Antigen binding molecules specifically binding psma and cd3 and medical uses thereof

Abstract

Provided are antigen binding molecules that specifically bind PSMA and CD3 and medical uses thereof. The antigen binding molecules can be used to treat tumor-related diseases.

Description

[0001] This application claims priority to Chinese patent application 202210116684.6, filed on February 7, 2022. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, to antigen-binding molecules and their applications. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] PSMA belongs to Glutamate carboxypeptidase II (GCPII), composed of 750 amino acids: 19 intracellularly, 24 transmembranely, and 707 extracellularly. The extracellular portion of PSMA consists of three domains: protease-like, apical, and C-terminal. All three domains are involved in substrate binding; the protease-like and apical domains bind directly to the substrate, while the C-terminal causes PSMA to form a dimer and exert its function. In prostate cancer patients, PSMA expression levels in tumor tissues are 100-1000 times higher than in normal prostate tissues. PSMA is also expressed on the surface of endometrial cancer cells, in the angiogenesis of gastric cancer (66.4%) and colorectal cancer (84.6%), in the angiogenesis of non-small cell lung cancer, and on some tumor cells. Therefore, PSMA can not only serve as a target for prostate cancer development but may also be applicable to other tumor types.

[0005] CD3 is an allo- or heterodimeric antigen expressed on T cells. Functional CD3 is formed by the dimerization of two of four different chains: ε, ζ, δ, and γ. The CD3 dimer arrangement includes 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 for therapeutic purposes. However, administration of anti-CD3 antibodies may trigger T cell activation and associated cytokine release. Excessive cytokine release leads to severe cytokine release syndrome (CRS), which is a significant challenge in the clinical use of anti-CD3 antibodies.

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

[0007] This disclosure provides an antigen-binding molecule that specifically binds to PSMA and CD3, and an antibody that specifically binds to PSMA.

[0008] In one aspect, this disclosure provides an antigen-binding molecule comprising 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 implementations, the antigen-binding molecule as described above, wherein

[0010] (i) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 77, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 78, or

[0011] (ii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 73, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 74, or

[0012] (iii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 75, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 76, or

[0013] (iv) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 79, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 80, or

[0014] (v) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 81, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 82, or

[0015] (vi) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 83, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 84, or

[0016] (vii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 85, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 86, or

[0017] (viii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 87, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 88, or

[0018] (ix) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 89, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 90, or

[0019] (x) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 91, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 92 or 152, or

[0020] (xi) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 93, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 94, or

[0021] (xii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in PSMA-VH respectively contain 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 PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 96.

[0022] In some implementations, the PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering rules.

[0023] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0024] (i) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 13, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 15, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 16, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 17, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, or

[0025] (ii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 1, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 4, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 6, or

[0026] (iii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 7, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 8, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 9, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 10, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 11, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 12, or

[0027] (iv) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 19, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 20, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 21, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, or

[0028] (v) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 25, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 27, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 28, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 29, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 30, or

[0029] (vi) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 31, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 32, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 33, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 34, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 35, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 36, or

[0030] (vii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 37, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 38, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 39, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 40, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 41, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 42, or

[0031] (viii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 43, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 44, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 45, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 46, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 47, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 48, or

[0032] (ix) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 49, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 50, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 51, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 52, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 53, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 54, or

[0033] (x) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 55, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 56, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 57, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 58 or 159, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 59, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 60, or

[0034] (xi) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 61, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 62, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 63, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 64, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 65, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 66, or

[0035] (xii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 67, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 68, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 69, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 70, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 71, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 72.

[0036] In some embodiments, the antigen-binding molecules as described in any of the preceding embodiments, wherein PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 are defined according to the Kabat numbering rules.

[0037] In some embodiments, the antigen-binding molecule, as described in any of the preceding embodiments, reacts at 25°C with a concentration of less than 1 × 10⁻⁶. -7 M, 5×10 -8 M, 1×10 -8 M's KD binding to human PSMA, wherein the KD is measured by surface plasmon resonance.

[0038] In some specific implementations, such as the antigen-binding molecule described in the preceding one, at 25°C, the concentration is less than 9 × 10⁻⁶. -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 M's KD binding to human PSMA, wherein the KD is measured by surface plasmon resonance.

[0039] In some embodiments, the antigen-binding molecule as described in the preceding one, wherein:

[0040] (i) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 77, 107, 108, 109, 110, 111 or 112, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 78, 113, 114 or 115, or

[0041] (ii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 73 or 99, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 74 or 100, or

[0042] (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

[0043] (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

[0044] (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

[0045] (vi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 83 or 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 84, 128, 129 or 130, or

[0046] (vii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 85, 131 or 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 86, 133, 134 or 135, or

[0047] (viii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 87, 136, 137 or 138, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 88, 139 or 140, or

[0048] (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

[0049] (x) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 91, 148, or 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 92, 150, 151, or 152, or

[0050] (xi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 93, 153, 154, 155 or 156, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 94, 157 or 158, or

[0051] (xii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 95, 277, 278 or 279, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 96, 280, 281, 282 or 283.

[0052] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0053] (i) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 115, or

[0054] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0055] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 77, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 78, or

[0056] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0057] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0058] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0059] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0060] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0061] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0062] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0063] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0064] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0065] (ii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 73, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 74, or

[0066] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 99, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 100, or

[0067] (iii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 75, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 76, or

[0068] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 104, or

[0069] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 104, or

[0070] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 104, or

[0071] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 105, or

[0072] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 105, or

[0073] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 105, or

[0074] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 106, or

[0075] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 106, or

[0076] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 106, or

[0077] (iv) The PSMA-VH contains the amino acid sequence SEQ ID NO: 79, and the PSMA-VL contains the amino acid sequence SEQ ID NO: 80, or

[0078] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 118, or

[0079] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 118, or

[0080] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 119, or

[0081] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 119, or

[0082] (v) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 81, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 82, or

[0083] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 121, or

[0084] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 122, or

[0085] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 123, or

[0086] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 124, or

[0087] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 125, or

[0088] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 126, or

[0089] (vi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 83, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 84, or

[0090] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 128, or

[0091] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 129, or

[0092] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 130, or

[0093] (vii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 85, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 86, or

[0094] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 133, or

[0095] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 134, or

[0096] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 135, or

[0097] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 133, or

[0098] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 134, or

[0099] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 135, or

[0100] (viii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 87, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 88, or

[0101] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 136, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 139, or

[0102] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 137, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 139, or

[0103] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 139, or

[0104] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 140, or

[0105] (ix) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 89, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 90, or

[0106] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0107] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0108] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0109] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0110] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0111] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0112] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0113] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0114] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 147, or

[0115] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 147, or

[0116] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 147, or

[0117] (x) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 91, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 92, or

[0118] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 150, or

[0119] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 150, or

[0120] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 151, or

[0121] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 151, or

[0122] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 152, or

[0123] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 152, or

[0124] (xi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 93, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 94; or

[0125] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0126] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0127] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0128] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0129] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0130] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0131] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0132] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0133] (xii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 95, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 96.

[0134] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0135] (i) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 115, or

[0136] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0137] (vi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 128.

[0138] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments includes an Fc region (including an IgG Fc region or an IgG1 Fc region). In some embodiments, the Fc region, compared to the wild-type Fc region, contains one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. In some embodiments, the Fc region is the 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 contains the amino acid sequence of SEQ ID NO: 175.

[0139] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments further 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 Fc2 each independently having one or more amino acid substitutions that reduce homodimerization of the Fc region.

[0140] The ordinal numbers “first,” “second,” “third,” “1,” “2,” “3,” etc. (such as “first subunit,” “Fc2,” “third chain,” “connector 2”) used in this disclosure are only used to distinguish different features, elements, components, or steps and are not intended to limit the number, order, or level.

[0141] In some embodiments, Fc1 and Fc2 each independently have one or more amino acid substitutions according to the pestle and mortar technique. In some embodiments, Fc1 has a protruding structure according to the pestle and mortar technique, and Fc2 has a porous structure according to the pestle and mortar technique. Conversely, the opposite is also true.

[0142] In some embodiments, the amino acid residue at position 366 of Fc1 is W; and the amino acid residue at position 366 of Fc2 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 is V, numbered according to the EU index. The reverse is also true.

[0143] In some embodiments, the amino acid residue at position 354 of Fc1 is C; and the amino acid residue at position 349 of Fc2 is C, numbered according to the EU index. The reverse is also true.

[0144] In some embodiments, Fc1 has the amino acid sequence of SEQ ID NO: 169, and Fc2 has the amino acid sequence of SEQ ID NO: 170.

[0145] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments includes an antigen-binding module that specifically binds to PSMA and an 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 scFv.

[0146] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises a first chain having the structure shown in formula (a) and a second chain having the structure shown in formula (b).

[0147] Equation (a) [PSMA-VH]-[Connector 1]-[PSMA-VL]-[Connector 2]-[CD3-VH]-[Connector 3]-[CD3-VL]-[Connector 4]-[Fc2],

[0148] Equation (b) [Fc1],

[0149] The structures shown in equations (a) and (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, such as...).Figure IE (Structure shown). It should be understood that Fc1 and Fc2 here function to form a dimer and are therefore interchangeable.

[0150] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0151] The antigen-binding molecule has: a first strand containing the amino acid sequence of SEQ ID NO: 191 and a second strand containing the amino acid sequence of SEQ ID NO: 169; or

[0152] The antigen-binding molecule has: a first strand containing the amino acid sequence of SEQ ID NO: 192 and a second strand containing the amino acid sequence of SEQ ID NO: 169; or

[0153] The antigen-binding molecule has: a first strand containing the amino acid sequence of SEQ ID NO: 198 and a second strand containing the amino acid sequence of SEQ ID NO: 169.

[0154] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, 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).

[0155] In some embodiments, the titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 210 to SEQ ID NO: 228, and the obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 229 to SEQ ID NO: 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.

[0156] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises an antigen-binding module that specifically binds to PSMA and an antigen-binding module that specifically binds to CD3. In some embodiments, the antigen-binding module that specifically binds to PSMA is a Fab; the antigen-binding module that specifically binds to CD3 is a replaced Fab comprising a Titin chain and an Obscurin chain (capable of forming a dimer).

[0157] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises a first chain having the structure shown in formula (c), a second chain having the structure shown in formula (d), a third chain having the structure shown in formula (e), and a fourth chain having the structure shown in formula (f).

[0158] Equation (c) [PSMA-VH]-[CH1]-[Fc1]

[0159] Equation (d)[PSMA-VL]-[CL]

[0160] Equation (e) [CD3-VH]-[connector 5]-[Obscurin chain]-[Fc2],

[0161] Equation (f) [CD3-VL]-[Connector 6]-[Titin chain],

[0162] The structures shown in equations (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 non-limiting examples, such as...). Figure 1A (Structure shown). It should be understood that Fc1 and Fc2 here function to form a dimer and are therefore interchangeable.

[0163] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0164] The antigen-binding molecule has: a first chain containing the amino acid sequence SEQ ID NO: 193, a second chain containing the amino acid sequence SEQ ID NO: 194, a third chain containing the amino acid sequence SEQ ID NO: 171, and a fourth chain containing the amino acid sequence SEQ ID NO: 172, or,

[0165] The antigen-binding molecule has: a first strand containing the amino acid sequence of SEQ ID NO: 201, a second strand containing the amino acid sequence of SEQ ID NO: 202, a third strand containing the amino acid sequence of SEQ ID NO: 171, and a fourth strand containing the amino acid sequence of SEQ ID NO: 172, or

[0166] The antigen-binding molecule has: a first chain containing the amino acid sequence of SEQ ID NO: 206, a second chain containing the amino acid sequence of SEQ ID NO: 207, a third chain containing the amino acid sequence of SEQ ID NO: 171, and a fourth chain containing the amino acid sequence of SEQ ID NO: 172, or

[0167] The antigen-binding molecule has: a first chain containing the amino acid sequence of SEQ ID NO: 284, a second chain containing the amino acid sequence of SEQ ID NO: 285, a third chain containing the amino acid sequence of SEQ ID NO: 171, and a fourth chain containing the amino acid sequence of SEQ ID NO: 172.

[0168] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises two antigen-binding modules that specifically bind PSMA and one antigen-binding module that specifically binds CD3. In some embodiments, the antigen-binding module that specifically binds PSMA is a Fab; the antigen-binding module that specifically binds CD3 is a replaced Fab comprising a Titin chain and an Obscurin chain (capable of forming a dimer).

[0169] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the antigen-binding molecule comprises a first chain having the structure shown in formula (c), two second chains having the structure shown in formula (d), a third chain having the structure shown in formula (g), and a fourth chain having the structure shown in formula (h).

[0170] Equation (c) [PSMA-VH]-[CH1]-[Fc1]

[0171] Equation (d)[PSMA-VL]-[CL]

[0172] Formula (g) [PSMA-VH]-[CH1]-[CD3-VH]-[linker 7HTitin chain]-[Fc2],

[0173] Formula (h) [CD3-VL]-[connector 8]-[Obscurin chain],

[0174] The structures shown in 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. (As a non-limiting example, such as...) Figure IB (Structure shown). It should be understood that Fc1 and Fc2 here function to form a dimer and are therefore interchangeable.

[0175] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0176] The antigen-binding molecule comprises: a first strand containing the amino acid sequence of SEQ ID NO: 180, two second strands containing the amino acid sequence of SEQ ID NO: 181, a third strand containing the amino acid sequence of SEQ ID NO: 182, and a fourth strand containing the amino acid sequence of SEQ ID NO: 174, or

[0177] The antigen-binding molecule comprises: a first strand containing the amino acid sequence of SEQ ID NO: 187, two second strands containing the amino acid sequence of SEQ ID NO: 188, a third strand containing the amino acid sequence of SEQ ID NO: 189, and a fourth strand containing the amino acid sequence of SEQ ID NO: 174, or

[0178] The antigen-binding molecule comprises: a first strand containing the amino acid sequence of SEQ ID NO: 193, two second strands containing the amino acid sequence of SEQ ID NO: 194, a third strand containing the amino acid sequence of SEQ ID NO: 195, and a fourth strand containing the amino acid sequence of SEQ ID NO: 174, or

[0179] The antigen-binding molecule has: a first chain containing the amino acid sequence of SEQ ID NO: 206, two second chains containing the amino acid sequence of SEQ ID NO: 207, a third chain containing the amino acid sequence of SEQ ID NO: 208, and a fourth chain containing the amino acid sequence of SEQ ID NO: 174.

[0180] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises two antigen-binding modules that specifically bind PSMA and two antigen-binding modules that specifically bind CD3. In some embodiments, the antigen-binding module that specifically binds PSMA is a Fab; the antigen-binding module that specifically binds CD3 is a replaced Fab comprising a Titin chain and an Obscurin chain (capable of forming a dimer).

[0181] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises two first strands having the structure shown in formula (j), two second strands having the structure shown in formula (d), and two third strands having the structure shown in formula (h).

[0182] Equation (j) [PSMA-VH]-[CH1]-[CD3-VH]-[connector 9]-[Titin chain]-[a subunit of Fc],

[0183] Equation (d) [PSMA-VLHCL],

[0184] Formula (h) [CD3-VL]-[connector 8]-[Obscurin chain],

[0185] The structures shown in equations (j), (d), and (h) are arranged from the N-terminus to the C-terminus, and linker 8 and linker 9 are the same or different peptide linkers. (As a non-limiting example, such as...) Figure 1C (Structure shown). It should be understood that Fc1 and Fc2 here function to form a dimer and are therefore interchangeable.

[0186] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0187] The antigen-binding molecule has: a first strand comprising two amino acid sequences containing the amino acid sequence SEQ ID NO: 183, a second strand comprising two amino acid sequences containing the amino acid sequence SEQ ID NO: 181, and a third strand comprising two amino acid sequences containing the amino acid sequence SEQ ID NO: 174, or

[0188] The antigen-binding molecule has: a first strand comprising two amino acid sequences containing the amino acid sequence SEQ ID NO: 185, a second strand comprising two amino acid sequences containing the amino acid sequence SEQ ID NO: 186, and a third strand comprising two amino acid sequences containing the amino acid sequence SEQ ID NO: 174, or

[0189] The antigen-binding molecule has: a first strand containing two amino acid sequences of SEQ ID NO: 196, a second strand containing two amino acid sequences of SEQ ID NO: 194, and a third strand containing two amino acid sequences of SEQ ID NO: 174, or

[0190] The antigen-binding molecule has: a first strand containing two amino acid sequences of SEQ ID NO: 203, a second strand containing two amino acid sequences of SEQ ID NO: 204, and a third strand containing two amino acid sequences of SEQ ID NO: 174, or

[0191] The antigen-binding molecule has: a first strand containing two amino acid sequences of SEQ ID NO: 286, a second strand containing two amino acid sequences of SEQ ID NO: 285, and a third strand containing two amino acid sequences of SEQ ID NO: 174.

[0192] In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments comprises two antigen-binding modules that specifically bind PSMA and two antigen-binding modules that specifically bind CD3. In some embodiments, the antigen-binding module that specifically binds PSMA is Fab, and the antigen-binding module that specifically binds CD3 is scFv.

[0193] In some embodiments, such as the antigen-binding molecule described in any of the preceding claims, said antigen-binding molecule comprises two first chains having the structure shown in formula (k) and two second chains having the structure shown in formula (d).

[0194] Equation (k) [PSMA-VH]-[CH1]-[CD3-VH]-[connector 9]-[CD3-VL]-[connector 10]-[a subunit of Fc],

[0195] Equation (d)[PSMA-VL]-[CL]

[0196] The structures shown in equations (k) and (d) are arranged from the N-terminus to the C-terminus, and linkers 9 and 10 are the same or different peptide linkers. (As a non-limiting example, such as...) Figure ID (Structure shown). It should be understood that Fcl and Fc2 here function to form a dimer and are therefore interchangeable.

[0197] In some implementations, such as the antigen-binding molecule described in the preceding one, wherein

[0198] The antigen-binding molecule has: two first strands containing the amino acid sequence SEQ ID NO: 184 and two second strands containing the amino acid sequence SEQ ID NO: 181, or

[0199] The antigen-binding molecule has: two first strands containing the amino acid sequence SEQ ID NO: 190 and two second strands containing the amino acid sequence SEQ ID NO: 188, or

[0200] The antigen-binding molecule has: two first strands containing the amino acid sequence SEQ ID NO: 197 and two second strands containing the amino acid sequence SEQ ID NO: 194, or

[0201] The antigen-binding molecule has: two first strands containing the amino acid sequence of SEQ ID NO: 199 and two second strands containing the amino acid sequence of SEQ ID NO: 200, or

[0202] The antigen-binding molecule has: two first strands containing the amino acid sequence SEQ ID NO: 205 and two second strands containing the amino acid sequence SEQ ID NO: 204, or

[0203] The antigen-binding molecule has: a first strand containing two amino acid sequences of SEQ ID NO: 209 and a second strand containing two amino acid sequences of SEQ ID NO: 207.

[0204] In another aspect, this disclosure also provides 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

[0205] (i) PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 77, and PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 78, or

[0206] (ii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 73, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 74, or

[0207] (iii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 75, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 76, or

[0208] (iv) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 79, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 80, or

[0209] (v) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 81, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 82, or

[0210] (vi) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 83, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 84, or

[0211] (vii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 85, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 86, or

[0212] (viii) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 87, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 88, or

[0213] (ix) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 89, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 90, or

[0214] (x) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 91, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 92 or 152, or

[0215] (xi) The PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in the PSMA-VH respectively contain the amino acid sequences of PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3 in SEQ ID NO: 93, and the PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in the PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 in SEQ ID NO: 94, or

[0216] (xii) PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 in PSMA-VH respectively contain 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 PSMA-VL respectively contain the amino acid sequences of PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 in SEQ ID NO: 96.

[0217] In some implementations, the PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering rules.

[0218] In some implementations, the isolated antibody, as described above, wherein

[0219] (i) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 13, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 15, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 16, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 17, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, or

[0220] (ii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 1, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 4, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 6, or

[0221] (iii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 7, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 8, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 9, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 10, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 11, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 12, or

[0222] (iv) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 19, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 20, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 21, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, or

[0223] (v) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 25, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 27, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 28, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 29, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 30, or

[0224] (vi) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 31, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 32, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 33, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 34, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 35, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 36, or

[0225] (vii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 37, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 38, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 39, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 40, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 41, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 42, or

[0226] (viii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 43, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 44, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 45, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 46, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 47, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 48, or

[0227] (ix) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 49, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 50, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 51, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 52, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 53, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 54, or

[0228] (x) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 55, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 56, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 57, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 58 or 159, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 59, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 60, or

[0229] (xi) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 61, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 62, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 63, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 64, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 65, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 66, or

[0230] (xii) The PSMA-VH comprises: PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 67, PSMA-HCDR2 containing the amino acid sequence of SEQ ID NO: 68, and PSMA-HCDR3 containing the amino acid sequence of SEQ ID NO: 69, and the PSMA-VL comprises: PSMA-LCDR1 containing the amino acid sequence of SEQ ID NO: 70, PSMA-LCDR2 containing the amino acid sequence of SEQ ID NO: 71, and PSMA-LCDR3 containing the amino acid sequence of SEQ ID NO: 72.

[0231] In some implementations, PSMA-HCDR1, PSMA-HCDR2, PSMA-HCDR3, PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3 are defined according to the Kabat numbering rules.

[0232] In some embodiments, the antibody is administered at EC50 concentrations 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, and 0.03 μg / mL. 50 Combined with PSMA, the EC 50 It is measured using ELISA.

[0233] In some implementations, the antibody, as described in the preceding one, wherein

[0234] (i) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 77, 107, 108, 109, 110, 111 or 112, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 78, 113, 114 or 115, or

[0235] (ii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 73 or 99, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 74 or 100, or

[0236] (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

[0237] (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

[0238] (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

[0239] (vi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 83 or 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 84, 128, 129 or 130, or

[0240] (vii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 85, 131 or 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 86, 133, 134 or 135, or

[0241] (viii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 87, 136, 137 or 138, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 88, 139 or 140, or

[0242] (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

[0243] (x) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 91, 148, or 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 92, 150, 151, or 152, or

[0244] (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

[0245] (xii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 95, 277, 278 or 279, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 96, 280, 281, 282 or 283.

[0246] In some implementations, the antibody, as described in the preceding one, wherein

[0247] (i) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 115, or

[0248] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0249] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 77, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 78, or

[0250] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0251] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0252] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0253] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0254] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 113, or

[0255] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 107, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0256] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 108, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0257] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 109, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0258] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 111, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0259] (ii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 73, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 74, or

[0260] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 99, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 100, or

[0261] (iii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 75, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 76, or

[0262] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 104, or

[0263] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 104, or

[0264] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 104, or

[0265] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 105, or

[0266] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 105, or

[0267] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 105, or

[0268] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 101, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 106, or

[0269] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 102, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 106, or

[0270] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 103, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 106, or

[0271] (iv) The PSMA-VH contains the amino acid sequence SEQ ID NO: 79, and the PSMA-VL contains the amino acid sequence SEQ ID NO: 80, or

[0272] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 118, or

[0273] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 118, or

[0274] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 116, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 119, or

[0275] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 117, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 119, or

[0276] (v) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 81, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 82, or

[0277] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 121, or

[0278] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 122, or

[0279] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 123, or

[0280] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 124, or

[0281] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 125, or

[0282] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 120, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 126, or

[0283] (vi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 83, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 84, or

[0284] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 128, or

[0285] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 129, or

[0286] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 130, or

[0287] (vii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 85, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 86, or

[0288] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 133, or

[0289] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 134, or

[0290] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 131, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 135, or

[0291] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 133, or

[0292] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 134, or

[0293] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 132, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 135, or

[0294] (viii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 87, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 88, or

[0295] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 136, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 139, or

[0296] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 137, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 139, or

[0297] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 139, or

[0298] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 138, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 140, or

[0299] (ix) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 89, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 90, or

[0300] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0301] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0302] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0303] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 145, or

[0304] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0305] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0306] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0307] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 144, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 146, or

[0308] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 141, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 147, or

[0309] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 142, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 147, or

[0310] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 143, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 147, or

[0311] (x) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 91, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 92, or

[0312] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 150, or

[0313] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 150, or

[0314] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 151, or

[0315] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 151, or

[0316] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 148, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 152, or

[0317] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 149, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 152, or

[0318] (xi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 93, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 94; or

[0319] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0320] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0321] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0322] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 157; or

[0323] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 153, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0324] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 154, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0325] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 155, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0326] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 156, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 158; or

[0327] (xii) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 95, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 96.

[0328] In some implementation schemes, antibodies, as described in the preceding one,

[0329] (i) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 112, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 115, or

[0330] The PSMA-VH contains the amino acid sequence of SEQ ID NO: 110, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 114, or

[0331] (vi) The PSMA-VH contains the amino acid sequence of SEQ ID NO: 127, and the PSMA-VL contains the amino acid sequence of SEQ ID NO: 128.

[0332] In some embodiments, the antibody, as described in any of the preceding embodiments, is a bispecific antibody. In some embodiments, the bispecific antibody specifically binds to PSMA and CD3.

[0333] In another aspect, this disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of the antigen-binding molecule or the antibody described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, 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 both PSMA and CD28.

[0334] In another aspect, this disclosure also provides isolated nucleic acids that encode the antigen-binding molecules or antibodies described in any of the foregoing.

[0335] In another aspect, this disclosure also provides a host cell containing the aforementioned nucleic acids. The host cell cannot develop into a plant or animal.

[0336] In another aspect, this disclosure also provides a method for treating a disease, the method comprising administering to a subject a therapeutically effective amount of any of the preceding antigen-binding molecules or any of the preceding antibodies or combinations thereof.

[0337] In another aspect, this disclosure also provides the use of any of the foregoing antigen-binding molecules or any of the foregoing antibodies or combinations thereof in the preparation of medicaments for the treatment or prevention of diseases.

[0338] In another aspect, this disclosure also provides the antigen-binding molecule or the antibody or a combination thereof described in any of the foregoing claims for use as a medicament. In some embodiments, the medicament is used to treat a disease.

[0339] In some embodiments, the disease described in any of the preceding embodiments is a proliferative disease, a tumor, or an immune disease. In some embodiments, the disease described in any of the preceding embodiments is selected from any of the following: prostate cancer, lung cancer, endometrial cancer, kidney cancer, bladder cancer, colorectal cancer (including colon and rectal cancer), and stomach cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is castration-resistant prostate cancer.

[0340] In some implementations, the aforementioned disease is a PSMA-related disease. In some implementations, the aforementioned disease is a disease of abnormal PSMA expression.

[0341] The antigen-binding molecules disclosed herein possess good therapeutic activity, safety, pharmacokinetic properties, and drug-likeness (such as stability). Attached Figure Description

[0342] Figure 1A : A structural diagram of Formatl.

[0343] Figure IB : Schematic diagram of Format2.

[0344] Figure 1C : Schematic diagram of Format3.

[0345] Figure ID : Schematic diagram of Format4.

[0346] Figure IE : Schematic diagram of Format5. Detailed Implementation

[0347] the term

[0348] The terminology used herein is for descriptive purposes 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 to which this disclosure pertains.

[0349] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of…”. For example, PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 1 explicitly covers the amino acid sequence PSMA-HCDR1 as shown in SEQ ID NO: 1.

[0350] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0351] The term “and / or”, such as “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.

[0352] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0353] 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 possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In one embodiment, an amino acid mutation is a non-conserved amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitutions include substitutions with non-naturally occurring amino acids or with derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used herein to refer to the same amino acid mutation. In this paper, the amino acid residue at a specific site can be represented by the format of position + amino acid residue. For example, 366W indicates that the amino acid residue at position 366 is W. T366W indicates that the amino acid residue at position 366 has mutated from T to W.

[0354] The term "antigen-binding molecule" is used in the broadest sense to encompass molecules that specifically bind antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of both, provided they exhibit the desired antigen-binding activity. The antigen-binding molecules described herein comprise a variable region (VH) and a variable region (VL), which together constitute the antigen-binding domain. Exemplarily, the antigen-binding molecules described herein are bispecific antigen-binding molecules (e.g., bispecific antibodies).

[0355] The term "antibody" is used in the broadest sense and encompasses a wide 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), as long as they exhibit the desired antigen-binding activity. For example, a natural IgG antibody is a heterotetrameric protein of approximately 150,000 Daltons, composed of two light chains and two heavy chains bound by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0356] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. Various structures of bispecific antibodies have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more valent bispecific antibodies. Based on whether the structure is symmetrical, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among them, antibody fragment-type bispecific antibodies, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by binding two or more Fab fragments into one molecule. They have lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration; typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively large molecular weight. The Fc fragment facilitates antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcAAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, and 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-(scF v)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585-608 (2019); ChenS1 et al., J Immunol Res. 2019 Feb 11; 2019: 4516041).

[0357] The term "variable region" or "variable domain" refers to the domain in an antigen-binding molecule that binds to the antigen. In this paper, the heavy chain variable region in the antigen-binding module that specifically binds PSMA is labeled PSMA-VH, and the light chain variable region is labeled PSMA-VL. The heavy chain variable region in the antigen-binding module that specifically binds CD3 is labeled CD3-VH, and the light chain variable region is labeled CD3-VL. Both VH and VL contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. In this study, the three CDR regions in PSMA-VH are labeled PSMA-HCDR1, PSMA-HCDR2, and PSMA-HCDR3; the three CDR regions in PSMA-VL are labeled PSMA-LCDR1, PSMA-LCDR2, and PSMA-LCDR3; the three CDR regions in CD3-VH are labeled CD3-HCDR1, CD3-HCDR2, and CD3-HCDR3; and the three CDR regions in CD3-VL are labeled CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3. Each VH and VL, from N-terminus to C-terminus, is labeled FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A single VH or VL may be sufficient to confer antigen binding specificity.

[0358] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering system (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 system, the "ABM" numbering system, the "contact" numbering system (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77(2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art. The numbering system disclosed herein is shown in Table 1 below.

[0359] Table 1. Relationship between CDR numbering systems

[0360] CDR IMGT Kabat AbM Chothia Contact HCDR1 27-38 31-35 26-35 26-32 30-35 HCDR2 56-65 50-65 50-58 52-56 47-58 HCDR3 105-117 95-102 95-102 95-102 93-101 LCDR1 27-38 24-34 24-34 24-34 30-36 LCDR2 56-65 50-56 50-56 50-56 46-55 LCDR3 105-117 89-97 89-97 89-97 89-96

[0361] Unless otherwise stated, the variable regions and CDR sequences in this disclosure embodiment are governed by the "Kabat" numbering rule. Although a numbering system (such as Kabat) is used to define amino acid residues in a specific implementation, the corresponding technical solutions of other numbering systems are considered equivalent.

[0362] The term "antibody fragment" refers to a molecule that is distinct from the complete antibody but contains a portion of the complete antibody that retains the antigen-binding ability of the complete antibody. 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), biantibodies, linear antibodies, single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0363] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgG1. Unless otherwise stated, Fc regions are numbered using the EU index.

[0364] The term "Titin chain" refers to a peptide segment or functional variant thereof of the Titin protein, consisting of 78-118 amino acids and containing a Titin Ig-like 152 domain. The Titin chain can bind to the Obscurin chain to form a dimerized complex.

[0365] The term "Obscurin chain" refers to a peptide segment or functional variant thereof of 87-117 amino acids containing an Obscurin Ig-like 1 domain on the Obscurin protein, or a peptide segment or functional variant thereof of 78-118 amino acids containing an Obscurin-like Ig-like 1 domain on the Obscurin-like 1 protein. The Obscurin chain can bind to the Titin chain to form a dimerized complex.

[0366] The Titin and Obscurin chains disclosed herein can be used to replace CH1 and CL in Fab to form a replaced Fab (Fab-S), which does not affect the binding of antigen-binding molecules to antigens or their epitopes.

[0367] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain originates from a specific source or species, while the remainder of the heavy and / or light chain originates from a different source or species.

[0368] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, humanization can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with a human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0369] The term "affinity" refers to the strength of the non-covalent interaction 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, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while the terms "kdis" or "kd" as used herein are intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).

[0370] The term "monoclonal antibody" refers to a group of antibodies or its members that are substantially homogeneous, meaning that the antibody molecules contained in the group have the same amino acid sequence, except for the possible presence of small amounts of naturally occurring mutations. In contrast, "polyclonal antibody" typically comprises a variety of different antibodies with different amino acid sequences in their variable domains, and they are generally specific to different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0371] The term "antigen" refers to a molecule or molecular moiety that can be selectively recognized or bound by an antigen-binding molecule (such as an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding molecules (such as antibodies).

[0372] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., through the tertiary folding of the antigen, a protein-like property), which allows non-consecutive amino acids to be spatially close. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. Epitopes contain 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 using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb. NY)).

[0373] The terms "specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or epitope. In some embodiments, the KD of the antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitope do not preclude cross-reactivity with other related antigens, such as cross-reactivity with corresponding antigens from other species (homologous) (e.g., humans or monkeys, such as the cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno) and the chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or the common marmoset (Callithrix jacchus) (commonmarmoset, marmoset).

[0374] The term "non-binding" means that the antibody cannot bind to an antigen or its epitope in the manner described above for specific binding. For example, when the antibody binds at approximately 1 × 10⁻⁶... -6 M or a larger equilibrium dissociation constant (KD) binds to the antigen or its epitope.

[0375] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to a target antigen or its epitope. Specific antigen-binding modules include the antigen-binding domain of an antibody, such as those containing a heavy chain variable region and a light chain variable region. The term "PSMA-specific antigen-binding module" refers to a module capable of binding to PSMA or its epitope with sufficient affinity, such that molecules containing this module can be used as diagnostic and / or therapeutic agents targeting PSMA. For example, a PSMA-specific antigen-binding module has the following equilibrium dissociation constant (KD): <approximately 1 × 10⁻⁶. -8 M or smaller, which is measured by surface plasmon resonance assay. The antigen-binding module includes antibody fragments as defined herein, such as Fab, replaced Fab, or scFv.

[0376] The term "linker" refers to a connecting unit that links two polypeptide fragments. In this document, linkers appearing in the same structural formula may be the same or different. Linkers may be peptide linkers, containing one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. Linkers used in this document may be the same or different. When a "-" appears in the structural formula, it indicates that the units on either side are directly connected by a covalent bond.

[0377] "Tm" is the dissolution and denaturation temperature (intrinsic fluorescence). When a protein denatures (due to heating or the action of a denaturing agent), its tertiary structure opens, and the microenvironment of aromatic amino acids changes, leading to a change in the emission fluorescence spectrum. In this disclosure, Tml refers to the temperature at which the fluorescence changes to half of its maximum value.

[0378] "Tonset" is the denaturation initiation temperature. It refers to the temperature at which a protein begins to denature, that is, the temperature at which its fluorescence value begins to change.

[0379] "Tagg" refers to the aggregation initiation temperature. Aggregates are detected at two wavelengths, 266 nm and 473 nm, using static light scattering to monitor the temperature at which the sample begins to aggregate. Tagg 266 refers to the aggregation initiation temperature detected at 266 nm.

[0380] The term “nucleic acid” is used interchangeably with the term “polynucleotide” herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). “Isolated” nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. An isolated nucleic acid encoding the antigen-binding molecule refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitution can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a degenerate base and / or a deoxyinosine residue.

[0381] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0382] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0383] The terms “fusion” or “linkage” refer to the direct or covalent connection of components (such as antigen-binding modules and Fc domains) via linkers.

[0384] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a nucleic acid sequence suitable for transforming host cells and containing a sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0385] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. In this text, the term includes mutant progeny that have the same function or biological activity as cells screened or selected in primary transformed cells. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but 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, young 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, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia amethanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula polymorpha*. The fungi include *Candida polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium sp.*, *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrellapatens*, and *Neurospora crassa*. The following species are included: *Pichia*, any *Saccharomyces*, *Hansenula polymorpha*, any *Kluyveromyces*, *Candida albicans*, any *Aspergillus*, *Trichoderma reesei*, *Chrysosporium lucknowense*, any *Fusarium*, *Yarrowia lipolytica*, and *Neurospora crassa*. The host cell of this patent does not include subject matter not authorized under patent law.

[0386] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0387] The term "pharmaceutical composition" means a mixture containing one or more antigen-binding molecules or antibodies described herein, along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0388] The term "pharmaceutically acceptable carrier, diluent, buffer, or excipient" refers to a component in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers, diluents, buffers, or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0389] The terms “subject” or “individual” include both 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, cattle, chickens, amphibians, and reptiles. Unless explicitly stated otherwise, the terms “patient” or “subject” are used interchangeably herein. As used herein, the term “cynomolgus monkey” refers to the cynomolgus monkey (Macacafascicularis). In some embodiments, the individual or subject is a human.

[0390] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0391] The term "sample" refers to a collection (such as fluid, cells, or tissue) separated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as culture media (including conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0392] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention intended to be applied to the individual being treated, and can be implemented for preventative purposes or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, the molecules disclosed herein are used to delay the onset of disease or slow its progression.

[0393] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a preventatively effective amount.

[0394] "Therapeutic effective amount" is an amount sufficient to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable symptom associated with the disease in any way.

[0395] A “preventive effective dose” is a dose that, when administered to a subject, will have a predetermined preventive 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 related symptoms. A complete therapeutic or preventive effect may not occur after a single dose, but may occur after a series of doses. Therefore, a therapeutic or preventive effective dose can be administered once or multiple times. Both “therapeutic effective doses” and “preventive effective doses” can vary depending on a variety of factors, such as an individual’s disease state, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status in the patient.

[0396] target molecules

[0397] The term “PSMA” should be interpreted broadly to encompass all forms of PSMA molecules at various stages in mammals, including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the PSMA gene (e.g., precursor PSMA, mature PSMA, membrane-expressed PSMA, PSMA splice variants, modified PSMA, or fragments thereof); the term also covers artificially prepared or in vitro expressed PSMA.

[0398] The term “CD3” should be interpreted broadly to encompass all forms of CD3 molecules at various stages in mammals, including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the BCMA gene (e.g., precursor CD3, mature CD3, membrane-expressed CD3, CD3 splice variants, modified CD3, or fragments thereof); the term also covers artificially prepared or in vitro expressed CD3.

[0399] The antigen-binding molecules disclosed herein

[0400] This disclosure provides antigen-binding molecules with a number of advantageous properties, such as high affinity for PSMA and PSMA-expressing cells, in vitro cytotoxic activity, therapeutic activity, safety, pharmacokinetic properties, and drugability (e.g., yield, purity, and stability).

[0401] Exemplary antigen-binding molecules

[0402] The antigen-binding molecules disclosed herein include bispecific antigen-binding molecules (e.g., bispecific antibodies) that specifically bind to PSMA and CD3, and anti-PSMA antibodies. Specifically, the antigen-binding molecules disclosed herein possess one or more of the following:

[0403] a. The antibody exhibits high affinity for PSMA. In some embodiments, the antibody is expressed at concentrations 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, and 0.03 μg / mL. 50 Combined with PSMA, the EC 50 It is measured using ELISA.

[0404] b. The antigen-binding molecule (such as a bispecific antibody) has a high affinity for PSMA. In some embodiments, the antigen-binding molecule exhibits an affinity of less than 1 × 10⁻⁶ at 25°C. -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 -9M, 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 M's KD binding to human PSMA, wherein the KD is measured by surface plasmon resonance.

[0405] c. High affinity for PSMA on cell surface. The antibody is available at EC50 concentrations 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, and 0.04 μg / mL. 50 The EC binds to cell surface PSMA. s0 It was measured by ELISA. The cells described were CHO cells overexpressing human PSMA.

[0406] d. In vitro specific killing activity against PSMA-expressing cells.

[0407] e. Induces the release of low levels of cytokines (IL6 and IFNγ).

[0408] f. Enhanced in vivo therapeutic activity.

[0409] This disclosure provides an antigen-binding molecule comprising 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.

[0410] This disclosure also provides an isolated antibody capable of specifically binding to PSMA, said antibody comprising 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. The antibody of this document is described below using antibody 15 as an example.

[0411] The PSMA-VH, an antigen-binding molecule that specifically binds to PSMA and CD3, or an anti-PSMA antibody, has the following amino acid sequences: PSMA-HCDR1 as shown in SEQ ID NO: 13, PSMA-HCDR2 as shown in SEQ ID NO: 14, and PSMA-HCDR3 as shown in SEQ ID NO: 15. The PSMA-VL has the following amino acid sequences: PSMA-LCDR1 as shown in SEQ ID NO: 16, PSMA-LCDR2 as shown in SEQ ID NO: 17, and PSMA-LCDR3 as shown in SEQ ID NO: 18.

[0412] In some embodiments, the PSMA-VH and / or PSMA-VL, as described above, are murine or humanized. In some embodiments, the PSMA-VH and / or PSMA-VL are humanized. In some embodiments, the FR1, FR2, and FR3 of the humanized PSMA-VH have at least 60%, 70%, or 80% sequence identity with the FR1, FR2, and FR3 of SEQ ID NO: 77, the FR4 of the humanized PSMA-VH has at least 80% or 90% sequence identity with the FR4 of SEQ ID NO: 77, the FR1, FR2, and FR3 of the humanized PSMA-VL have at least 60%, 70%, or 80% sequence identity with the FR1, FR2, and FR3 of SEQ ID NO: 78, and / or the FR4 of the humanized PSMA-VL has at least 80% or 90% sequence identity with the FR4 of SEQ ID NO: 78. In some embodiments, the PSMA-VH has FR1, FR2, FR3 derived from IGHV1-3*01 and FR4 derived from IGHJ1*01, and is either 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 derived from IGKV1-27*01 and FR4 derived from IGKJ2*01, and is either 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 aforementioned variable regions and CDRs are defined according to the Kabat numbering rules.

[0413] In some embodiments, the antigen-binding molecule or antibody as described in any of the preceding embodiments, wherein the amino acid sequence of said PSMA-VH has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 77, 107, 108, 109, 110, 111, or 112, and the amino acid sequence of said PSMA-VL has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 78, 113, 114, or 115. In some embodiments, the amino acid sequence of said PSMA-VH is as shown in SEQ ID NO: 77, 107, 108, 109, 110, 111, or 112, and the amino acid sequence of said PSMA-VL is as shown in SEQ ID NO: 78, 113, 114, or 115. In some embodiments, the amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 107, 108, 109, 110, 111 or 112, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 113, 114 or 115.

[0414] In some implementations, such as the antigen-binding molecule or antibody described in any of the preceding embodiments, wherein

[0415] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 112, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 115, or

[0416] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 110, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or

[0417] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 77, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 78, or

[0418] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 107, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or

[0419] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 108, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or

[0420] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 109, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or

[0421] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 110, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or

[0422] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 111, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or

[0423] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 107, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or

[0424] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 108, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or

[0425] The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 109, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or

[0426] 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.

[0427] In some embodiments, the antigen-binding molecule or antibody as described in any of the preceding claims, wherein the amino acid sequence of said PSMA-VH is as shown in SEQ ID NO: 112, and the amino acid sequence of said PSMA-VL is as shown in SEQ ID NO: 115, or

[0428] 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.

[0429] In some embodiments, the antigen-binding molecule as described above, the CD3-VH has: the amino acid sequence as shown in SEQ ID NO: 160, CD3-HCDR1, the amino acid sequence as shown in SEQ ID NO: 161, and CD3-HCDR3, the amino acid sequence as shown in SEQ ID NO: 162; and the CD3-VL has: the amino acid sequence as shown in SEQ ID NO: 163, CD3-LCDR1, the amino acid sequence as shown in SEQ ID NO: 164, and CD3-LCDR3, the amino acid sequence as shown in SEQ ID NO: 165.

[0430] In some embodiments, the CD3-VH and / or CD3-VL of the antigen-binding molecule as described in any of the preceding embodiments are murine or humanized. In some embodiments, the CD3-VH and / or CD3-VL are humanized. In some embodiments, the amino acid sequence of the CD3-VH is as shown in SEQ ID NO: 166, and the amino acid sequence of the CD3-VL is as shown in SEQ ID NO: 167. In some embodiments, the variable region and CDR described above are defined according to the Kabat numbering rules.

[0431] According to the technical solutions of antibody series 2, 13, 19, 27, 31, 41, 43, 46, 50, 71 and 73 disclosed in the embodiments of this article, these antibodies have a similar range of technical solutions to antibody 15 described above.

[0432] Structure of antigen-binding molecules

[0433] The bispecific antigen-binding molecules disclosed herein are not limited to a specific molecular structure, as long as they possess the desired antigen-binding function. For example, the bispecific antigen-binding molecules described herein can be bivalent (1+1), trivalent (2+1), or tetravalent (2+2). The antigen-binding module in the antigen-binding molecule can be any antibody fragment with antigen-binding activity, fused via a peptide linker. The peptide linkers disclosed herein (e.g., linkers 1 to 11) can be any suitable peptide chain, as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker can be a flexible peptide of 1-50 or 3-20 amino acid residues. In some embodiments, each peptide linker independently has an L1-(GGGGS)n-L2 structure, wherein 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, and 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 peptide linker is 3-15 amino acid residues in length. In some embodiments, each peptide linker independently has a (GGGGS)n structure, wherein n is 1, 2, or 3. In some embodiments, 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 as shown in SEQ ID NO: 176; the amino acid sequence of linker 2 is as shown 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 as shown in SEQ ID NO: 173.

[0434] For example, the antigen-binding molecule disclosed herein comprises a first chain having the structure shown in formula (ai) and a second chain having the structure shown in formula (b).

[0435] Formula (ai)

[0436] [PSMA-VH]-[GGGGSGGGGSGGGGS]-[PSMA-VL]-[SGGGGS]-[CD3-VH]-[GGGGSGGGGSGGGGS]-[CD3-VL]-[GGG]-[Fc2],

[0437] Equation (b) [Fcl],

[0438] The structures shown in equations (ai) and (b) are arranged from the N end to the C end.

[0439] Exemplary bivalent antigen-binding molecules have:

[0440] The antigen-binding molecule has: a first strand with an amino acid sequence as shown in SEQ ID NO: 192 and a second strand with an amino acid sequence as shown in SEQ ID NO: 169; or

[0441] The antigen-binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 191 and a second chain with an amino acid sequence as shown in SEQ ID NO: 169.

[0442] Variants of antigen-binding molecules

[0443] In some embodiments, amino acid sequence variants of the antigen-binding molecules provided herein are included. For example, it may be desirable 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 suitable 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 within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

[0444] Replace, insert, and delete variants

[0445] In some embodiments, antigen-binding molecule variants with one or more amino acid substitutions are provided. Substitutions can be made in CDR and FR. Conservative substitutions are shown in Table 2 under the heading “Preferred Substitutions.” More substantial variations are provided in Table 2 under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into antibodies of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0446] Table 2. Substitution of amino acids

[0447]

[0448]

[0449] Based on common side chain characteristics, amino acids can be grouped as follows:

[0450] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0451] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0452] (3) Acidic: Asp, Glu;

[0453] (4) Alkaline: His, Lys, Arg;

[0454] (5) Residues that affect chain orientation: Gly, Pro;

[0455] (6) Aromatic: Trp, Tyr, Phe.

[0456] Non-conservative substitution refers to replacing a member of one category with a member of another category.

[0457] One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity). CDRs can be altered (e.g., substituted), for example, to improve antibody affinity. Such alterations can be made to CDR “hotspots,” residues encoded by codons that undergo mutations at a high frequency during somatic maturation, and / or residues that contact the antigen, while testing the binding affinity of the resulting variant VH or VL. In some implementations of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling.

[0458] In some embodiments, substitution, insertion, or deletion may occur within one or more CDRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) may be made to CDRs that do not materially reduce binding affinity. In some 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.

[0459] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan 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 a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0460] Amino acid sequence insertions include: peptides with one residue fused to the amino and / or carboxyl terminus or with a length of 100 or more residues; and intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of an enzyme (or a peptide that prolongs the serum half-life of the antibody) fused to the N or C terminus.

[0461] Fab transformation

[0462] In one aspect, in the antigen-binding molecule disclosed herein, one of the antigen-binding modules that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 is a replaced Fab, said replaced Fab comprising a heavy chain variable region, a light chain variable region, a titin chain, and an obscurin chain. In the replaced Fab, the original CHI and CL of the Fab are replaced by the titin chain and the obscurin chain. Exemplarily, the sequences of the titin chain and the obscurin chain are shown in Tables 3-1 and 3-2.

[0463] Table 3-1. Amino acid sequence of the titin chain

[0464]

[0465]

[0466] Table 3-2. Amino acid sequence of the obscurin chain

[0467]

[0468]

[0469]

[0470] Renovation of Fc District

[0471] In one aspect, the Fc region of the antigen-binding molecule disclosed herein contains one or more amino acid substitutions that reduce its binding to an Fc receptor, such as an Fcγ receptor, and reduce or eliminate effector function. Natural IgG Fc regions, specifically IgG1 or IgG4 Fc regions, may cause the antigen-binding molecule disclosed herein to target cells expressing Fc receptors rather than cells expressing antigens. The modified Fc region of this disclosure exhibits reduced binding affinity to Fc receptors and / or reduced effector function. In some embodiments, the modified Fc region exhibits a 50%, 80%, 90%, or 95% or more reduction in binding affinity to Fc receptors compared to the natural Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the modified Fc region exhibits reduced affinity for complement (e.g., C1q) compared to the native Fc region. In some embodiments, the modified Fc region does not exhibit reduced affinity for neonatal Fc receptors (FcRn) compared to the native Fc region. In some embodiments, the modified Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell initiation.

[0472] For the IgG1 Fc region, amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 replace potentially degraded effector functions. In some embodiments, the Fc region is the human IgG1 Fc region, and amino acid residues at positions 234 and 235 are designated A, numbered according to the EU index. For the IgG4 Fc region, amino acid residues at positions 228 replace potentially degraded effector functions.

[0473] Antigen-binding molecules can also contain disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit. To increase the serum half-life of antigen-binding molecules, mutations in 252Y, 254T, and 256E can be introduced.

[0474] When an antigen-binding molecule contains different binding modules fused to the two subunits of the Fc region, undesirable homodimerization may occur. To improve yield and purity, it is advantageous to introduce modifications that promote heterodimerization into the Fc region of the antigen-binding molecule disclosed herein. In some embodiments, the Fc region of this disclosure comprises a modification according to the knock-in-hole (KIH) technique, which involves introducing a protrusion (knob) at the interface of the first subunit and a hole (hole) at the interface of the second subunit. This allows the protrusion to be positioned within the hole, promoting heterodimer formation and inhibiting homodimer production. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first subunit with a larger side chain (e.g., tyrosine or tryptophan). The hole is created at the interface of the second subunit by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and hole structures are prepared by altering the nucleic acid encoding the polypeptide; optional amino acid substitutions are shown in the table below.

[0475] Table 4. KIH mutation combinations

[0476]

[0477] Besides the mortar and pestle technique, other techniques for modifying the CH3 domain of heavy chains to achieve heterodimerization are also known in the art, such as WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO 007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 and WO 013 / 096291.

[0478] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, the composition of a complete antibody may include an antibody population with all K447 residues and / or G446+K447 residues removed. In some embodiments, the composition of a complete antibody may include an antibody population without the removal of K447 residues and / or G446+K447 residues. In some embodiments, the composition of a complete antibody has an antibody population consisting of a mixture of antibodies with and without K447 residues and / or G446+K447 residues.

[0479] Recombination method

[0480] Antigen-binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecule are provided.

[0481] In the case of natural antibodies, natural antibody fragments, or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required: one for the light chain or a fragment thereof, and one for the heavy chain or a fragment thereof. These nucleic acids encode the amino acid sequence containing the antibody VL and / or the amino acid sequence containing the antibody VH (e.g., the light and / or heavy chains of the antibody). These nucleic acids can be expressed on the same expression vector or on different expression vectors.

[0482] In the case of bispecific antibodies with heterodimeric heavy chains, for example, four nucleic acids are required: one for the first light chain, one for the first heavy chain containing the Fc region polypeptide of the first heteromonomer, one for the second light chain, and one for the second heavy chain containing the Fc region polypeptide of the second heteromonomer. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors, typically on two or three expression vectors, meaning that one vector can contain more than one of these nucleic acids.

[0483] In one embodiment, this disclosure provides isolated nucleic acids encoding antibodies as described above. Such nucleic acids can independently encode any of the aforementioned polypeptide chains. In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In yet another aspect, this disclosure provides host cells comprising such nucleic acids. In one embodiment, a method for preparing an antigen-binding molecule is provided, wherein the method includes culturing host cells comprising nucleic acids encoding said antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering said antibody from the host cells (or host cell culture medium).

[0484] To generate antigen-binding molecules through recombination, nucleic acids encoding proteins are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains), or generated through recombination methods or obtained through chemical synthesis.

[0485] 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 function are not required. After expression, antibodies can be separated from bacterial cell paste in soluble fractions and can be further purified.

[0486] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in antibodies with partial or complete human glycosylation patterns. Suitable host cells for expressing (glycosylated) antibodies can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US6040498, US6420548, US7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (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, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0487] Diagnostic and therapeutic compositions

[0488] In some embodiments, the antigen-binding molecules provided in this disclosure can be used to detect the presence of PSMA and / or CD3 in biological samples. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some embodiments, the biological sample comprises cells or tissues, such as tumor tissue.

[0489] In one embodiment, an antigen-binding molecule is provided for use in a diagnostic or detection method. In another aspect, a method is provided for detecting the presence of PSMA and / or CD3 in a biological sample. In some embodiments, the method includes contacting the biological sample with an antigen-binding molecule under suitable conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods can be in vitro or in vivo. In one embodiment, antigen-binding molecules are used to select subjects suitable for treatment; for example, PSMA and / or CD3 are biomarkers used for patient selection.

[0490] Exemplary conditions, such as tumors or cancers, can be diagnosed using the antigen-binding molecules disclosed herein.

[0491] In some embodiments, a labeled antigen-binding molecule is provided. The label includes, but is not limited to, labels or modules for direct detection (such as fluorescent, chromogenic, electronically dense, chemiluminescent, and radioactive labels) and modules for indirect detection (e.g., modules for indirect detection via enzyme reactions or molecular interactions, such as enzymes or ligands).

[0492] In another aspect, pharmaceutical compositions comprising the said antigen-binding molecule are provided, for example, for any of the following treatment methods. In one aspect, the pharmaceutical composition comprises any antigen-binding molecule provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any antigen-binding molecule provided herein and at least one additional therapeutic agent.

[0493] The pharmaceutical compositions of antigen-binding molecules described in this disclosure are prepared by mixing such antigen-binding molecules, having the desired purity, with one or more optional pharmaceutically acceptable carriers, wherein the pharmaceutical composition is in the form of a lyophilized composition or an aqueous solution. Formulations intended for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane.

[0494] Treatment methods and routes of administration

[0495] Any antigen-binding molecules provided in this article can be used for treatment.

[0496] In another aspect, this disclosure provides the use of antigen-binding molecules in the manufacture or preparation of medicaments. In one embodiment, the medicament is used to treat tumors or cancer. And the medicament is present in an effective amount for the aforementioned disease. In some embodiments, the effective amount is a daily dose or a weekly dose. In one such embodiment, the use further includes administering to a subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). The “subject” according to any of the above embodiments can be a human being.

[0497] In another aspect, a pharmaceutical composition comprising the said antigen-binding molecule is provided, for example, for any of the pharmaceutical uses or treatment methods described above. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0498] The antigen-binding molecule disclosed herein can be used alone or in combination with other agents for treatment. For example, the antigen-binding molecule disclosed herein can be administered co-administered with at least one other therapeutic agent.

[0499] The antigen-binding molecules (and any other therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0500] The antigen-binding molecules disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the medical practitioner. The antigen-binding molecules may optionally be formulated with one or more agents currently used for the prevention or treatment of the said disease. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. These are generally used at the same dosage and route of administration as described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and via any route determined empirically / clinically as appropriate.

[0501] For the prevention or treatment of disease, the appropriate dose of the antigen-binding molecule disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and duration of the disease, whether it is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either as a single dose or as part of a series of treatments. Depending on the type and severity of the disease, an antigen-binding molecule of about 1 μg / kg to 15 mg / kg may be a candidate initial dose for administration to the patient, whether, for example, by a single or multiple separate administrations or by continuous infusion. A typical daily dose may be in the range of about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. Accordingly, for a body weight of 50 kg, an exemplary unit daily dose is 50 μg to 5 g.

[0502] Products

[0503] In another aspect of this disclosure, an article of manufacture is provided comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule of this disclosure. The label or package insert indicates that the use of the composition is for the treatment of a selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing a composition, wherein the composition contains an antigen-binding molecule of this disclosure; and (b) a second container containing a composition, wherein the composition contains additional cytotoxic agents or other therapeutic agents. The article of manufacture in this embodiment of the present disclosure may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including additional buffers, diluents, filters, needles, and syringes.

[0504] As an example, the product is prepared in the form of a medicine kit.

[0505] Examples and Test Cases

[0506] The present disclosure is further described below with reference to examples and test cases, but these examples and test cases are not intended to limit the scope of the disclosure. Experimental methods in the examples and test cases of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0507] Example 1. Antigen-binding molecules containing Titin / Obscurin chains

[0508] The Titin / Obscurin chain disclosed herein can be derived from any suitable polypeptide, including polypeptides derived from WO2021139758A1 (included herein in its entirety by reference) and CN202110527339.7 and the patent that holds priority thereto (included herein in its entirety by reference). A bispecific antibody was constructed, wherein CL is the constant region of the kappa light chain in WO2021139758A1. The amino acid sequences of the Titin and Obscurin chains are shown in Tables 3-1 and 3-2. The linker sequences include GGGGS (SEQ ID NO: 173), ASTKG (SEQ ID NO: 270), or RTVAS (SEQ ID NO: 271). The amino acid sequences of Fc1, Fc2, and CH1 in this embodiment are shown below.

[0509] >Fc1(knob, SEQ ID NO: 272)

[0510]

[0511] >Fc2(hole, SEQ ID NO: 273)

[0512]

[0513] >CH1 (SEQ ID NO: 168).

[0514] 1.1 DI Bispecific Antibody

[0515] Referring to Example 5 of WO2021139758A1, bispecific antibodies against hNGF and hRANKL, DI-2 to DI-20, were constructed, comprising the first heavy chain, second heavy chain, first light chain, and second light chain as described below:

[0516] The first chain: from the N end to the C end, it is as follows: [VH1-I]-[connector 1]-[Obscurin chain]-[Fc2],

[0517] The first light chain, from the N end to the C end, is as follows: [VL1-I] - [connector 2] - [Titin chain].

[0518] The second chain: from the N-terminus to the C-terminus, is: [VH2-D]-[CH1]-[Fc1], and

[0519] The second light chain consists of the following sequence from the N-terminus to the C-terminus: [VL2-D]-[CL];

[0520] In this embodiment, VH1-I and VL1-I represent the heavy chain variable region and light chain variable region of 10 in WO2021139758A1, respectively, while VH2-D and VL2-D represent the heavy chain variable region and light chain variable region of DO in WO2021139758A1, respectively. The structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the DI bispecific antibody are shown in the table below.

[0521] Table 5. Correspondence between Obscurin chain / Titin chain and linker in DI bispecific antibodies

[0522]

[0523] Note: The Titin and Obscurin chain numbers in the table are shown in Tables 3-1 and 3-2.

[0524] The binding activity of bispecific antibodies DI-2 to DI-20 to their antigens was detected using the method described in Test Example 4 of WO2021139758A1. Thermostability of the antibodies was also studied. Methods: The antibody concentration was diluted to 5 mg / mL with PBS, and its thermostability was determined using a high-throughput differential scanning fluorometer (UNCHAINED, Unit). The results showed that the binding activity of the modified bispecific antibodies to the antigens did not change significantly; furthermore, compared with DI-2, the Tm1 (°C) and Tonset (°C) of DI-4 to DI-8, DI-10 to DI-16, and DI-20 were significantly improved, indicating superior thermostability of the bispecific antibodies.

[0525] Table 6. Detection of binding activity of DI bispecific antibodies

[0526]

[0527]

[0528] Table 7. Results of thermostability test of DI bispecific antibodies

[0529] Number Tm1 (°C) Tonset (°C) Number Tm1 (°C) Tonset (°C) DI-2 55.6 48.3 DI-11 57.35 - DI-4 60.1 52.493 DI-12 59.9 51.726 DI-5 61 51.967 DI-13 61 50.988 DI-6 60.8 53.012 DI-14 61.2 52.191 DI-7 60.34 52.003 DI-15 60.41 50.558 DI-8 60.61 50.425 DI-16 61.5 50.691 DI-10 60.2 52.766 DI-20 60.7 51.859

[0530] A solution containing DI bispecific antibody was prepared using a buffer solution of 10 mM acetic acid, pH 5.5, and 9% sucrose. The solution was incubated at 40°C for four weeks. After incubation, the antibody concentration was concentrated back to the initial concentration, and precipitation was observed. The results showed that precipitation occurred in the DI-2 bispecific antibody group, and DI-3 to DI-7 exhibited better stability compared to DI-2.

[0531] Table 8. Precipitation of DI bispecific antibodies

[0532] Number Starting concentration Concentration concentrated to at 4th week Precipitation of solution DI-2 20 mg / ml 20 mg / ml Precipitation occurred DI-3 20 mg / ml 20 mg / ml No precipitation DI-4 60 mg / ml 60 mg / ml No precipitation DI-5 25 mg / ml 25 mg / ml No precipitation DI-6 60 mg / ml 60 mg / ml No precipitation DI-7 16 mg / ml 16 mg / ml No precipitation

[0533] 1.2 PL Bispecific Antibody

[0534] Construct bispecific antibodies against hPDL1 and hCTLA4: PL-1 to PL-19, comprising a first heavy chain, a second heavy chain, a first light chain, and a second light chain as described below:

[0535] The first chain: from the N end to the C end, it is as follows: [VH1-P]-[connector 1]-[Obscurin chain]-[Fc1],

[0536] The first light chain, from the N end to the C end, is as follows: [VL1-P]-[connector 2]-[Titin chain].

[0537] The second chain: from the N-terminus to the C-terminus, is: [VH2-L]-[CH1]-[Fc2], and

[0538] The second light chain consists of the following sequence from the N-terminus to the C-terminus: [VL2-L]-[CL];

[0539] Among them, 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 shown below.

[0540] VH2-L (SEQ ID NO: 274)

[0541] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS

[0542] VL2-L (SEQ ID NO: 275)

[0543] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0544] The structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the PL bispecific antibody in this embodiment are shown in the table below.

[0545] Table 9. Correspondence between Obscurin chain / Titin chain and linker in PL bispecific antibody

[0546]

[0547] Note: The Titin and Obscurin chain numbers in the table are shown in Tables 3-1 and 3-2.

[0548] The binding activity of the PL bispecific antibody was detected using the ELISA method described in Test Example 4 of WO2021139758A1. The hPDL1 and hCTLA4 antigens were purchased from Sino Biology. The thermostability of the antibody was studied. Methods: The antibody concentration was diluted to 1.4-3 mg / mL with PBS, and its thermostability was determined using a high-throughput differential scanning fluorometer (UNCHAINED, Unit). The results showed that the PL bispecific antibody still exhibited good binding activity to the antigen; furthermore, compared with PL-1, the Tml (°C), Tagg 266 (°C), and Tonset (°C) of PL-2 to PL-19 were significantly improved, indicating superior thermostability of the bispecific antibody.

[0549] Table 10. Detection of binding activity of PL bispecific antibody

[0550]

[0551] Table 11. Results of thermostability test of PL bispecific antibody

[0552]

[0553] 1.3 HJ Bispecific Antibody

[0554] Construct HJ bispecific antibodies against hIL5 and hTSLP: HJ-3 to HJ11, comprising a first heavy chain, a second heavy chain, a first light chain, and a second light chain as described below:

[0555] The first chain: from the N end to the C end, it is as follows: [VH1-H]-[connector 1]-[Titin chain]-[Fc1]

[0556] The first light chain, from N to C, consists of: [VL1-H] - [connector 2] - [Obscurin chain].

[0557] The second chain: from the N-terminus to the C-terminus, is: [VH2-J]-[CH1]-[Fc2], and

[0558] The second light chain consists of the following sequence from the N-terminus to the C-terminus: [VL2-J]-[CL];

[0559] In this embodiment, 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. The structures of Obscurin chain, Titin chain, linker 1, and linker 2 in the HJ bispecific antibody are shown in the table below.

[0560] Table 12. Correspondence between Obscurin chain / Titin chain and linker in HJ bispecific antibodies

[0561]

[0562] The antigen-binding activity of the HJ bispecific antibody was detected according to the method in Test Example 4 of WO2021139758A1. The thermostability of the antibody was studied. Method: A diluted solution of the HJ bispecific antibody was prepared using a 10mM acetic acid pH 5.5 buffer solution with 9% sucrose. The bispecific antibody was then concentrated by ultrafiltration 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 incubated in a 40℃ incubator. The SEC purity of the samples was measured on day 0 (before the start of incubation at 40℃, D0), day 7 (day 7 of incubation at 40℃, D7), day 14 (day 14 of incubation at 40℃, D14), day 21 (day 21 of incubation at 40℃, D21), and day 28 (day 28 of incubation at 40℃, D28). After 28 days of incubation at 40℃, samples were immediately taken to test the CE-SDS purity. Experimental results show that the binding activity of the HJ bispecific antibodies constructed in this study to the antigen did not change significantly; and, compared with HJ-3, the HJ-5 to HJ-11 bispecific antibodies have better thermal stability.

[0563] Table 13-1. Detection of binding activity of HJ bispecific antibodies

[0564]

[0565] Table 13-2. Results of accelerated stability test of HJ bispecific antibody

[0566]

[0567] Example 2. Screening and identification of anti-human PSMA hybridoma antibodies

[0568] This disclosure describes the preparation of a monoclonal antibody against human PSMA using hybridoma technology. The resulting antibody binds specifically to human PSMA with high affinity and can cross-react with cynomolgus monkey PSMA; the antibody also exhibits good binding activity to both human PSMA and cynomolgus monkey PSMA on cell surfaces.

[0569] Human PSMA-ECD-his, LnCap cells, or ChoK1-human PSMA cells can be used as immunoassay reagents, or human PSMA-ECD-his and LnCap cells can be used as cross-immunoassay reagents. Gold Adjuvant(Sigma CatNo.T2684) and Thermo Alum (Thermo Cat No. 77161) was used as an adjuvant for cross-immunization of mice. After primary immunization and seven booster immunizations, mice with high antibody titers in their serum (numbers 1-1, 1-2, 2-1, 2-5, 3-3, 3-4, 3-5, and 4-2#, titer > 307.2K) were selected for spleen cell fusion. After fusion, the hybridoma culture supernatant was analyzed based on the hybridoma cell growth density, and antibodies specifically binding to cell surface PSMA were screened. Highly active monoclonal hybridoma cell lines were obtained. Logarithmically growing hybridoma cells were collected, and RNA was extracted using NucleoZol (MN) (following the kit instructions) and reverse transcribed (PrimeScript). TM Reverse Transcriptase (Takara, cat#2680A). The cDNA obtained from reverse transcription was amplified by PCR using mouseIg-Primer Set (Novagen, TB326 Rev.B0503) and then sequenced. The amino acid sequences of the CDR and variable regions of the screened monoclonal hybridoma cell lines are as follows:

[0570] Table 14. CDR of PSMA antibodies

[0571]

[0572]

[0573]

[0574] >2. Mouse-derived heavy chain variable region (SEQ ID NO: 73)

[0575]

[0576] >2. Mouse-derived light chain variable region (SEQ ID NO: 74)

[0577]

[0578] >13. Mouse heavy chain variable region (SEQ ID NO: 75)

[0579]

[0580] >13. Mouse-derived light chain variable region (SEQ ID NO: 76)

[0581]

[0582] >15. Mouse heavy chain variable region (SEQ ID NO: 77)

[0583]

[0584]

[0585] >15. Mouse-derived light chain variable region (SEQ ID NO: 78)

[0586]

[0587] >19. Mouse heavy chain variable region (SEQ ID NO: 79)

[0588]

[0589] >19. Mouse-derived light chain variable region (SEQ ID NO: 80)

[0590]

[0591] >27. Mouse heavy chain variable region (SEQ ID NO: 81)

[0592]

[0593] >27. Mouse-derived light chain variable region (SEQ ID NO: 82)

[0594]

[0595] >31. Mouse heavy chain variable region (SEQ ID NO: 83)

[0596]

[0597] >31. Mouse-derived light chain variable region (SEQ ID NO: 84)

[0598]

[0599] >41. Mouse heavy chain variable region (SEQ ID NO: 85)

[0600]

[0601] >41. Mouse-derived light chain variable region (SEQ ID NO: 86)

[0602]

[0603] >43. Mouse heavy chain variable region (SEQ ID NO: 87)

[0604]

[0605]

[0606] >43. Mouse-derived light chain variable region (SEQ ID NO: 88)

[0607]

[0608] >46 Mouse heavy chain variable region (SEQ ID NO: 89)

[0609]

[0610] >46 Mouse-derived light chain variable region (SEQ ID NO: 90)

[0611]

[0612] >50 Mouse heavy chain variable region (SEQ ID NO: 91)

[0613]

[0614] >50 murine light chain variable regions (SEQ ID NO: 92)

[0615]

[0616] >71. Mouse heavy chain variable region (SEQ ID NO: 93)

[0617]

[0618] >71. Mouse-derived light chain variable region (SEQ ID NO: 94)

[0619]

[0620] >73 Mouse heavy chain variable region (SEQ ID NO: 95)

[0621]

[0622] >73 Mouse-derived light chain variable region (SEQ ID NO: 96)

[0623]

[0624] Note: The underlined area is the CDR area obtained according to the Kabat numbering rules.

[0625] A chimeric antibody is obtained by combining the variable region sequence of a murine anti-PSMA antibody with the constant regions shown in SEQ ID NO: 97 and SEQ ID NO: 98. For example, Chi13 represents a chimeric antibody comprising 13 murine heavy chain variable regions, light chain variable regions, and the constant regions.

[0626] >hIgG1: CH1-Fc (SEQ ID NO: 97)

[0627]

[0628] >CL(SEQ ID NO: 98)

[0629]

[0630] Example 3. Humanization design of anti-human PSMA monoclonal antibody

[0631] Humanization of murine monoclonal antibodies was performed according to methods published in numerous publications in the field. In short, based on the typical VH / VL CDR structure of the obtained murine antibody, homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) were searched from the human germline database. The CDR region of the murine antibody was transplanted onto the human template, and some residues of VL and VH were mutated to replace the constant region of the murine antibody with the human constant region, resulting in the final humanized molecule.

[0632] 3-1. Humanization of Antibody 2

[0633] For the humanized antibody of mouse antibody 2, FR1, FR2, and FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region; FR1, FR2, and FR3 of IGKV6-21*02 and FR4 of IGKJ2*01 were selected as templates for the light chain framework region. Optionally, an amino acid residue at position 49 of the variable region of the light chain of the humanized antibody was substituted, specifically, the mutation was K49Y (according to the Kabat numbering system, the K at position 49 was mutated to Y). The sequence of the antibody variable region is as follows:

[0634] >hu2H1(SEQ ID NO: 99)

[0635]

[0636] >hu2L1(SEQ ID NO: 100)

[0637]

[0638] 3-2. Humanization of Antibody 13

[0639] For the humanized antibody of murine antibody 13, FR1, FR2, and FR3 of IGHV2-26*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 of 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 of the light chain variable region of the humanized antibody were substituted.

[0640] Table 15-1. Mutations of humanized antibody 13

[0641]

[0642] The sequence of the antibody variable region is as follows:

[0643] >hu13H1 (SEQ ID NO: 101)

[0644]

[0645] >hu13H2(SEQ ID NO: 102)

[0646]

[0647] >hu13H3 (SEQ ID NO: 103)

[0648]

[0649] >hu13L1 (SEQ ID NO: 104)

[0650]

[0651] >hu13L2 (SEQ ID NO: 105)

[0652]

[0653] >hu13L3 (SEQ ID NO: 106)

[0654]

[0655] 3-3. Humanization of Antibody 15

[0656] The humanized antibody of mouse antibody 15 uses FR1, FR2, and FR3 of IGHV1-3*01 and FR4 of IGHJ1*01 as templates for the heavy chain framework region; and FR1, FR2, and FR3 of IGKV1-27*01 and FR4 of IGKJ2*01 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 of the heavy chain variable region of the humanized antibody are substituted; and / or amino acid residues at positions 43, 60, and / or 100 of the light chain variable region of the humanized antibody are substituted.

[0657] Table 15-2. Mutations of humanized antibody 15

[0658]

[0659] The sequence of the antibody variable region is as follows:

[0660] >hu15H1 (SEQ ID NO: 107)

[0661]

[0662] >hu15H2 (SEQ ID NO: 108)

[0663]

[0664] >hu15H3 (SEQ ID NO: 109)

[0665]

[0666] >hu15H4 (SEQ ID NO: 110)

[0667]

[0668] >hu15H5(SEQ ID NO: 111)

[0669]

[0670] >hu15H6 (SEQ ID NO: 112)

[0671]

[0672] >hu15L1 (SEQ ID NO: 113)

[0673]

[0674] >hu15L2 (SEQ ID NO: 114)

[0675]

[0676] >hu15L3 (SEQ ID NO: 115)

[0677]

[0678] 3-4. Humanization of Antibody 19

[0679] For the humanized antibody of murine antibody 19, FR1, FR2, and FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 of the heavy chain variable region of the humanized antibody was substituted; and / or the amino acid residue at position 71 of the light chain variable region of the humanized antibody was substituted.

[0680] Table 15-3. Mutations of humanized antibody 19

[0681]

[0682] The sequence of the antibody variable region is as follows:

[0683] >hu19H1 (SEQ ID NO: 116)

[0684]

[0685] >hu19H2 (SEQ ID NO: 117)

[0686]

[0687] >hu19L1 (SEQ ID NO: 118)

[0688]

[0689] >hu19L2 (SEQ ID NO: 119)

[0690]

[0691] 3-5. Humanization of antibody 27

[0692] For the humanized antibody derived from murine antibody 27, FR1, FR2, and FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 on the variable region of the light chain of the humanized antibody were substituted.

[0693] Table 15-4. Mutations of humanized antibody 27

[0694]

[0695] The sequence of the antibody variable region is as follows:

[0696] >hu27H1 (SEQ ID NO: 120)

[0697]

[0698] >hu27L1 (SEQ ID NO: 121)

[0699]

[0700] >hu27L2 (SEQ ID NO: 122)

[0701]

[0702]

[0703] >hu27L3 (SEQ ID NO: 123)

[0704]

[0705] >hu27L4 (SEQ ID NO: 124)

[0706]

[0707] >hu27L5 (SEQ ID NO: 125)

[0708]

[0709] >hu27L6 (SEQ ID NO: 126)

[0710]

[0711] 3-6. Humanization of antibody 31

[0712] For the humanized antibody of mouse antibody 31, FR1, FR2, and FR3 of IGHV3-7*01 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region; FR1, FR2, and FR3 of IGKV1-39*01 and FR4 of IGKJ4*01 were selected as templates for the light chain framework region. Optionally, amino acid residue at position 94 of the heavy chain variable region of the humanized antibody was substituted; and / or amino acid residues at positions 43, 45, 48, and / or 70 of the light chain variable region of the humanized antibody were substituted.

[0713] Table 15-5. Mutations of humanized antibody 31

[0714]

[0715] The sequence of the antibody variable region is as follows:

[0716] >hu31H1 (SEQ ID NO: 127)

[0717]

[0718] >hu31L1 (SEQ ID NO: 128)

[0719]

[0720] >hu31L2 (SEQ ID NO: 129)

[0721]

[0722]

[0723] >hu31L3 (SEQ ID NO: 130)

[0724]

[0725] 3-7. Humanization of Antibody 41

[0726] For the humanized antibody of mouse antibody 41, FR1, FR2, and FR3 of IGHV1-18*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 of 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 of the light chain variable region of the humanized antibody were substituted.

[0727] Table 15-6. Mutations of humanized antibody 41

[0728]

[0729] The sequence of the antibody variable region is as follows:

[0730] >hu41H1 (SEQ ID NO: 131)

[0731]

[0732] >hu41H2(SEQ ID NO: 132)

[0733]

[0734] >hu41L1 (SEQ ID NO: 133)

[0735]

[0736] >hu41L2(SEQ ID NO: 134)

[0737]

[0738] >hu41L3 (SEQ ID NO: 135)

[0739]

[0740] 3-8. Humanization of antibody 43

[0741] For the humanized antibody of murine antibody 43, FR1, FR2, and FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 of the heavy chain variable region of the humanized antibody were substituted; and / or amino acid residue at position 43 of the light chain variable region of the humanized antibody was substituted.

[0742] Table 15-7. Mutations of humanized antibody 43

[0743]

[0744] The sequence of the antibody variable region is as follows:

[0745] >hu43H1 (SEQ ID NO: 136)

[0746]

[0747] >hu43H2(SEQ ID NO: 137)

[0748]

[0749] >hu43H3(SEQ ID NO: 138)

[0750]

[0751] >hu43L1 (SEQ ID NO: 139)

[0752]

[0753] >hu43L2 (SEQ ID NO: 140)

[0754]

[0755] 3-9. Humanization of antibody 46

[0756] For the humanized antibody of murine antibody 46, FR1, FR2, and FR3 of IGHV3-21*01 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 on the heavy chain variable region of the humanized antibody were substituted; and / or amino acid residues at positions 60, 85, and / or 87 on the light chain variable region of the humanized antibody were substituted.

[0757] Table 15-8. Mutations of humanized antibody 46

[0758]

[0759] >hu46H1 (SEQ ID NO: 141)

[0760]

[0761] >hu46H2(SEQ ID NO: 142)

[0762]

[0763] >hu46H3 (SEQ ID NO: 143)

[0764]

[0765] >hu46H4 (SEQ ID NO: 144)

[0766]

[0767] >hu46L1 (SEQ ID NO: 145)

[0768]

[0769] >hu46L2 (SEQ ID NO: 146)

[0770]

[0771] >hu46L3 (SEQ ID NO: 147)

[0772]

[0773] 3-10. Humanization of Antibody 50

[0774] For the humanized antibody of mouse antibody 50, FR1, FR2, and FR3 of IGHV1-18*01 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 of the heavy chain variable region of the humanized antibody were substituted; and / or amino acid residues at positions 2, 3, and / or 28 of the light chain variable region of the humanized antibody were substituted.

[0775] Table 15-9. Mutations in humanized antibody 50

[0776]

[0777] >hu50H1 (SEQ ID NO: 148)

[0778]

[0779] >hu50H2 (SEQ ID NO: 149)

[0780]

[0781] >hu50L1 (SEQ ID NO: 150)

[0782]

[0783] >hu50L2 (SEQ ID NO: 151)

[0784]

[0785] >hu50L3 (SEQ ID NO: 152)

[0786]

[0787] >hu50L3 LCDR1RSSQSILHSYGNTYLE (SEQ ID NO: 159).

[0788] 3-11. Humanization of Antibody 71

[0789] For the humanized antibody of mouse antibody 71, FR1, FR2, and FR3 of IGHV3-30*09 and FR4 of IGHJ1*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 of the heavy chain variable region of the humanized antibody were substituted; and / or amino acid residues at positions 2 and / or 4 of the light chain variable region of the humanized antibody were substituted.

[0790] Table 15-10. Mutations of humanized antibody 71

[0791]

[0792] >hu71H1 (SEQ ID NO: 153)

[0793]

[0794] >hu71H2(SEQ ID NO: 154)

[0795]

[0796] >hu71H3 (SEQ ID NO: 155)

[0797]

[0798] >hu71H4 (SEQ ID NO: 156)

[0799]

[0800] >hu71L1 (SEQ ID NO: 157)

[0801]

[0802] >hu71L2(SEQ ID NO: 158)

[0803]

[0804] The heavy chain variable regions and light chain variable regions of each group described above are combined with the constant regions shown in SEQ ID NO: 97 and SEQ ID NO: 98 to obtain complete humanized antibodies. 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 so on.

[0805] 3-12. Humanization of Antibody 73

[0806] For the humanized antibody of murine antibody 73, FR1, FR2, and FR3 of IGHV1-69*02 and FR4 of IGHJ6*01 were selected as templates for the heavy chain framework region; FR1, FR2, and 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 on 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 on the light chain variable region of the humanized antibody were substituted.

[0807] Table 15-11. Mutations of humanized antibody 73

[0808]

[0809] >hu73H1 (SEQ ID NO: 277)

[0810]

[0811] >hu73H2(SEQ ID NO: 278)

[0812]

[0813] >hu73H3(SEQ ID NO: 279)

[0814]

[0815] >hu73L1 (SEQ ID NO: 280)

[0816]

[0817] >hu73L2(SEQ ID NO: 281)

[0818]

[0819] >hu73L3(SEQ ID NO: 282)

[0820]

[0821] >hu73L4 (SEQ ID NO: 283)

[0822]

[0823] Example 4. Preparation of anti-PSMA-CD3 bispecific antibody

[0824] The CD3-binding molecule disclosed herein can be derived from any suitable antibody. Specifically, the embodiments disclosed herein use S107E.

[0825] Table 16. CDR of S107E

[0826]

[0827] The S107E variable region sequence is as follows:

[0828] >S107E-VH (SEQ ID NO: 166)

[0829]

[0830] >S107E-VL (SEQ ID NO: 167)

[0831]

[0832] This disclosure includes the following molecular structures.

[0833] Format 1 is an asymmetric molecular structure, containing

[0834] Chain 1: VH(anti-PSMA)-IgG1(CH1)-IgG1Fc(Knob, L234A / L235A / S354C / T366W);

[0835] Chain 2: VL (anti-PSMA)-CL;

[0836] Chain 3 (Ob-hole): VH(S107E)-linker α-Obscurin-IgG1Fc (Hole, L234A / L235A / Y349C / T366S / L368A / Y407V); and

[0837] Chain 4 (VL-Titin): VL(S107E)-connector a-Titin.

[0838] Its schematic diagram is as follows Figure 1AAs shown (Ob represents Obscurin, the same applies below).

[0839] >IgG1(CH1)(SEQ ID NO: 168)

[0840]

[0841] >CL(SEQ ID NO: 98)

[0842] >IgG1Fc (Knob, L234A / L235A / S354C / T366W) (SEQ ID NO: 169)

[0843]

[0844] >IgG1Fc (Hole, L234A / L235A / Y349C / T366S / L368A / Y407V) (SEQ ID NO: 170)

[0845]

[0846] >Ob-hole (SEQ ID NO: 171)

[0847]

[0848] >VL-Titin: VL(S107E)-connector a-Titin (SEQ ID NO: 172)

[0849]

[0850] >Connector a (SEQ ID NO: 173) GGGGS

[0851] Note: Single-underlined regions are CDR regions of CD3-binding domains obtained according to Kabat numbering rules; double-underlined regions are Titin or Obscurin sequences; italics indicate constant regions. The same applies below.

[0852] Format 2 is an asymmetric molecule, containing

[0853] Chain 1: VH(anti-PSMA)-IgG1(CH1)-IgG1Fc(Knob, L234A / L235A / S354C / T366W);

[0854] Chain 2 (two chains): VL (anti-PSMA)-CL;

[0855] Chain 3: VH(anti-PSMA)-IgG1(CH1)-VH(S107E)-linker a-Titin-IgG1Fc(Hole, L234A / L235A / Y349C / T366S / L368A / Y407V);

[0856] Chain 4 (VL-Ob): VL(S107E) - connector a-Ob. Its schematic diagram is shown below. Figure IB As shown.

[0857] >VL-Ob: VL(S107E)-connector a-Ob (SEQ ID NO: 174)

[0858]

[0859] Format3 is a symmetrical molecule containing

[0860] Chain 1 (two chains): VH(anti-PSMA)-IgG1(CH1)-VH(S107E)-linker a-Titin-IgG1(AA)Fc;

[0861] Chain 2 (two chains): VL (anti-PSMA)-CL; and

[0862] Chain 4 (two chains) is VL-Ob: VL(S107E) - connector a-Ob. Its schematic diagram is shown below. Figure 1C As shown.

[0863] >IgG1(AA)Fc(SEQ ID NO: 175)

[0864]

[0865] Format 4 is a symmetrical molecule containing 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; the light chain is VL(anti-PSMA)-CL. A schematic diagram is shown below. Figure ID As shown.

[0866] > Connector b (SEQ ID NO: 176)

[0867] GGGGSGGGGSGGGGS

[0868] >Connector c

[0869] GGG.

[0870] Format5 is an asymmetric molecule, containing

[0871] 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

[0872] Chain 3 (Fc(Knob)): IgG1Fc(Knob, L234A / L235A / S354C / T366W), its schematic diagram is shown below. Figure IE As shown.

[0873] >IgG1Fc (Hole, Y349C / T366S / L368A / Y407V) (SEQ ID NO: 170)

[0874] >IgG1Fc (Knob, L234A / L235A / S354C / T366W) (SEQ ID NO: 169)

[0875] > Connector d (SEQ ID NO: 178)

[0876] SGGGGS

[0877] >Connector c: GGG.

[0878] Based on the amino acid sequences of 2, 13, 15, 19, 27, 31, 43, 46, 73 and humanized antibodies, the following bispecific antibodies were constructed.

[0879] Table 17. Bispecific antibodies disclosed herein

[0880]

[0881]

[0882] In this designation, 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 so on. The specific amino acid sequence is as follows:

[0883] >hu2H1-F2-1(SEQ ID NO: 180)

[0884]

[0885] >hu2L1-CL (SEQ ID NO: 181)

[0886]

[0887]

[0888] >hu2H1-F2-3(SEQ ID NO:182)

[0889]

[0890] >hu2H1-F3-1(SEQ ID NO:183)

[0891]

[0892] >hu2H1-F4-1(SEQ ID NO:184)

[0893]

[0894]

[0895] >hu13H1-F3-1(SEQ ID NO:185)

[0896]

[0897] >hu13L1-CL(SEQ ID NO:186)

[0898]

[0899] >hu15H4-F2-1(SEQ ID NO:187)

[0900]

[0901]

[0902] >hu15L2-CL(SEQ ID NO:188)

[0903]

[0904] >hu15H4-F2-3(SEQ ID NO:189)

[0905]

[0906] >hu15H4-F4-1(SEQ ID NO:190)

[0907]

[0908]

[0909] >hu15H4L2-F5-1 (SEQ ID NO: 191)

[0910]

[0911] >hu15H6L3-F5-1 (SEQ ID NO: 192)

[0912]

[0913] >hu19H2-F1-1 also known as hu19H2-F2-1 (SEQ ID NO: 193)

[0914]

[0915] >hu19L2-CL (SEQ ID NO: 194)

[0916]

[0917] >hu19H2-F2-3 (SEQ ID NO: 195)

[0918]

[0919] >hu19H2-F3-1 (SEQ ID NO: 196)

[0920]

[0921]

[0922] >hu19H2-F4-1 (SEQ ID NO: 197)

[0923]

[0924] >hu19H2L2-F5-1 (SEQ ID NO: 198)

[0925]

[0926]

[0927] >hu27H1-F4-1 (SEQ ID NO: 199)

[0928]

[0929] >hu27L6-CL (SEQ ID NO: 200)

[0930]

[0931] >hu31H1-F1-1(SEQ ID NO: 201)

[0932]

[0933]

[0934] >hu31L1-CL(SEQ ID NO: 202)

[0935]

[0936] >hu43H3-F3-1(SEQ ID NO: 203)

[0937]

[0938] >hu43L2-CL(SEQ ID NO: 204)

[0939]

[0940] >hu43H3-F4-1(SEQ ID NO: 205)

[0941]

[0942]

[0943] >hu46H2-F1-1 also known as hu46H2-F2-1(SEQ ID NO: 206)

[0944]

[0945] >hu46L2-CL(SEQ ID NO: 207)

[0946]

[0947] >hu46H2-F2-3(SEQ ID NO: 208)

[0948]

[0949]

[0950] >hu46H2-F4-1(SEQ ID NO: 209)

[0951]

[0952] >73VH-F1-1(SEQ ID NO: 284)

[0953]

[0954] >73VL-CL (SEQ ID NO: 285)

[0955]

[0956] >73VH-F3-1 (SEQ ID NO: 286)

[0957]

[0958]

[0959] The positive control molecule used in this disclosure is Aca-Mab, whose amino acid sequence is shown in SEQ ID NO: 382 in WO2017134158A1.

[0960] Test case

[0961] Cho-human PSMA and Cho-monkey PSMA overexpressing cell lines, hamster ovary cells Cho-K1, human prostate cancer cells Lncap (ATCC), human prostate cancer cells 22RV1 (ATCC), and human prostate cancer cells PC-3 (purchased from the Chinese Academy of Sciences Cell Bank) were obtained through methods described in other previous patents.

[0962] Test Example 1. Binding activity of anti-PSMA antibody to PSMA

[0963] To test the binding ability of the PSMA-targeting humanized antibody disclosed herein to PSMA antigen, this test example used ELISA to detect the binding of the PSMA-targeting humanized antibody to human PSMA antigen (purchased from R&D SYSTEM, 4234-ZN-010). Specifically, PSMA antigen was coated onto 96-well plates at a ratio of 1 μg / well and incubated overnight at 4°C. After discarding the supernatant, the plates were blocked with 5% skim milk for 2 h. Different concentrations of the humanized antibody were prepared using 1% BSA, starting at 10 μg / mL and diluted 4-fold for 12 concentrations. After washing the blocked 96-well plates twice, the prepared PSMA antigen was added, and the plates were incubated at 4°C for 1 h. After washing four times, the plates were incubated with Strepidation-HRP secondary antibody (1:10000) at 4°C for 1 h. The reaction was terminated with 0.1M sulfuric acid after TMB color development. The ELISA results are shown in the table below.

[0964] Table 18. ELISA binding of humanized antibodies to PSMA antigen

[0965]

[0966] Note: Top indicates maximum absorbance.

[0967] The results showed that the humanized and chimeric PSMA antibodies screened in this disclosure had good binding interactions with the PSMA antigen.

[0968] Test Example 2. Antibody binding activity to PSMA

[0969] This test case used flow cytometry to detect the binding activity of the antibody with the stable cell line Cho-hPSMA overexpressing human PSMA. The full-length gene encoding human PSMA was cloned into the mammalian cell expression vector pCDH, and HEK293T cells were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91, and pCDH-human PSMA. The virus was packaged using CRL-11268. After 48 hours of transfection, the virus was collected to infect CHO-K1 cells. After two weeks of pressure selection, cell subcloning was performed, and the cell line Cho-hPSMA, which highly expressed PSMA, was obtained by FACS detection.

[0970] Experiment 1: Cho-hPSMA cells were cultured in F12 medium containing 10% FBS. The culture medium inoculated with cells was placed in a 37°C, 5% CO2 incubator for 2 days, with a cell count of 1×10⁶ cells per well. 5 Cells were added to cell culture plates and centrifuged and washed. Antibody was serially diluted, with 100 μL of antibody solution added to each well of the cell culture plate. The plates were incubated at 4°C for 1 hour, then washed. 100 μL of AF488-Goat Anti-human IgG Fc fluorescent secondary antibody dilution buffer (1:400) was added to each well of the cell culture plate, and the plates were incubated at 4°C for 1 hour, then washed. 100 μL of PBS was added to each well of the cell culture plate, and the plates were read. The cell-binding activity of the antibody is shown in the table below.

[0971] Table 19-1. Binding activity of humanized antibodies and chimeric antibodies to Cho-hPSMA

[0972] Antibody EC 50 (μg / mL) Antibody EC 50 (μg / mL) hu13H1L1 0.9474 hu13H3L2 1.100 hu13H2L1 1.055 hu13H1L3 1.012 hu13H3L1 1.052 hu13H2L3 1.142 hu13H1L2 0.9808 hu13H3L3 1.495 hu13H2L2 1.088 Chi13 1.213

[0973] Experiment 2: Cells were cultured using the same method as in Experiment 1 and seeded into cell culture plates. Antibodies were then serially diluted, with 100 μL of antibody solution added to each well of the cell culture plate. After incubation at 4°C for 1 hour, the cells were washed. 100 μL / well of HRP-Goat Anti-human IgG (H+L) secondary antibody dilution buffer (1:8000) was added to each well, and the cells were incubated at 4°C for 1 hour, followed by centrifugation and washing. The reaction was terminated with 0.1 M sulfuric acid after TMB staining, and the OD450 value was measured. The cell-binding activity of the antibody is shown in the table below.

[0974] Table 19-2. Binding activity of humanized antibodies and chimeric antibodies to Cho-hPSMA (ECG) 50 (μg / mL)

[0975]

[0976]

[0977] Experiment 3: The binding activity of the bispecific antibody was detected using the method described in Experiment 1. The binding activity of the antibody with cells under various concentration conditions is shown in the table below.

[0978] Table 19-3. Binding activity (fluorescence intensity) of bispecific antibodies to Cho-hPSMA

[0979]

[0980]

[0981] Experiment 4: The binding activity of the bispecific antibody was detected using the method described in Experiment 2. The binding activity of the antibody with cells under various concentration conditions is shown in the table below.

[0982] Table 19-4. Binding activity (absorbance) of bispecific antibodies to Cho-hPSMA

[0983] Antibody concentration (nM) 27-F4 43-F3 43-F4 2-F3 2-F4 Aca-Mab 100 NA 1.3903 1.4031 1.8077 1.3578 0.768 25 1.0463 0.7209 1.0401 1.63 1.5745 0.4532 6.25 0.7315 0.4406 0.5267 1.1226 1.2698 0.3044 1.5625 0.4194 0.2266 0.2541 0.5479 0.5673 0.1901

[0984] Experiment 5: Antibodies were affinity-captured using a Protein A biosensor chip, and then human PSMA was passed through the chip surface. Binding and dissociation curves were obtained in real-time using a Biacore T200 instrument at 25°C. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated using Glycine 1.5. A 1:1 model was used for data fitting. Human PSMA antigen was purchased from Acro (catalog numbers PSA-H52H3; Lys44-Ala750); hCD3D & CD3E complex antigen was purchased from Sino Biological (catalog number CT038-H2508H). The binding and dissociation of each antibody are shown in the table below.

[0985] Table 19-5. Detection of the binding activity of bispecific antibodies to human PSMA using surface plasmon resonance method.

[0986]

[0987] The results showed that the anti-PSMA antibody and bispecific antibody disclosed herein had a good binding effect with PSMA, which was stronger than that of Aca-mab.

[0988] Test Example 3. In vitro cytotoxicity of the bispecific antibody disclosed herein

[0989] This test case investigated the cytotoxic activity of the disclosed bispecific antibody as a T-cell adaptor molecule against tumor cells. The target-specific cytotoxic activity of the disclosed bispecific antibody was detected using the PSMA-expressing cell line Lncap (human prostate cancer cells, metastatic) as the target cells. Fresh PBMCs (purchased from Xuanfeng Biotechnology Co., Ltd.) were centrifuged at 300g for 10 min, the supernatant was discarded, and the cells were resuspended in phenol red-free 1640 + 4% FBS. After centrifugation again, the cells were resuspended, counted, and the cell number was adjusted to 1.5 × 10⁻⁶ cells / mL. 6 Add 50 μL of the target cells per well, at a concentration of 1000 cells / mL. Collect the target cells, centrifuge at 1000 rpm for 3 min, resuspend, count, and adjust the cell count to 3 × 10⁶ cells / mL. 5 Cells were diluted with phenol red-free complete medium to a starting concentration of 400 nM (4× final concentration), and 10 8-fold dilutions were performed, with 25 μL added to each well. Cells were then incubated at 37°C in a 5% CO2 incubator for 48 h. Before detection, 10 μL of medium was aspirated from two wells containing only target cells, and 10 μL of Lysis Solution (10×) was added. After lysis for 45 min, the culture plate was removed, centrifuged at 1000 rpm for 3 min, and 50 μL of supernatant was transferred to a new 96-well plate (#3590). 50 μL of dissolved CytoTox was added at a 1:1 ratio. After incubating with Reagent at room temperature for 0.5 h, 50 μL of stop solution was added, and the absorbance (490 nm) was detected using FlexStation 3 (Molecular Devices).

[0990] Table 20. Cytotoxic activity of bispecific antibodies against Lncap

[0991]

[0992] The results showed that the bispecific antibody disclosed herein had a strong tumor-killing effect on Lncap cells expressing PSMA, EC 50 The value was much lower than that of the control antibody Aca-Mab.

[0993] Test Example 4. Cytokine Release Levels of the Bispecific Antibody Disclosed

[0994] CD3 T cell adaptor molecules can cause a cytokine storm. Therefore, when developing CD3 T cell adaptor molecules, it is necessary to keep cytokines, especially IL-6, which is unrelated to drug efficacy but can cause side effects, at low levels.

[0995] This test case investigated the release levels of cytokines IFNγ and IL6 of the disclosed bispecific antibody when killing the PSMA-expressing Lncap cell line. Fresh PBMCs (purchased from Xuanfeng Biotechnology Co., Ltd.) were centrifuged at 300g for 10 min, the supernatant was discarded, and the cells were resuspended in phenol red-free 1640 + 4% FBS. After centrifugation again, the cells were resuspended, counted, and the cell number was adjusted to 1.5 × 10⁻⁶ cells / year. 6 Add 50 μL of the target cells per well, at a concentration of 1000 cells / mL. Collect the target cells, centrifuge at 1000 rpm for 3 min, resuspend, count, and adjust the cell count to 3 × 10⁶ cells / mL. 5 Cells were diluted with 25 μL per well at an E:T ratio of 10:1. The antibody was diluted with phenol red-free complete medium to an initial concentration of 400 nM (4 × final concentration), and then diluted 8-fold (10 gradients) at 25 μL per well. Cells were then incubated at 37°C in a 5% CO2 incubator for 48 h.

[0996] IL6 was detected using an ELISA method. The cell samples were centrifuged at 1000 rpm for 3 min, and 50 μL of cell supernatant was collected and diluted 6-fold with a universal sample diluent. The IL6 standard was also diluted 6-fold. The diluted sample or different concentrations of standard (100 μL / well) were added to the detection plate. The wells were sealed with sealing tape and incubated at 37°C for 90 min. After washing 5 times, biotinylated antibody working solution (100 μL / well) was added, and the wells were sealed with fresh sealing tape. The plate was incubated at 37°C for 60 min, and then washed 5 times. Enzyme conjugate working solution (100 μL / well) was added, and the wells were sealed with fresh sealing tape. The plate was incubated at 37°C for 30 min, and then washed 5 times. 100 μL of chromogenic substrate (TMB) was added to each well, and the plate was incubated at 37°C in the dark for 8 min. 100 μL of stop solution was added to each well, and the mixture was stirred. The OD450 value was measured immediately (within 3 minutes).

[0997] IFNγ was detected using the HTRF method. The cell samples were centrifuged at 1000 rpm for 3 min, and 50 μL of cell supernatant was collected. The assay kit was equilibrated to room temperature, and the two detection antibodies in the kit were diluted 20-fold using detection buffer. 16 μL of the 20-fold diluted sample and IFNγ standard were added to a 384-well plate, along with 4 μL of the corresponding diluted detection antibody. The plate was sealed with sealant, vortexed to mix, centrifuged at 1000 rpm for 1 min, incubated overnight at room temperature, centrifuged again at 1000 rpm for 1 min, and the sealant was removed. The absorbance values ​​at 665 nm and 620 nm were read using a Pherastar multi-plate reader, and the data were processed and analyzed using Graphpad Prism 5.

[0998] Table 21-1. IFN-γ release in Lncap cytotoxicity assay

[0999]

[1000] Table 21-2. IL-6 release in Lncap cytotoxicity assay

[1001]

[1002] The results showed that the bispecific antibody disclosed herein released lower levels of IL6 and IFNγ than Aca-Mab, suggesting that the bispecific antibody disclosed herein has better safety.

[1003] Biological evaluation of in vivo activity

[1004] Test Example 5. Efficacy of the disclosed bispecific antibody in a 22RV1 subcutaneous xenograft model.

[1005] This disclosure uses a human prostate cancer 22RV1 cell-human PBMC NCG mouse xenograft tumor model to evaluate the antitumor activity of the PSMA-CD3 bispecific antibody.

[1006] Male NCG mice, 4-5 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[1007] Human prostate cancer 22RV1 cells were purchased from ATCC.

[1008] 22RV1 cells were added to RPMI-1640 medium containing 10% FBS and cultured in a humidified incubator at 37°C with 5% CO2. 22RV1 cells in the logarithmic growth phase were collected, resuspended in RPMI-1640 medium containing 50% Matrigel, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / cells. 7 / mL. Under aseptic conditions, 0.1 mL of cell suspension was injected subcutaneously into the right back of mice, at a concentration of 2 × 10⁹ / mL. 6 / 0.1mL / mouse. One day after tumor cell inoculation, under aseptic conditions, 0.1mL of human PBMC suspension was injected intraperitoneally into mice at a concentration of 8×10⁻⁶. 6 / 0.1mL / mouse. When the average tumor volume reaches 100mm... 3 At approximately 10:00 AM, animals were randomly divided into groups based on tumor volume, ensuring that the difference in tumor volume between groups was less than 10% of the mean. Grouping was recorded as Day 0, and medication was initiated based on animal body weight. Administered immediately after grouping, intraperitoneally twice weekly for six consecutive weeks (IP, BIW×3).

[1009] Tumor inhibition rate (%) = (1-(Ti-T0) / (Ci-C0))×100%, where Ti and Ci are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[1010] Table 22. Efficacy of bispecific antibodies in the 22RV1 subcutaneous xenograft model

[1011] Dosing group Dosing dose (mg / kg) Tumor volume (Day 21) mm 3 ]] Tumor inhibition rate % Blank (PBS) / 1977.87 / 31-F1 0.75 415.94 83.18 31-F1 3 464.92 80.57 43-F4 1 345.77 86.91 43-F4 4 181.52 95.66

[1012] Experimental results showed that the disclosed bispecific antibodies 31-F1 and 43-F4 exhibited good efficacy under the specified reagent dosage conditions. No drug-related animal deaths or other significant drug-related adverse reactions were observed during the experiment.

[1013] Test Example 6. Efficacy of the disclosed bispecific antibody in a C4-2 subcutaneous xenograft model.

[1014] This disclosure uses a human prostate cancer C4-2 cell-mediated human PBMC NCG mouse xenograft tumor model to evaluate the antitumor activity of PSMA-CD3 bispecific antibody.

[1015] Male NCG mice, 7-8 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[1016] Human prostate cancer C4-2 cells, purchased from ATCC.

[1017] C4-2 cells were cultured in DMEM / Ham's F12K (4:1) medium containing 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO2. Logarithmic growth phase C4-2 cells were collected, resuspended in RPMI-1640 medium containing 50% Matrigel, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / year. 7 / mL. Under aseptic conditions, 0.1 mL of cell suspension was injected subcutaneously into the right back of mice, at a concentration of 2 × 10⁹ / mL. 6 / 0.1mL / mouse. On the 3rd day after tumor cell inoculation, under aseptic conditions, 0.1mL of human PBMC suspension was injected intraperitoneally into mice, at a concentration of 1×10⁻⁶. 7 / 0.1mL / mouse. On day 12 post-inoculation of tumor cells (mean tumor volume reached 51.60 mm), 3 Animals were randomly divided into groups based on tumor volume (approximately 10% of the mean), and administration was initiated according to the animals' body weight. Administration was administered immediately after grouping, twice weekly via intraperitoneal injection, for a total of four administrations (IP, BIW×2).

[1018] Table 23. Efficacy of bispecific antibodies in the C4-2 subcutaneous xenograft model (day 10 after administration)

[1019] Dosing group Dosing dose (mg / kg) Tumor volume (mm3 3 )]]> Tumor inhibition rate % Blank (PBS) / 1882.65 / Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate % Blank (PBS) Aca-Mab NA Biological evaluation of in vivo activity Dosing group Dosing dose (mg / kg) Tumor inhibition rate 1 363.89 82.96 15-F5-1 1 111.27 96.74 15-F5-1 0.2 112.05 96.69 15-F5-2 0.2 170.32 93.50

[1020] Experimental results showed that the bispecific antibodies 15-F5-1 and 15-F5-2 had stronger tumor-suppressive effects than Aca-Mab in the C4-2 model; even low doses of bispecific antibodies could achieve significantly stronger tumor-suppressive effects than high doses of Aca-Mab.

Claims

1. An antigen-binding molecule that specifically binds to PSMA and CD3; comprising: One or two antigen-binding domains that specifically bind to PSMA, and One or two antigen-binding domains that specifically bind to CD3; The antigen-binding domain that specifically binds to PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL. The antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL; wherein (i) The PSMA-VH comprises: PSMA-HCDR1 with the amino acid sequence shown in SEQ ID NO: 13, PSMA-HCDR2 with the amino acid sequence shown in SEQ ID NO: 14, and PSMA-HCDR3 with the amino acid sequence shown in SEQ ID NO: 15, and The PSMA-VL has the following amino acid sequences: PSMA-LCDR1 with the amino acid sequence shown in SEQ ID NO: 16, PSMA-LCDR2 with the amino acid sequence shown in SEQ ID NO: 17, and PSMA-LCDR3 with the amino acid sequence shown in SEQ ID NO:

18.

2. The antigen-binding molecule according to claim 1, wherein: (i) The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 112, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 115, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 110, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 77, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 78, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 107, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 108, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 109, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 110, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 111, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 107, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 108, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 109, and the amino acid sequence of PSMA-VL is shown 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.

3. The antigen-binding molecule according to claim 2, wherein: (i) The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 112, and the amino acid sequence of the PSMA-VL is shown 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.

4. The antigen-binding molecule according to claim 1, wherein... The CD3-VH has the following amino acid sequences: CD3-HCDR1 (SEQ ID NO: 160), CD3-HCDR2 (SEQ ID NO: 161), and CD3-HCDR3 (SEQ ID NO: 162); and the CD3-VL has the following amino acid sequences: CD3-LCDR1 (SEQ ID NO: 163), CD3-LCDR2 (SEQ ID NO: 164), and CD3-LCDR3 (SEQ ID NO: 165).

5. The antigen-binding molecule according to claim 4, wherein... The amino acid sequence of CD3-VH is shown in SEQ ID NO: 166, and the amino acid sequence of CD3-VL is shown in SEQ ID NO:

167.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises an Fc region, and the Fc region is an IgG Fc region.

7. The antigen-binding molecule according to claim 6, wherein the Fc region is the IgG1 Fc region.

8. The antigen-binding molecule of claim 7, wherein the Fc region comprises one or more amino acid substitutions capable of reducing the binding of the Fc region to the Fcγ receptor.

9. The antigen-binding molecule according to claim 8, wherein the Fc region is the human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index.

10. The antigen-binding molecule according to any one of claims 1 to 5, wherein 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, wherein Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region.

11. The antigen-binding molecule according to claim 10, wherein Fc1 has a protruding structure according to the mortar and pestle technique, and Fc2 has a porous structure according to the mortar and pestle technique; or The Fc2 has a protruding structure according to the pestle and mortar technique, and the Fc1 has a hole structure according to the pestle and mortar technique.

12. The antigen-binding molecule according to claim 11, 1) The amino acid residue W at position 366 of Fc1 is... The amino acid residue at position 366 of Fc2 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 is V, numbered according to the EU index; or 2) The amino acid residue W at position 366 of Fc2; and The amino acid residue at position 366 of Fc1 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 is V, and the numbering is based on the EU index.

13. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises: An antigen-binding domain that specifically binds to PSMA, and An antigen-binding domain that specifically binds to CD3; The antigen-binding domain that specifically binds to PSMA and the antigen-binding domain that specifically binds to CD3 are scFv; The antigen-binding molecule comprises: A first chain having the structure shown in equation (a), and A second chain having the structure shown in equation (b); Equation (a) [PSMA-VH]-[Connector 1]-[PSMA-VL]-[Connector 2]-[CD3-VH]-[Connector 3]-[CD3-VL]-[Connector 4]-[Fc2], Equation (b) [Fc1], The structures shown in formulas (a) and (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.

14. The antigen-binding molecule according to claim 13, wherein the antigen-binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 192 and a second chain with an amino acid sequence as shown in SEQ ID NO: 169; or The antigen-binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 191 and a second chain with an amino acid sequence as shown in SEQ ID NO:

169.

15. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding domain specifically binding PSMA or the antigen-binding domain specifically binding CD3 each independently comprises a titin chain and an obscurin chain. The amino acid sequence of the titin chain is shown in SEQ ID NO: 226, and the amino acid sequence of the obscurin chain is shown in SEQ ID NO:

264.

16. The antigen-binding molecule of claim 15, wherein the antigen-binding molecule comprises two antigen-binding domains that specifically bind PSMA and one antigen-binding domain that specifically binds CD3. The antigen-binding domain that specifically binds to PSMA is Fab; the antigen-binding domain that specifically binds to CD3 is a substituted Fab containing a Titin chain and an Obscurin chain. The antigen-binding molecule comprises a first strand having the structure shown in formula (c), two second strands having the structure shown in formula (d), a third strand having the structure shown in formula (g), and a fourth strand having the structure shown in formula (h). Equation (c) [PSMA-VH]-[CH1]-[Fc1], Equation (d) [PSMA-VL]-[CL] Formula (g) [PSMA-VH]-[CH1]-[CD3-VH]-[connector 7]-[Titin chain]-[Fc2], Formula (h) [CD3-VL]-[connector 8]-[Obscurin chain], The structures shown in 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.

17. The antigen-binding molecule according to claim 16, The antigen-binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO: 187, a second chain with two amino acid sequences as shown in SEQ ID NO: 188, a third chain with an amino acid sequence as shown in SEQ ID NO: 189, and a fourth chain with an amino acid sequence as shown in SEQ ID NO:

174.

18. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises two antigen-binding domains that specifically bind PSMA and two antigen-binding domains that specifically bind CD3, wherein the antigen-binding domains that specifically bind PSMA are Fab and the antigen-binding domains that specifically bind CD3 are scFv. The antigen-binding molecule comprises two first chains having the structure shown in formula (k) and two second chains having the structure shown in formula (d). Equation (k) [PSMA-VH]-[CH1]-[CD3-VH]-[connector 10]-[CD3-VL]-[connector 11]-[a subunit of Fc], Equation (d) [PSMA-VL]-[CL] The structures shown in formulas (k) and (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.

19. The antigen-binding molecule according to claim 18, The antigen-binding molecule has: a first chain with two amino acid sequences as shown in SEQ ID NO: 190 and a second chain with two amino acid sequences as shown in SEQ ID NO:

188.

20. An 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) The PSMA-VH has: PSMA-HCDR1 with the amino acid sequence shown in SEQ ID NO: 13, PSMA-HCDR2 with the amino acid sequence shown in SEQ ID NO: 14, and PSMA-HCDR3 with the amino acid sequence shown in SEQ ID NO: 15, and the PSMA-VL has: PSMA-LCDR1 with the amino acid sequence shown in SEQ ID NO: 16, PSMA-LCDR2 with the amino acid sequence shown in SEQ ID NO: 17, and PSMA-LCDR3 with the amino acid sequence shown in SEQ ID NO:

18.

21. The antibody according to claim 20, wherein... (i) The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 112, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 115, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 110, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 77, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 78, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 107, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 108, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 109, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 110, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 111, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 113, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 107, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 108, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 114, or The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 109, and the amino acid sequence of PSMA-VL is shown 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.

22. The antibody according to claim 21, (i) The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 112, and the amino acid sequence of the PSMA-VL is shown 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.

23. The antibody according to any one of claims 20-22, wherein the antibody is a bispecific antibody; the bispecific antibody specifically binds to PSMA and CD3.

24. A pharmaceutical composition comprising: A therapeutically effective amount of the antigen-binding molecule as described in any one of claims 1 to 19 or the isolated antibody as described in any one of claims 20 to 23, and One or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition further comprises at least one second therapeutic agent.

26. The pharmaceutical composition of claim 25, wherein the second therapeutic agent is an antibody capable of specifically binding to CD28.

27. An isolated nucleic acid encoding an antigen-binding molecule as claimed in any one of claims 1 to 19 or an isolated antibody as claimed in any one of claims 20 to 23.

28. A host cell comprising the isolated nucleic acid as described in claim 27.

29. The use of the antigen-binding molecule according to any one of claims 1 to 19, or the isolated antibody according to any one of claims 20 to 23, or the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating a disease, wherein the disease is prostate cancer.

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