chains (chain 2). The heavy chain is VH(anti-PSMA)-IgG1(CH1)-VH(S107E)-linker b-VL(S107E)-linker c-IgG1(AA)Fc, and the light chain is VL(anti-PSMA)-CL, the schematic of which is shown in FIG. 1D.

[0230] >Linker b (SEQ ID NO: 176) [ka] >Linker c GGG.

[0231] Format5 is a molecule with an asymmetric structure and contains: Chain 1: VH(anti-PSMA)-linker b-VL(anti-PSMA)-linker d-VH(S107E)-linker b-VL(S107E)-linker c-IgG1Fc (Hole, L234A / L235A / Y349C / T366S / L368A / Y407V), and Chain 3 (Fc (Knob)): IgG1 Fc (Knob, L234A / L235A / S354C / T366W), a schematic diagram of which is shown in Figure 1E.

[0232] >IgG1Fc (Hole, Y349C / T366S / L368A / Y407V) (SEQ ID NO: 170) >IgG1Fc (Knob, L234A / L235A / S354C / T366W) (SEQ ID NO: 169) >Linker d (SEQ ID NO: 178) [ka] >Linker c:GGG.

[0233] Based on the amino acid sequences of 2, 13, 15, 19, 27, 31, 43, 46, 73 and the humanized antibody, the following bispecific antibodies were constructed.

[0234] [Table 17-1] [Table 17-2]

[0235] Among them, 2-F2 indicates that the molecule uses the variable region of Antibody 2 and its humanized antibody as the PSMA binding domain and Format 2 as the molecular structure. hu2H1-F2-1 indicates that the chain uses hu2H1 as the heavy chain variable region and Format 2 as the molecular structure, and the others are inferred in this manner. The specific amino acid sequences are as follows: >hu2H1-F2-1 (SEQ ID NO: 180) [ka] >hu2L1-CL (SEQ ID NO: 181) [ka] >hu2H1-F2-3 (SEQ ID NO: 182) [ka] >hu2H1-F3-1 (SEQ ID NO: 183) [ka] >hu2H1-F4-1 (SEQ ID NO: 184) [ka] >hu13H1-F3-1 (SEQ ID NO: 185) [ka] >hu13L1-CL (SEQ ID NO: 186) [ka] >hu15H4-F2-1 (SEQ ID NO: 187) [ka] >hu15L2-CL (SEQ ID NO: 188) [ka] >hu15H4-F2-3 (SEQ ID NO: 189) [ka] >hu15H4-F4-1 (SEQ ID NO: 190) [ka] >hu15H4L2-F5-1 (SEQ ID NO: 191) [ka] >hu15H6L3-F5-1 (SEQ ID NO: 192) [ka] >hu19H2-F1-1, which may also be hu19H2-F2-1 (SEQ ID NO: 193) [ka] >hu19L2-CL (SEQ ID NO: 194) [ka] >hu19H2-F2-3 (SEQ ID NO: 195) [ka] >hu19H2-F3-1 (SEQ ID NO: 196) [ka] >hu19H2-F4-1 (SEQ ID NO: 197) [ka] >hu19H2L2-F5-1 (SEQ ID NO: 198) [ka] >hu27H1-F4-1 (SEQ ID NO: 199) [ka] >hu27L6-CL (SEQ ID NO: 200) [ka] >hu31H1-F1-1 (SEQ ID NO: 201) [ka] >hu31L1-CL (SEQ ID NO: 202) [ka] >hu43H3-F3-1 (SEQ ID NO: 203) [ka] >hu43L2-CL (SEQ ID NO: 204) [ka] >hu43H3-F4-1 (SEQ ID NO: 205) [ka] >hu46H2-F1-1, which may also be hu46H2-F2-1 (SEQ ID NO: 206) [ka] >hu46L2-CL (SEQ ID NO: 207) [ka] >hu46H2-F2-3 (SEQ ID NO: 208) [ka] >hu46H2-F4-1 (SEQ ID NO: 209) [ka] >73VH-F1-1 (SEQ ID NO: 284) [ka] >73VL-CL (SEQ ID NO: 285) [ka] >73VH-F3-1 (SEQ ID NO: 286) [ka]

[0236] The positive control molecule used in the present disclosure is Aca-Mab, the amino acid sequence of which is set forth in SEQ ID NO: 382 in WO2017134158A1. Test Example

[0237] The above-mentioned other patents used the above-mentioned methods to obtain Cho-human PSMA and Cho-monkey PSMA overexpressing cell lines, which were obtained by purchasing hamster ovary cells Cho-K1, human prostate cancer cells Lncap (ATCC), human prostate cancer cells 22RV1 (ATCC) and human prostate cancer cells PC-3 (Chinese Academy of Sciences Cell Bank).

[0238] Test Example 1. Binding activity of anti-PSMA antibodies to PSMA In order to test the ability of the PSMA-targeting humanized antibody according to the present disclosure to bind to the PSMA antigen, this test example detected the binding of the PSMA-targeting humanized antibody to human PSMA antigen (purchased from R&D SYSTEM, 4234-ZN-010) by ELISA. Specifically, a 96-well plate was coated with PSMA antigen at 1 μg / well and left overnight at 4° C. After discarding the supernatant, the plate was blocked with 5% skim milk for 2 h. Each humanized antibody was prepared at different concentrations with 1% BSA, with an initial concentration of 10 μg / mL, and diluted 4-fold to 12 concentrations. After washing the blocked 96-well plate twice, the prepared PSMA antigen was added, incubated at 4° C. for 1 h, washed four times, and then incubated with Strepidation-HRP secondary antibody (1:10000) at 4° C. for 1 h, developed with TMB, and then stopped with 0.1 M sulfuric acid. The ELISA results are shown in the table below.

[0239] [Table 18]

[0240] Note: Top indicates maximum absorbance.

[0241] As a result, the PSMA humanized antibodies and chimeric antibodies screened in the present disclosure have been shown to have good binding activity with the PSMA antigen.

[0242] Test Example 2. Binding activity of antibodies to PSMA In this test example, the binding activity of the antibody to the stable transformed cell line Cho-hPSMA overexpressing human PSMA was detected by flow cytometry. The full-length gene encoding human PSMA was cloned into the mammalian cell expression vector pCDH, and three plasmids, pVSV-G, pCMV-dR8.91 and pCDH-human PSMA, were co-transfected into HEK293T cells (ATCC® CRL-11268) to package the virus. After 48 hours of transfection, the virus was collected and infected into CHO-K1 cells. After pressure screening for 2 weeks, the cells were subcloned and detected by FACS to obtain the cell line Cho-hPSMA with high expression of PSMA.

[0243] Experiment 1: Cho-hPSMA was cultured in F12 medium with 10% FBS. The medium in which the cells were inoculated was placed in an incubator at 37℃ and 5% CO2 for 2 days, and the cells were added to the cell plate at 1×105 cells / well, centrifuged, and washed. The antibodies were serially diluted, and the antibody solution was added to the cell plate at 100μL / well, incubated at 4℃ for 1h, and then washed. AF488-Goat Anti-human IgG Fc fluorescent secondary antibody dilution solution (1:400) was added to the cell plate at 100μL / well, incubated at 4℃ for 1h, and then washed. PBS was added to the cell plate at 100μL / well, and the plate was read. The cell binding activity of the antibodies is shown in the table below.

[0244] [Table 19-1]

[0245] Experiment 2: Cells were cultured in the same manner as in Experiment 1, and then inoculated into cell plates. Furthermore, the antibodies were serially diluted, and the antibody solution was added to the cell plates at 100 μL / well, incubated at 4°C for 1 h, and then washed. 100 μL / well of HRP-Goat Anti-human IgG(H+L) secondary antibody dilution solution (1:8000) was added to the cell plates, incubated at 4°C for 1 h, and then washed by centrifugation. After color development with TMB, the reaction was stopped with 0.1 M sulfuric acid, and the OD450 value was detected. The cell binding activity of the antibodies is shown in the table below.

[0246] [Table 19-2]

[0247] Experiment 3: The binding activity of the bispecific antibody was detected by the method of Experiment 1. The binding activity to cells under each antibody concentration condition is shown in the table below.

[0248] [Table 19-3]

[0249] Experiment 4: The binding activity of the bispecific antibody was detected by the method of Experiment 2. The binding activity to cells under each antibody concentration condition is shown in the table below.

[0250] [Table 19-4]

[0251] Experiment 5: The antibody was affinity captured by Protein A biosensor chip, and human PSMA was applied to the chip surface. The Biacore T200 instrument detected the reaction signal in real time at 25℃ to obtain the binding-dissociation curve. After the dissociation of each experimental cycle was completed, the biosensor chip was washed and regenerated with Glycine 1.5. The data fitting model was 1:1 model. Here, human PSMA antigen was purchased from Acro (product number PSA-H52H3, Lys44-Ala750), and hCD3D&CD3E complex antigen was purchased from Sino Biological (product number CT038-H2508H). The binding-dissociation status of each antibody is shown in the table below.

[0252] [Table 19-5]

[0253] As a result, the anti-PSMA antibodies and bispecific antibodies disclosed herein have been shown to have good binding activity to PSMA, and are stronger than Aca-mab.

[0254] Test Example 3. In vitro cytotoxicity of bispecific antibodies according to the present disclosure In this test example, the tumor cell killing activity of the bispecific antibody according to the present disclosure was studied using the bispecific antibody according to the present disclosure as a T cell bridging molecule. The target-specific cytotoxic activity of the bispecific antibody according to the present disclosure was detected using the PSMA-expressing cell line Lncap (human prostate cancer cell, metastatic type) as the target cell. Fresh PBMC (purchased from Xuanfeng Biological Co., Ltd.) was centrifuged at 300 g for 10 min, the supernatant was discarded, and the cell number was resuspended in phenol red-free 1640 + 4% FBS, centrifuged again, resuspended, and counted to obtain a cell number of 1.5 x 10 6 The target cells were collected, centrifuged at 1000 rpm for 3 min, resuspended, and counted to obtain a cell number of 3 × 10 5The antibody was adjusted to 1000 / mL and 25μL was added to each well, making the E:T ratio 10:1. The antibody was diluted with phenol red-free complete medium to an initial concentration of 400nM (4x final concentration), diluted 8-fold with 10 gradients, and 25μL was added to each well. The cells were then cultured for 48h in an incubator at 37℃ and 5%CO2. Before detection, 10μL of medium was aspirated from the two wells containing only the target cells, and 10μL of Lysis Solution (10x) was added. After 45min of lysis, the culture plate was removed and centrifuged at 1000rpm for 3min. 50μL of the supernatant was aspirated and placed in a new 96-well plate (#3590), and the dissolved CytoTox 96 was added at a ratio of 1:1. (登録商標) 50 μL of the reagent was added and incubated at room temperature for 0.5 hours, after which 50 μL of a stop solution was added and the absorbance (490 nm) was detected using FlexStation 3 (Molecular Devices).

[0255] [Table 20]

[0256] As a result, the bispecific antibody of the present disclosure has a relatively strong tumor-killing effect against Lncap cells expressing PSMA, and 50 It is shown that the values ​​are much lower than those of the control antibody Aca-Mab.

[0257] Test Example 4. Cytokine release levels of bispecific antibodies according to the present disclosure CD3 T cell bridge molecules induce a cytokine storm. Therefore, when developing CD3 T cell bridge molecules, it is necessary to maintain low levels of cytokines, especially IL6, a factor that is unrelated to efficacy but causes side effects.

[0258] This test example investigated the release levels of cytokines IFNγ and IL6 when the bispecific antibody of the present disclosure killed the PSMA-expressing cell line Lncap. Fresh PBMCs (purchased from Xuanfeng Biological Co., Ltd.) were centrifuged at 300 g for 10 min, the supernatant was discarded, and resuspended in phenol red-free 1640 + 4% FBS, centrifuged again, resuspended, and counted to obtain a cell number of 1.5 × 10 6 The target cells were collected, centrifuged at 1000 rpm for 3 min, resuspended, and counted to obtain a cell number of 3 × 10 5 The antibody was diluted with phenol red-free complete medium to an initial concentration of 400 nM (4x final concentration), diluted 8-fold in a 10-fold gradient, and added to each well at 25 μL. The cells were then cultured in a 37°C, 5% CO2 incubator for 48 h.

[0259] IL6 was detected by ELISA. The above cell samples were centrifuged at 1000 rpm for 3 min, 50 μL of cell supernatant was collected and diluted 6-fold with a general dilution solution for specimens, and the IL6 standard was also diluted 6-fold. The diluted samples or standards of different concentrations were added to the detection plate (100 μL / well), the reaction wells were sealed with plate sealing adhesive paper, and incubated at 37°C for 90 min. The plate was then washed 5 times, and the biotinylated antibody working solution was added (100 μL / well). The reaction wells were sealed with new plate sealing adhesive paper. The wells were sealed and incubated at 37°C for 60 min, then the plate was washed five times, enzyme complex working solution (100 μL / well) was added, the reaction wells were sealed with new plate sealing adhesive paper, incubated at 37°C for 30 min, then the plate was washed five times, chromogenic substrate (TMB) was added at 100 μL / well, incubated at 37°C for 8 min in the dark, reaction stop solution was added at 100 μL / well, and mixed uniformly. The OD450 value was immediately measured (within 3 min).

[0260] HTRF method was adopted for the detection of IFNγ. The above cell samples were centrifuged at 1000 rpm for 3 min, 50 μL of cell supernatant was collected, the detection reagent kit was equilibrated to room temperature, and the two detection antibodies in the reagent kit were diluted (20 times diluted) with detection buffer. 16 μL of the 20 times diluted sample and IFNγ standard were taken and placed in a 384-well plate, 4 μL of the diluted corresponding detection antibody was added, the sealing material was attached, the mixture was evenly shaken, centrifuged at 1000 rpm for 1 min, incubated overnight at room temperature, and then centrifuged at 1000 rpm for 1 min, the sealing material was removed, and the absorbance values ​​at 665 nm and 620 nm were read by a PHERAstar multifunction plate reader, and the data was processed and analyzed by Graphpad Prism 5.

[0261] [Table 21-1] [Table 21-2]

[0262] As a result, it was shown that the bispecific antibody according to the present disclosure released lower levels of IL6 and IFNγ than Aca-Mab, suggesting that the bispecific antibody according to the present disclosure has better safety. Biological evaluation of in vivo activity

[0263] Test Example 5. Efficacy of bispecific antibodies according to the present disclosure in a 22RV1 subcutaneously transplanted tumor model The present disclosure evaluated the antitumor activity of PSMA-CD3 biantibody using a human prostate cancer 22RV1 cell human PBMC NCG mouse xenograft tumor model.

[0264] NCG mice were male, 4-5 weeks old, and purchased from Jiangsu Jisui Yaokang Biotechnology Co., Ltd.

[0265] Human prostate cancer 22RV1 cells were purchased from ATCC.

[0266] 22RV1 cells were added to RPMI-1640 medium containing 10% FBS and cultured in a 5% CO2, 37°C saturated humidity incubator. 22RV1 cells in the logarithmic growth phase were collected and resuspended in RPMI-1640 medium containing 50% Matrigel to a cell concentration of 2 × 10 7 Under aseptic conditions, 0.1 mL of the cell suspension was inoculated subcutaneously into the right dorsum of each mouse, giving an inoculation concentration of 2 × 10 6 One day after tumor cell inoculation, 0.1 mL of the human PBMC suspension was intraperitoneally injected into the mouse under aseptic conditions, with an injection concentration of 8 × 10 6 The average tumor volume was 100 mm 3 When the tumor volume reached 100%, the animals were randomly divided into groups according to tumor volume so that the tumor volume difference between each group was less than 10% of the mean value. The day of grouping was marked as Day 0, and administration began according to the weight of the animals. Administration was started immediately after grouping, intraperitoneally twice a week, and six consecutive doses were administered (IP, BIW x 3).

[0267] Tumor inhibition rate (%) = (1-(Ti-T0) / (Ci-C0)) × 100%, where Ti and Ci are the tumor volumes of the treatment group and control group at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0268] [Table 22]

[0269] The experimental results showed that the bispecific antibodies 31-F1 and 43-F4 according to the present disclosure exhibited good efficacy at the doses tested, and no drug-related animal deaths or other obvious drug-related toxicities or side effects were observed during the experiment.

[0270] Test Example 6. Efficacy of bispecific antibodies according to the present disclosure in a C4-2 subcutaneously transplanted tumor model The present disclosure evaluated the anti-tumor activity of PSMA-CD3 bispecific antibodies using a human prostate cancer C4-2 cell human PBMC NCG mouse xenograft tumor model.

[0271] NCG mice were male, 7-8 weeks old, and purchased from Jiangsu Jisui Yaokang Biotechnology Co., Ltd.

[0272] Human prostate cancer C4-2 cells were purchased from ATCC.

[0273] C4-2 cells were cultured in DMEM / Ham's F12K (4:1) medium containing 10% fetal bovine serum in a humidity-saturated incubator at 37°C with 5% CO. C4-2 cells in logarithmic growth phase were harvested and resuspended in RPMI-1640 medium containing 50% Matrigel to a cell concentration of 2 × 10 7 Under aseptic conditions, 0.1 mL of the cell suspension was inoculated subcutaneously into the right dorsum of each mouse, giving an inoculation concentration of 2 × 10 6 On the third day after tumor cell inoculation, 0.1 mL of the human PBMC suspension was intraperitoneally injected into the mouse under aseptic conditions, with the injection concentration being 1 × 10 7 The tumor volume was 0.1 mL / mouse. On the 12th day after tumor cell inoculation (mean tumor volume was 51.60 mm 3 When tumor volume reached 100%, animals were randomly grouped according to tumor volume and started to be dosed according to animal weight, so that the tumor volume difference between each group was less than 10% of the mean value. Dosing was started immediately after grouping, intraperitoneally twice a week, and four consecutive doses were administered (IP, BIW x 2).

[0274] [Table 23]

[0275] Experimental results showed that the bispecific antibodies 15-F5-1 and 15-F5-2 had stronger tumor inhibitory effects than Aca-Mab in the C4-2 model, and even low doses of the bispecific antibodies could achieve significantly stronger tumor inhibitory effects than high doses of Aca-Mab.

Claims

1. An antigen-binding molecule that specifically binds to PSMA and CD3, at least one antigen binding module that specifically binds to PSMA; and at least one antigen-binding module that specifically binds to CD3; The antigen binding module that specifically binds to PSMA comprises a heavy chain variable region PSMA-VH and a light chain variable region PSMA-VL; The antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein: (i) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 77, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 78, respectively; or (ii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 73, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 74, respectively; or (iii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 75, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 76, respectively; or (iv) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 79, respectively; and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 80, respectively; or (v) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 81, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 82, respectively; or (vi) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 83, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 84, respectively; or (vii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 85, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 86, respectively; or (viii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 87, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 88, respectively; or (ix) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 89, respectively; and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 90, respectively; or (x) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 91, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 92 or 152, respectively; or (xi) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 93, respectively; and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 94, respectively; or (xii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 95, respectively; and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 96, respectively; Preferably, (i) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (ii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:4, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:6; or (iii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; or (iv) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:22, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:23, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:24; or (v) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:28, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:29, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:30; or (vi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 33; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 36; or (vii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 39; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (viii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 45; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:46, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:47, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:48; or (ix) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 49, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 51; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:52, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:53, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:54; or (x) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 57; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58 or 159, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 59, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 60; or (xi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 61, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 63; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:64, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:65, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:66; or (xii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 67, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 69; and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 72; More preferably, The antigen-binding molecule was dissolved at 25°C at a concentration of 1 × 10 －８ binds to human PSMA with a KD of less than M, as measured by surface plasmon resonance. Antigen-binding molecules.

2. (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, 110, 77, 107, 108, 109, or 111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115, 114, 78, or 113; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73 or 99, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74 or 100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, 101, 102, or 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76, 104, 105, or 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 79, 116, or 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 80, 118, or 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81 or 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82, 121, 122, 123, 124, 125, or 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127 or 83, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128, 84, 129, or 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, 131, or 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86, 133, 134, or 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, 136, 137, or 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88, 139, or 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, 141, 142, 143, or 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90, 145, 146, or 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, 148, or 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92, 150, 151, or 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, 153, 154, 155, or 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94, 157, or 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, 277, 278, or 279, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96, 280, 281, 282, or 283; Preferably, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:77, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:78; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:99, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 79, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 80; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 121; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 122; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 123; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 124; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 125; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 83, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 84; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 129; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 136, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 137, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96; More preferably, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128; The antigen-binding molecule of claim 1.

3. The antigen binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein CD3-HCDR1, CD3-HCDR2, and CD3-HCDR3 in the CD3-VH comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2, and CD3-HCDR3 in SEQ ID NO: 166, respectively, and CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 in the CD3-VL comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 in SEQ ID NO: 167, respectively; Preferably, the CD3-VH has a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the CD3-VL has a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165; More preferably, the CD3-VH comprises the amino acid sequence of SEQ ID NO: 166, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 167; The antigen-binding molecule of claim 1.

4. The antigen-binding molecule comprises an Fc region, and the Fc region is preferably an IgG Fc region, and IgG １ More preferably, an Fc region, Preferably, the Fc region comprises one or more amino acid substitutions capable of reducing binding of the Fc region to an Fcγ receptor; More preferably, the Fc region is human IgG １ an Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index; The antigen-binding molecule of claim 1.

5. the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 and the Fc2 each independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; Preferably, the Fc1 has a knob structure with knob-in-hole technology, and the Fc2 has a hole structure with knob-in-hole technology; or The Fc2 has a knob structure based on knob-in-hole technology, and the Fc1 has a hole structure based on knob-in-hole technology; More preferably, 1) the Fc1 has an amino acid residue of W at position 366; and the Fc2 has an amino acid residue at position 366 that is S, an amino acid residue at position 368 that is A, and an amino acid residue at position 407 that is V, numbered according to the EU index; or 2) the Fc2 comprises an amino acid residue at position 366 that is W; and the Fc1 has an amino acid residue at position 366 that is S, an amino acid residue at position 368 that is A, and an amino acid residue at position 407 that is V, numbered according to the EU index; The antigen-binding molecule of claim 1.

6. The antigen-binding molecule one antigen binding module that specifically binds to PSMA; one antigen-binding module that specifically binds to CD3; the antigen-binding module that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 are scFvs; Preferably, The antigen-binding molecule one first chain having a structure shown in formula (a); and one second chain having the structure shown in formula (b), Formula (a) [PSMA-VH]-[Linker 1]-[PSMA-VL]-[Linker 2]-[CD3-VH]-[Linker 3]-[CD3-VL]-[Linker 4]-[Fc2], Formula (b) [Fc1], The structures represented by formula (a) and formula (b) are arranged from the N-terminus to the C-terminus, and the linker 1, linker 2, linker 3, and linker 4 are the same or different peptide linkers; More preferably, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 192 and one second chain comprising the amino acid sequence of SEQ ID NO: 169; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 191 and one second chain comprising the amino acid sequence of SEQ ID NO: 169; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 198 and one second chain comprising the amino acid sequence of SEQ ID NO:

169. The antigen-binding molecule of claim 5 .

7. the antigen binding module that specifically binds to PSMA or the antigen binding module that specifically binds to CD3 each independently comprises a titin chain and an obscurin chain; Preferably, The Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 210 to 228, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 229 to 269; More preferably, The Titin chain comprises the amino acid sequence of SEQ ID NO: 226, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO:

264. The antigen-binding molecule of claim 1.

8. the antigen-binding molecule comprises one antigen-binding module that specifically binds to PSMA and one antigen-binding module that specifically binds to CD3; the antigen binding module that specifically binds to PSMA is a Fab; the antigen-binding module that specifically binds to CD3 is a substituted Fab containing a titin chain and an obscurin chain; Preferably, The antigen-binding molecule comprises one first chain having the structure represented by formula (c), one second chain having the structure represented by formula (d), one third chain having the structure represented by formula (e), and one fourth chain having the structure represented by formula (f); Formula (c) [PSMA-VH]-[CH1]-[Fc1], Formula (d) [PSMA-VL]-[CL], Formula (e) [CD3-VH]-[linker 5]-[Obscurin chain]-[Fc2], Formula (f) [CD3-VL]-[linker 6]-[Titin chain], The structures represented by formulas (c), (d), (e), and (f) are arranged from the N-terminus to the C-terminus, and the linker 5 and the linker 6 are the same or different peptide linkers; More preferably, the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 201, one second chain comprising the amino acid sequence of SEQ ID NO: 202, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 172; or the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 193, one second chain comprising the amino acid sequence of SEQ ID NO: 194, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 172; or the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 206, one second chain comprising the amino acid sequence of SEQ ID NO: 207, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 172; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 284, one second chain comprising the amino acid sequence of SEQ ID NO: 285, one third chain comprising the amino acid sequence of SEQ ID NO: 171, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

172. The antigen-binding molecule of claim 7.

9. the antigen-binding molecule comprises two antigen-binding modules that specifically bind to PSMA and one antigen-binding module that specifically binds to CD3; the antigen-binding module that specifically binds to PSMA is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab that includes a titin chain and an obscurin chain; Preferably, The antigen-binding molecule comprises one first chain having a structure represented by formula (c), two second chains having a structure represented by formula (d), one third chain having a structure represented by formula (g), and one fourth chain having a structure represented by formula (h); Formula (c) [PSMA-VH]-[CH1]-[Fc1], Formula (d) [PSMA-VL]-[CL], Formula (g) [PSMA-VH]-[CH1]-[CD3-VH]-[linker 7]-[Titin chain]-[Fc2], Formula (h) [CD3-VL]-[linker 8]-[Obscurin chain], The structures represented by formulas (c), (d), (g), and (h) are arranged from the N-terminus to the C-terminus, and the linker 7 and the linker 8 are the same or different peptide linkers; More preferably, the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 180, two second chains comprising the amino acid sequence of SEQ ID NO: 181, one third chain comprising the amino acid sequence of SEQ ID NO: 182, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 174; or the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 187, two second chains comprising the amino acid sequence of SEQ ID NO: 188, one third chain comprising the amino acid sequence of SEQ ID NO: 189, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 174; or the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 193, two second chains comprising the amino acid sequence of SEQ ID NO: 194, one third chain comprising the amino acid sequence of SEQ ID NO: 195, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 206, two second chains comprising the amino acid sequence of SEQ ID NO: 207, one third chain comprising the amino acid sequence of SEQ ID NO: 208, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

174. The antigen-binding molecule of claim 7.

10. the antigen-binding molecule comprises two antigen-binding modules that specifically bind to PSMA and two antigen-binding modules that specifically bind to CD3, wherein the antigen-binding modules that specifically bind to PSMA are Fabs, and the antigen-binding module that specifically binds to CD3 is a substituted Fab comprising a titin chain and an obscurin chain; Preferably, The antigen-binding molecule comprises two first chains having a structure represented by formula (j), two second chains having a structure represented by formula (d), and two third chains having a structure represented by formula (h), Formula (j) [PSMA-VH]-[CH1]-[CD3-VH]-[linker 9]-[Titin chain]-[one subunit of Fc]; Formula (d) [PSMA-VL]-[CL], Formula (h) [CD3-VL]-[linker 8]-[Obscurin chain], The structures represented by formulas (j), (d), and (h) are arranged from the N-terminus to the C-terminus, and the linker 8 and the linker 9 are the same or different peptide linkers; More preferably, The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 183, two second chains comprising the amino acid sequence of SEQ ID NO: 181, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or the antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 185, two second chains comprising the amino acid sequence of SEQ ID NO: 186, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or the antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 196, two second chains comprising the amino acid sequence of SEQ ID NO: 194, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or the antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 203, two second chains comprising the amino acid sequence of SEQ ID NO: 204, and two third chains comprising the amino acid sequence of SEQ ID NO: 174; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 286, two second chains comprising the amino acid sequence of SEQ ID NO: 285, and two third chains comprising the amino acid sequence of SEQ ID NO:

174. The antigen-binding molecule of claim 7.

11. the antigen-binding molecule comprises two antigen-binding modules that specifically bind to PSMA and two antigen-binding modules that specifically bind to CD3, wherein the antigen-binding modules that specifically bind to PSMA are Fabs and the antigen-binding modules that specifically bind to CD3 are scFvs; Preferably, The antigen-binding molecule comprises two first chains having a structure represented by formula (k) and two second chains having a structure represented by formula (d), Formula (k) [PSMA-VH]-[CH1]-[CD3-VH]-[linker 10]-[CD3-VL]-[linker 11]-[one subunit of Fc]; Formula (d) [PSMA-VL]-[CL], The structures represented by formula (k) and formula (d) are arranged from the N-terminus to the C-terminus, and the linker 10 and the linker 11 are the same or different peptide linkers; More preferably, the antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 184 and two second chains comprising the amino acid sequence of SEQ ID NO: 181; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 190 and two second chains comprising the amino acid sequence of SEQ ID NO: 188; or the antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 197 and two second chains comprising the amino acid sequence of SEQ ID NO: 194; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 199 and two second chains comprising the amino acid sequence of SEQ ID NO: 200; or the antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 205 and two second chains comprising the amino acid sequence of SEQ ID NO: 204; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 209 and two second chains comprising the amino acid sequence of SEQ ID NO:

207. The antigen-binding molecule of claim 1.

12. 1. An isolated antibody capable of specifically binding to PSMA, said antibody comprising a heavy chain variable region PSMA-VH and a light chain variable region PSMA-VL, wherein: (i) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3, respectively, in SEQ ID NO: 77, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3, respectively, in SEQ ID NO: 78; or (ii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 73, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 74, respectively; or (iii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 75, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 76, respectively; or (iv) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3, respectively, in SEQ ID NO: 79, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3, respectively, in SEQ ID NO: 80; or (v) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 81, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 82, respectively; or (vi) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 83, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 84, respectively; or (vii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 85, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 86, respectively; or (viii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 87, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 88, respectively; or (ix) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 89, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 90, respectively; or (x) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 91, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 92 or 152, respectively; or (xi) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 93, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 94, respectively; or (xii) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in said PSMA-VH comprise the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 95, respectively, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in said PSMA-VL comprise the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 96, respectively; Preferably, (i) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 15, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18; or (ii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (iii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:7, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:8, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:9, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:10, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:11, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:12; or (iv) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (v) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:25, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:27, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:28, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:29, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:30; or (vi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 33, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 36; or (vii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 37, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 42; or (viii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 45, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 46, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 48; or (ix) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:49, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:50, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO:51, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:52, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:53, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:54; or (x) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 57, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 58 or 159, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 59, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 60; or (xi) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 61, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 66; or (xii) the PSMA-VH has a PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 67, a PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 69, and the PSMA-VL has a PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 70, a PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 72; More preferably, The antibody was diluted to 1 × 10 －８ binds to human PSMA with a KD of less than M, as measured by surface plasmon resonance. Isolated antibodies.

13. (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, 110, 77, 107, 108, 109, or 111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115, 114, 78, or 113; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73 or 99, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74 or 100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, 101, 102, or 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76, 104, 105, or 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 79, 116, or 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 80, 118, or 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81 or 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82, 121, 122, 123, 124, 125, or 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127 or 83, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128, 84, 129, or 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, 131, or 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86, 133, 134, or 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, 136, 137, or 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88, 139, or 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, 141, 142, 143, or 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90, 145, 146, or 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, 148, or 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92, 150, 151, or 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, 153, 154, 155, or 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94, 157, or 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, 277, 278, or 279, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96, 280, 281, 282, or 283; Preferably, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:77, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:78; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 113; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or (ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 73, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 74; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO:99, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:100; or (iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 75, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 76; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 104; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 105; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 106; or (iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 79, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 80; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 118; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 119; or (v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 81, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 82; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 121; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 122; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 123; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 124; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 125; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 126; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 83, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 84; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 129; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 130; or (vii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 85, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 86; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 133; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 134; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 135; or (viii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 87, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 88; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 136, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 137, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 139; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 140; or (ix) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 89, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 90; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 145; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 146; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 147; or (x) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 91, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 92; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 150; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 151; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 152; or (xi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 93, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 94; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 157; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 158; or (xii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 95, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 96; More preferably, (i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 115; or the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 114; or (vi) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 128; 13. The isolated antibody of claim 12.

14. the antibody is a bispecific antibody; Preferably, the bispecific antibody specifically binds to PSMA and CD3.

13. The isolated antibody of claim 12.

15. 1. A pharmaceutical composition comprising: A therapeutically effective amount of an antigen-binding molecule according to any one of claims 1 to 11 or an isolated antibody according to any one of claims 12 to 14; one or more pharmaceutically acceptable vectors, diluents, buffers or excipients; Preferably, the pharmaceutical composition further comprises at least one second therapeutic agent; More preferably, the second therapeutic agent is an antibody capable of specifically binding to CD28. Pharmaceutical compositions.

16. Encoding the antigen-binding molecule of any one of claims 1 to 11 or the isolated antibody of any one of claims 12 to 14. Isolated nucleic acids.

17. 17. The isolated nucleic acid of claim 16, host cell.

18. Use of an antigen-binding molecule according to any one of claims 1 to 11 or an isolated antibody according to any one of claims 12 to 14 in the manufacture of a medicament for treating a disease, comprising: the disease is a proliferative disease, a tumor, or an immune disease; Preferably, the disease is any one selected from prostate cancer, lung cancer, endometrial cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer. use.

19. 16. A pharmaceutical composition according to claim 15 for treating a disease, comprising: the disease is a proliferative disease, a tumor, or an immune disease; Preferably, the disease is any one selected from prostate cancer, lung cancer, endometrial cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer. Pharmaceutical compositions.