Anti-CD3l1 antibody-drug conjugate and use thereof in preparation of Anti-tumor drug

Abstract

A conjugate (ADC) of a CD3L1 humanized antibody and an anti-tumor small-molecule drug, and the use of the conjugate in the preparation of an anti-tumor drug. The antibody-drug conjugate has an antigen epitope affinity equivalent to that of the CD3L1 humanized antibody, has endocytosis activity and the ability to release the drug in lysosomes, and has a tumor inhibition effect.

Description

Anti-CD3L1 antibody-drug conjugates and their use in the preparation of antitumor drugs Technical Field

[0001] This invention relates to the field of biomedicine, specifically to anti-CD3L1 antibody-drug conjugates, their preparation methods, and their use in the preparation of antitumor drugs. Background Technology

[0002] Inositol 1,4,5-triphosphate receptor-interacting protein-like 1 (ITPRIPL1) is a single-transmembrane protein normally expressed primarily in testicular tissue. Previous studies have shown that ITPRIPL1 is highly expressed on the surface of various tumor cells and, as a natural ligand of CD3ε (CD3ε ligand 1, CD3L1), binds to CD3ε on the surface of T cells, inhibiting calcium ion influx and downstream ZAP70 phosphorylation, thus hindering T cell activation and killing function and promoting tumor immune escape. Antibodies targeting CD3L1 can effectively inhibit the growth of various tumor types in vivo and can serve as excellent targets for tumor therapy.

[0003] Antibody-drug conjugates (ADCs) are complexes formed by conjugating monoclonal antibodies targeting specific antigens to varying amounts and types of cytotoxic drugs (payloads) via linkers. ADCs combine the advantages of antibody targeting with the highly efficient killing activity of cytotoxic drugs. By recognizing tumor-specific antigens, antibodies deliver cytotoxic drugs to tumor cells, achieving rapid and precise killing. ADCs have already achieved several breakthroughs in the field of cancer treatment.

[0004] ADC drug design is influenced by various factors, including target specificity and endocytosis, drug-linked polymerase chain stability, and cytotoxic drug killing efficiency. Given the crucial role of CD3L1 in tumorigenesis and development, its rarity in normal tissues, and the rapid endocytosis of cells after binding to antibodies, CD3L1 possesses the characteristics of an excellent target for ADC drugs. However, no ADC drugs targeting CD3L1 have been reported to date. Summary of the Invention

[0005] This invention relates to an antibody-drug conjugate that specifically targets CD3L1. It has the characteristics of specifically targeting CD3L1, efficiently internalizing into tumor cells, releasing cytotoxic drugs in lysosomes, rapidly killing tumor cells, inhibiting tumor growth and promoting tumor cell apoptosis, and has antitumor activity that is significantly superior to positive control drugs against HER2-ADC.

[0006] On the one hand, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU) n

[0007] Wherein: A is an antibody that specifically binds to CD3L1 or its antigen-binding fragment; U is a therapeutic agent, and each U may be the same or different; L is a linker, and each L may be the same or different; n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0008] In some embodiments, the U is selected from the group consisting of cytotoxic molecules, immune enhancers, immunosuppressants, cell inhibitors, chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic agents, anti-metastatic agents, targeted anticancer agents, other anticancer agents, and radioisotopes.

[0009] In some embodiments, the U is selected from microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, DNA alkylating agents, RNA polymerase inhibitors, RNA cleavage enzyme inhibitors, Bcl-xL inhibitors, antimitotic agents, sodium-potassium ion pump ATPase inhibitors, matrix metalloproteinase inhibitors, and / or immune agonists.

[0010] In some embodiments, the microtubule inhibitor is selected from saurustoxin and olritastatin cytotoxic molecules (e.g., MMAE, MMAF), and / or maytansine cytotoxic molecules (e.g., DM1, DM4).

[0011] In some embodiments, the DNA damaging agent includes those selected from chachiin, pyromycin, atrazomycin derivatives (e.g., PBD), and / or camptothecin and camptothecin derivatives (e.g., SN-38, Dxd).

[0012] In some implementations, the U is selected from DXd and MMAE.

[0013] In some embodiments, the linker is covalently linked to an amino or thiol residue on the antibody or its antigen-binding fragment.

[0014] In some implementations, the connectors include shardable connectors and non-shardable connectors.

[0015] In some embodiments, the cleavable linker comprises a peptide unit containing 2-20 amino acids. In some preferred embodiments, the peptide unit is selected from any one or a combination of multiples of -valine-citrulline-, -glycine-glycine-phenylalanine-glycine-, -valine-alanine-, -valine-lysine-, -valine-arginine-, -phenylalanine-citrulline-, -phenylalanine-lysine-, and -phenylalanine-arginine-.

[0016] In some embodiments, the cleavable linker comprises a structure selected from maleimide-hexanoyl (MC), maleimide-butyric acid (MB), or p-aminobenzyloxycarbonyl (PAB).

[0017] In some embodiments, the cleavable linker is selected from maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleimide-hexanoyl-valine-alanine (MC-VA), maleimide-butyric acid-valine-citrulline (MB-VC), glycine-glycine-phenylalanine-glycine (GGFG), maleimide-hexanoyl-glycine-glycine-phenylalanine-glycine (MC-GGFG), or valine-citrulline-p-aminobenzyloxycarbonyl (VC-PAB).

[0018] In some embodiments, the cleavable linker is a linker that can cleave under acidic conditions. In some preferred embodiments, the cleavable linker is a linker that can cleave in endosomes or lysosomes.

[0019] In some embodiments, the antibody that specifically binds to CD3L1 or its antigen-binding fragment is capable of binding to the amino acid sequence of CD3L1, such as SEQ ID NO:1 or SEQ ID NO:2.

[0020] In some embodiments, the antibody that specifically binds to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13. In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14.

[0021] In some embodiments, the antibody that specifically binds to CD3L1 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region including HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains an amino acid sequence as shown in SEQ ID NO:5, HCDR2 contains an amino acid sequence as shown in SEQ ID NO:6 and HCDR3 contains an amino acid sequence as shown in SEQ ID NO:7.

[0022] In some embodiments, the antibody that specifically binds to CD3L1 includes a heavy chain variable region and a light chain variable region, the light chain variable region including LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 contains an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16.

[0023] In some embodiments, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:7.

[0024] In some embodiments, the amino acid sequence of LCDR1 is shown in SEQ ID NO:8 or SEQ ID NO:15, the amino acid sequence of LCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:10 or SEQ ID NO:16.

[0025] In some embodiments, the antibody that specifically binds to CD3L1 comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, HCDR3 as shown in SEQ ID NO:7, LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:10.

[0026] In some embodiments, the antibody that specifically binds to CD3L1 comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, HCDR3 as shown in SEQ ID NO:7, LCDR1 as shown in SEQ ID NO:15, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:16.

[0027] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences.

[0028] In some embodiments, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences.

[0029] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4.

[0030] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:11, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:12.

[0031] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14.

[0032] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:17, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:18.

[0033] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:20.

[0034] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:21, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:22.

[0035] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:23, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:24.

[0036] In some embodiments, the antibody that specifically binds to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45. In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, and SEQ ID NO:46.

[0037] In some embodiments, the antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48, and HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49.

[0038] In some embodiments, the antibody that specifically binds to CD3L1 includes a heavy chain variable region and a light chain variable region, the light chain variable region including LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 contains an amino acid sequence as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52.

[0039] In some embodiments, the amino acid sequence of HCDR1 is shown as SEQ ID NO:27, SEQ ID NO:40 or SEQ ID NO:47, the amino acid sequence of HCDR2 is shown as SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41 or SEQ ID NO:48, and the amino acid sequence of HCDR3 is shown as SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42 or SEQ ID NO:49.

[0040] In some embodiments, the amino acid sequence of LCDR1 is shown as SEQ ID NO:30, SEQ ID NO:43 or SEQ ID NO:50, the amino acid sequence of LCDR2 is shown as SEQ ID NO:31, SEQ ID NO:9 or SEQ ID NO:51, and the amino acid sequence of LCDR3 is shown as SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44 or SEQ ID NO:52.

[0041] In some embodiments, the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO:29, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:32.

[0042] In some embodiments, the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:35, HCDR3 as shown in SEQ ID NO:36, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:37.

[0043] In some embodiments, the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:40, HDCR2 as shown in SEQ ID NO:41, HCDR3 as shown in SEQ ID NO:42, LCDR1 as shown in SEQ ID NO:43, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:44.

[0044] In some embodiments, the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:47, HDCR2 as shown in SEQ ID NO:48, HCDR3 as shown in SEQ ID NO:49, LCDR1 as shown in SEQ ID NO:50, LCDR2 as shown in SEQ ID NO:51, and LCDR3 as shown in SEQ ID NO:52.

[0045] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:45, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences.

[0046] In some embodiments, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences.

[0047] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:25, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26.

[0048] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:33, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:34.

[0049] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:39.

[0050] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:45, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:46.

[0051] In some embodiments, the antibody that specifically binds to CD3L1 is a monoclonal antibody.

[0052] In some embodiments, the antibody that specifically binds to CD3L1 is a chimeric antibody or a humanized antibody.

[0053] In some embodiments, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0054] A is an antibody or its antigen-binding fragment that specifically binds to CD3L1, wherein,

[0055] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or

[0056] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:7; and / or

[0057] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:16; and / or

[0058] The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to the amino acid sequence shown therein; and / or

[0059] The light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to these sequences.

[0060] The U is a therapeutic agent, and each U may be the same or different, and it is selected from MMAE or DXd;

[0061] The L is a connector, and each L can be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and

[0062] n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0063] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0064] in:

[0065] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:19 ... The amino acid sequence shown in SEQ ID NO:23 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it.The amino acid sequence is 9%; U is a therapeutic agent, and it is MMAE; L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0066] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0067] in:

[0068] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:19 ... The amino acid sequence shown in SEQ ID NO:23 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it.The amino acid sequence is 9%; U is a therapeutic agent, and it is DXd; L is a linker, and it is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0069] In some embodiments, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0070] in:

[0071] A is an antibody or its antigen-binding fragment that specifically binds to CD3L1, wherein,

[0072] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or

[0073] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 with the amino acid sequence shown in SEQ ID NO:5, HCDR2 with the amino acid sequence shown in SEQ ID NO:6, and HCDR3 with the amino acid sequence shown in SEQ ID NO:7; and / or

[0074] The antibody that specifically binds to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 with an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16; and / or

[0075] The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to the amino acid sequence shown therein; and / or

[0076] The light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to these sequences.

[0077] The U is a therapeutic agent, and each U may be the same or different, and it is selected from MMAE or DXd;

[0078] The L is a connector, and each L can be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and

[0079] n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0080] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0081] in:

[0082] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:5. HCDR3 as shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1 with an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 with an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; and / or, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:7; The amino acid sequences shown in NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, or amino acid sequences with an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% compared to them; the U is a therapeutic agent, and it is MMAE; the L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0083] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0084] in:

[0085] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:5. HCDR3 as shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1 with an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 with an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; and / or, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:7; The amino acid sequences shown in NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, or amino acid sequences with an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% compared to them; the U is a therapeutic agent and is DXd; the L is a linker and is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0086] In some embodiments, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0087] in:

[0088] A is an antibody or its antigen-binding fragment that specifically binds to CD3L1, wherein,

[0089] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or

[0090] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or

[0091] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or

[0092] The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:45, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to the amino acid sequence shown therein; and / or

[0093] The light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to them;

[0094] The U is a therapeutic agent, and each U may be the same or different, and it is selected from MMAE or DXd;

[0095] The L is a connector, and each L can be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and

[0096] n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0097] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0098] in:

[0099] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28, SEQ ID NO:45, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:45, SEQ ID NO:39, SEQ ID NO:46 ... The amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48, and the HCDR3 contains an amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the light chain variable region including LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 contains an amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 contains an amino acid sequence shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region contains an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the heavy chain variable region contains an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49 ...36, SEQ ID NO:49, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49, SEQ ID The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.9% of the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence selected from, as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; the U is a therapeutic agent, and it is MMAE; the L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0100] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0101] in:

[0102] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:46. The amino acid sequence shown in NO:48, and the HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49 ... The amino acid sequence shown in NO:45 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it.The amino acid sequence is 9%; U is a therapeutic agent, and it is DXd; L is a linker, and it is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0103] In some embodiments, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0104] in:

[0105] A is an antibody or its antigen-binding fragment that specifically binds to CD3L1, wherein,

[0106] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or

[0107] The antibody or its antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47; HCDR2 with an amino acid sequence as shown in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48; and HCDR3 with an amino acid sequence as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or

[0108] The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:43 or SEQ ID NO:50, LCDR2 with an amino acid sequence as shown in SEQ ID NO:31, SEQ ID NO:9 or SEQ ID NO:51, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44 or SEQ ID NO:52; and / or

[0109] The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:45, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to the amino acid sequence shown therein; and / or

[0110] The light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to them;

[0111] The U is a therapeutic agent, and each U may be the same or different, and it is selected from MMAE or DXd;

[0112] The L is a connector, and each L can be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and

[0113] n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0114] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0115] in:

[0116] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in the amino acid sequence SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR1 in the amino acid sequence SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:46. HCDR2 as shown in NO:48, and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with amino acid sequences as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 with amino acid sequences as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 with amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region comprises HCDR3 selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49 ... The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.9% of the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence selected from, as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; the U is a therapeutic agent, and it is MMAE; the L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0117] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0118] in:

[0119] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR1 in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:46. HCDR2 as shown in NO:48, and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with amino acid sequences as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 with amino acid sequences as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 with amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region comprises HCDR3 selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49 ... The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.9% of the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; the U is a therapeutic agent, and it is DXd; the L is a linker, and it is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0120] In some embodiments, the antibody-drug conjugate can be efficiently internalized into target cells and release the therapeutic agent within the cells.

[0121] On the other hand, this disclosure provides a method for preparing the above-mentioned antibody-drug conjugate, wherein the method is capable of linking the antibody or its antigen-binding fragment to the therapeutic agent and achieving a stable drug / antibody ratio.

[0122] In another aspect, this disclosure also provides a pharmaceutical composition having the aforementioned antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0123] On the other hand, this disclosure also provides the use of the above-mentioned antibody-drug conjugates, or antibody-drug conjugates or pharmaceutical compositions prepared by the aforementioned methods, in the preparation of antitumor drugs.

[0124] In some preferred embodiments, the tumor is a tumor that has not responded well to treatment with antibody-drug conjugates targeting HER2.

[0125] On the other hand, this disclosure also provides a kit comprising a container, a formulation placed in the container, and optional instructions; wherein the formulation comprises the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition.

[0126] On the other hand, this disclosure also provides the use of the aforementioned antibody-drug conjugates, or antibody-drug conjugates or pharmaceutical compositions prepared by the aforementioned methods, in the preparation of products for the treatment or prevention of tumors.

[0127] In some embodiments, the tumor is a solid tumor or hematologic malignancy that expresses CD3L1.

[0128] In some embodiments, the tumor is a CD3L1-positive tumor, including melanoma, sarcoma, lung cancer, breast cancer, thyroid cancer, nervous system tumors, kidney cancer, urothelial carcinoma, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, head and neck cancer, leukemia, lymphoma, and other CD3L1-positive associated tumors.

[0129] In another aspect, this disclosure also provides the use of the aforementioned antibody-drug conjugates, or antibody-drug conjugates prepared by the aforementioned methods, or the aforementioned pharmaceutical compositions in the preparation of medicaments for treating diseases related to CD3L1 expression.

[0130] Furthermore, this disclosure also provides an antibody or antigen-binding fragment thereof suitable for antibody-drug conjugates specifically targeting CD3L1, i.e., an antibody or antigen-binding fragment thereof specifically binding to CD3L1, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46.

[0131] In another aspect, this disclosure also provides an antibody or antigen-binding fragment thereof suitable for antibody-drug conjugates specifically targeting CD3L1, i.e., an antibody or antigen-binding fragment thereof specifically binding to CD3L1, wherein the antibody specifically binding to CD3L1 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:27, SEQ ID NO:40 or SEQ ID NO:47, wherein HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41 or SEQ ID NO:48, and wherein HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42 or SEQ ID NO:49.

[0132] In another aspect, this disclosure also provides an antibody or antigen-binding fragment thereof suitable for antibody-drug conjugates specifically targeting CD3L1, i.e., an antibody or antigen-binding fragment thereof specifically binding to CD3L1, wherein the antibody specifically binding to CD3L1 comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:43 or SEQ ID NO:50, wherein LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:31, SEQ ID NO:9 or SEQ ID NO:51, and wherein LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44 or SEQ ID NO:52.

[0133] In some embodiments, the amino acid sequence of HCDR1 is shown as SEQ ID NO:27, SEQ ID NO:40 or SEQ ID NO:47, the amino acid sequence of HCDR2 is shown as SEQ ID NO:28, SEQ ID NO:35 or SEQ ID NO:41 or SEQ ID NO:48, and the amino acid sequence of HCDR3 is shown as SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42 or SEQ ID NO:49.

[0134] In some embodiments, the amino acid sequence of LCDR1 is shown as SEQ ID NO:30, SEQ ID NO:43 or SEQ ID NO:50, the amino acid sequence of LCDR2 is shown as SEQ ID NO:31, SEQ ID NO:9 or SEQ ID NO:51, and the amino acid sequence of LCDR3 is shown as SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44 or SEQ ID NO:52.

[0135] In some embodiments, the present disclosure discloses an antibody that specifically binds to CD3L1 or an antigen-binding fragment thereof, wherein the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO:29, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:32.

[0136] In some embodiments, the present disclosure discloses an antibody that specifically binds to CD3L1 or an antigen-binding fragment thereof, wherein the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:35, HCDR3 as shown in SEQ ID NO:36, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:37.

[0137] In some embodiments, the present disclosure discloses an antibody that specifically binds to CD3L1 or an antigen-binding fragment thereof, wherein the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:40, HDCR2 as shown in SEQ ID NO:41, HCDR3 as shown in SEQ ID NO:42, LCDR1 as shown in SEQ ID NO:43, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:44.

[0138] In some embodiments, the present disclosure discloses an antibody that specifically binds to CD3L1 or an antigen-binding fragment thereof, wherein the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:47, HDCR2 as shown in SEQ ID NO:48, HCDR3 as shown in SEQ ID NO:49, LCDR1 as shown in SEQ ID NO:50, LCDR2 as shown in SEQ ID NO:51, and LCDR3 as shown in SEQ ID NO:52.

[0139] In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:45, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences.

[0140] In some embodiments, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences.

[0141] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:25, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26.

[0142] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:33, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:34.

[0143] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:39.

[0144] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:45, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:46.

[0145] In some embodiments, the antibody that specifically binds to CD3L1 is a monoclonal antibody.

[0146] In some embodiments, the antibody that specifically binds to CD3L1 is a chimeric antibody or a humanized antibody.

[0147] In some embodiments, the antibody that specifically binds to CD3L1 or its antigen-binding fragment is capable of binding to the amino acid sequence of CD3L1, such as SEQ ID NO:1 or SEQ ID NO:2. Beneficial effects

[0148] This disclosure provides an antibody-drug conjugate targeting CD3L1. The antibody-drug conjugate possesses the characteristics of recognizing tumor cells, binding to tumor cells, efficiently internalizing into tumor cells, and releasing the drug in lysosomes. It reliably transfers antitumor small molecule drugs into tumor cells and exerts tumor-killing activity, enhancing the antitumor effect of CD3L1 antibodies while reducing the dosage of the antitumor small molecule drug compared to administering the small molecule compound alone. This mitigates the effects of the small molecule drug on normal cells, expands the therapeutic window, and achieves higher safety. This disclosure innovatively links an antitumor small molecule drug to a humanized CD3L1 antibody via a linker structure to form an antibody-drug conjugate, achieving excellent antitumor efficacy and safety. Attached Figure Description

[0149] This disclosure can be more fully understood with reference to the following figures.

[0150] Figure 1 shows the drug distribution and metabolism of the humanized CD3L1 antibody 5H conjugated with Cy7 fluorescence in 6-week-old male tumor-bearing nude mice at different time points (24h, 48h, 72h, 96h) after injection.

[0151] Figure 2 shows the drug / antibody ratio and aggregation rate analysis for 5H-MMAE, anti-HER2-MMAE, and control IgG1-MMAE. Figure 2(A) shows the drug / antibody ratio analysis by hydrophobic interaction chromatography (HIC); Figure 2(B) shows the aggregation rate analysis by size exclusion chromatography (SEC).

[0152] Figure 3 shows the affinity curves of CD3L1 humanized antibody 5H and its drug conjugate 5H-MMAE with the CD3L1 antigen, determined by ELISA and flow cytometry. Figure 3(A) shows the binding of 5H and 5H-MMAE to the CD3L1-his recombinant protein detected by ELISA; Figure 3(B) shows the binding of 5H and 5H-MMAE to CD3L1-positive cells RD detected by flow cytometry.

[0153] Figure 4 shows the expression level of CD3L1 in different tumor cells detected by flow cytometry. The fluorescence intensity of 5H-MMAE staining in different tumor cells is compared with that of control IgG1-MMAE staining; the horizontal axis represents different cells, and the vertical axis represents the geometric mean fluorescence intensity.

[0154] Figure 5 shows the endocytosis effect of CD3L1 humanized antibody 5H coupled with pH-sensitive dye and control IgG1 antibody in RD cells as detected by confocal fluorescence microscopy.

[0155] Figure 6 shows the colocalization of the CD3L1 humanized antibody-drug conjugate 5H-MMAE with RD cell endosomes and lysosomes at different time points (0, 10, 30, 60, 120, 240, 480 min) as detected by confocal fluorescence microscopy. Blue fluorescence indicates the cell nucleus, green fluorescence indicates 5H-MMAE, and red fluorescence indicates labeled antibodies for different organelles.

[0156] Figure 7 shows the endocytosis efficiency of CD3L1 humanized antibody-drug conjugates 5H-MMAE and anti-HER2-MMAE detected by flow cytometry. The horizontal axis represents time, and the vertical axis represents the geometric mean fluorescence intensity. Figure 7(A) shows the endocytosis efficiency of 5H-MMAE in RD cells, and Figure 7(B) shows the endocytosis efficiency of anti-HER2-MMAE in A375 cells.

[0157] Figure 8 shows the cytotoxicity of control IgG1-MMAE, humanized CD3L1 antibody 5H and its drug conjugate 5H-MMAE against tumor cells with different CD3L1 expression levels.

[0158] Figure 9 shows the cytotoxicity assay comparing the killing effects of different clone numbers of CD3L1 antibodies and HER2 antibodies conjugates with MMAE on different types of tumor cells in vitro. The table shows the corresponding IC50 values. 50 value.

[0159] Figure 10 shows a comparison of the killing efficiency of CD3L1 humanized antibody-drug conjugate 5H-DXd and anti-HER2-DXd against different tumor cells in a cytotoxicity assay.

[0160] Figure 11 compares the cytotoxicity of 5H-DXd and anti-HER2-DXd against different types of tumor cells using a cytotoxicity assay. The table shows the corresponding IC50 values. 50 value.

[0161] Figure 12 shows the cytotoxicity assay comparing the killing effects of different clone numbers of CD3L1 antibody and HER2 antibody conjugate with DXd on different tumor cells. The table shows the corresponding IC50 values.

[0162] Figure 13 compares the cytotoxic effects of 5H-DM1 and anti-HER2-DM1 on different types of tumor cells using a cytotoxicity assay. The table shows the corresponding IC50 values. 50 value.

[0163] Figure 14 shows the cytotoxicity assay comparing the killing effects of different clone numbers of CD3L1 antibody and HER2 antibody conjugate with DM1 on different tumor cell lines. The table shows the corresponding IC50 values.

[0164] Figure 15 shows a comparison of the efficacy of equal doses of control IgG1-MMAE, CD3L1 humanized antibody 5H, and its drug conjugate 5H-MMAE in different nude mouse xenograft models. Figure 15(A) is the RD xenograft model, Figure 15(B) is the RKO xenograft model, and Figure 15(C) is the A375 xenograft model.

[0165] Figure 16 shows a comparison of the efficacy of equal doses of anti-HER2-DXd and anti-CD3L1-DXd in a nude mouse A375 xenograft model.

[0166] Figure 17 shows the efficacy comparison results of equal doses of control Isotype mAb-DXd, anti-HER2-DXd, anti-CD3L1-DXd, and PBS solvent control in different nude mouse xenograft models. Figure 17(A) shows the LoVo xenograft model, Figure 17(B) shows the SW480 xenograft model, and Figure 17(C) shows the SW1116 xenograft model.

[0167] Figure 18 shows the immunohistochemical staining of cleaved-caspase 3 in tumor tissue samples treated with CD3L1 humanized antibody 5H and its drug conjugate 5H-MMAE. Figure 18(A) is a schematic diagram of cleaved-caspase 3 positive cell staining in the two groups of samples, and Figure 18(B) is a statistical graph of the percentage of cleaved-caspase 3 positive cells in the two groups of samples. Detailed Implementation

[0168] While the present invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that verify the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all materials and reagents used in the examples of this invention are commercially available products.

[0169] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0170] The terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as including at least a certain number of embodiments, this should also be understood as disclosing a group that preferably consists only of those embodiments.

[0171] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0172] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described in this invention can be implemented in a different order than that described or illustrated in this invention.

[0173] The term “and / or” is considered as a specific disclosure of each of the two specified features or components having or not having the other. Thus, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0174] The terms “for example” and “that is” are used only as examples and are not intended to be limiting, and should not be interpreted as referring only to those items explicitly listed in the specification.

[0175] Terms such as "or more," "at least," and "more than," for example, "at least one," should be understood to include, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more of the stated values. This also includes any larger numbers or fractions in between.

[0176] Conversely, the term "not exceeding" includes every value less than the stated value. For example, "not exceeding 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 5 4, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also includes any smaller numbers or fractions in between.

[0177] The terms "multiple," "at least two," "two or more," and "at least the second" should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also includes any larger numbers or fractions in between.

[0178] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. The abbreviations for amino acid residues are the standard three-letter or single-letter symbols used in the art to refer to the 20 commonly used L-amino acids.

[0179] In this specification and claims, the singular form includes the plural form of the object it refers to, unless it is clearly and explicitly limited to one object. The term "or" may be used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0180] In this article, the terms “CD3L1” and “ITPRIPL1” are used interchangeably, both referring to a ligand of CD3, which is a very important target in fields such as tumor therapy. For related research, please refer to, for example, the paper (Deng, Shouyan et al., ITPRIPL1 binds CD3ε to impede T cell activation and enable tumor immune evasion, Cell, Volume 187, Issue 9, 2305-2323.e33).

[0181] As used herein, the term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. Typically, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0182] The light chain and heavy chain variable regions each contain a "framework" region interspersed with three hypervariable regions (also known as "complementarity-determining regions" or "CDRs"). A "complementarity-determining region" or "CDR" or "hypervariable region" (which can be used interchangeably with "HVR" in this text) is a region within the antibody variable domain that is sequence-hypervariant and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. The heavy and light chain CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0183] However, it should be noted that the boundaries of the CDRs for the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences for the variable region of the same antibody may differ under different assignment systems. The antibody CDR assignment system used in this invention is IMGT, and for example, the "Annotate" function at http: / / www.abysis.org / abysis / can be used for numbering and labeling.

[0184] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0185] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0186] Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camel-derived antibodies, affinity molecules, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), and any of the above antigen-binding fragments.

[0187] Mouse antibodies exhibit high immunogenicity in humans, leading to decreased therapeutic efficacy with repeated administration. The primary immunogenicity is mediated by the heavy chain constant region. However, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided by chimeric or humanized versions of the antibodies.

[0188] "Chimeric antibodies" are antibodies whose different parts originate from different animal species, for example, antibodies having a variable region derived from mouse antibodies and a constant region from human immunoglobulins. Chimeric antibodies are obtained by linking the variable regions of the mouse heavy and light chains to the constant regions of the human heavy and light chains (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are generated by linking the constant region of the human κ light chain to the variable region of the mouse light chain. In another preferred embodiment, chimeric antibodies are generated by linking the constant region of the human λ light chain to the variable region of the mouse light chain.

[0189] The term "humanized antibody" is intended to refer to antibodies obtained by grafting a CDR sequence derived from another mammalian species, such as a mouse lineage, onto a human frame sequence. Further frame region modifications can be performed on the human frame sequence.

[0190] The term "monoclonal antibody" refers to an antibody molecule preparation with a single molecular composition that exhibits single binding specificity and affinity for a specific epitope.

[0191] The antibodies of this disclosure can be generated using various techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While hybridoma techniques are preferred, other techniques for generating monoclonal antibodies can be used in principle, such as viral or oncogene transformation of B lymphocytes or phage display using antibody gene libraries, somatic cell hybridization, and, for example, genetic engineering recombination techniques. For instance, DNA molecules encoding the heavy and light chain genes of the antibodies of this disclosure can be obtained through chemical synthesis or PCR amplification, the resulting DNA molecules can be inserted into an expression vector, transfected into host cells, and then cultured under specific conditions to express the antibodies of this disclosure.

[0192] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92: 9348 (1995), see also Rossie et al. Am. J. Clin. Pathol. 124: 295 (2005)). Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known.

[0193] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, or combinations thereof. For example, monoclonal antibodies can be produced using hybridomas and well-established biochemical and genetic engineering techniques, as described in detail in An, Zhiqiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed. 2009; Shireet et al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, 1st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0194] It should be understood that the selected binding sequence can be further modified, for example, to increase the affinity for the target, humanize the target binding sequence, improve its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing the modified target binding sequence are also antibodies of this disclosure.

[0195] To obtain hybridomas that produce antibodies of this disclosure, such as the human monoclonal antibodies of this disclosure, spleen cells and / or lymph node cells from immunized mice can be isolated and fused into a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, for example, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0196] The antibodies disclosed herein can also be generated in host cells transfected with tumors using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In some embodiments, DNA encoding a partial or full-length light and heavy chain, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the gene is operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene.

[0197] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In some embodiments, the variable region is used to generate a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, such that the VH segment is operatively linked to the CH segment within the vector and the VL segment is operatively linked to the CL segment within the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the N-terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0198] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. Antibodies disclosed herein can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells (which can assemble and secrete antibodies with appropriate folding and immunological activity).

[0199] As used herein, “antigen-binding molecule,” “antigen-binding fragment,” or “antibody fragment” means any molecule that contains an antigen-binding fragment (e.g., a CDR) of an antibody derived from that molecule. Antigen-binding molecules may include antigen complementarity-determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments formed from antigen-binding molecules, dAb, linear antibodies, scFv antibodies, and multispecific antibodies.

[0200] As used herein, the term "antigen" refers to any molecule that elicits an immune response or can be bound by an antibody or antigen-binding molecule. An immune response may involve antibody production or activation of specific immune-active cells, or both. Those skilled in the art will readily understand that any macromolecule (including almost all proteins or peptides) can act as an antigen. Antigens can be endogenously expressed, i.e., expressed from genomic DNA, or can be recombinantly expressed. Antigens may be specific to certain tissues, such as cancer cells, or they may be widely expressed. Furthermore, fragments of larger molecules can act as antigens.

[0201] As used herein, in some embodiments, antigen-binding molecules, scFvs, antibodies, or fragments thereof directly block binding sites on ligands or indirectly alter the binding affinity of ligands (e.g., by altering the structure or energy of the ligand). In some embodiments, antigen-binding molecules, scFvs, antibodies, or fragments thereof prevent the proteins bound to them from performing their biological functions.

[0202] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. Proteins or peptides contain at least two amino acids and there is no limit to the maximum number of amino acids that can contain the sequence of a protein or peptide. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which have many types). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0203] As used herein, the term "specific binding" or "specific binding to" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen.

[0204] As used herein, the ability to “inhibit binding,” “block binding,” or “compete for the same epitope” refers to the ability of an antibody to inhibit the binding of two molecules to any detectable extent. In some embodiments, an antibody that blocks the binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In some embodiments, this inhibition may be greater than 20%, 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0205] As used in this article, the term "K" a "K" is intended to represent the association rate of a specific antibody-antigen interaction, while the term "K" used in this article... d"K" is intended to represent the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "K" is... D "or "K D The "value" is intended to represent the dissociation constant of a specific antibody-antigen interaction, which ranges from K. d With K a The ratio (i.e., K) d / K a The K of the antibody is obtained and expressed as molar concentration (M). D The value can be determined using methods well-established in the art.

[0206] As used in this article, the term "high affinity" refers to an antibody that has a affinity of 1 × 10⁻⁶ against the target antigen. -7 M or lower, preferably 5×10 -8 M or lower, or even better, 1×10 -8 M or lower, or even better, 5×10 -9 M or lower, and even more preferably 1×10 -9 M or lower K D Antibodies with high antibody value.

[0207] The term "chimeric antigen receptor" (CAR) comprises an extracellular domain, a transmembrane domain, and possibly an intracellular domain. The extracellular domain consists of protein domains that recognize and bind to specific antigens. Chimeric antigen receptors can be expressed on immune cells and regulate their interaction with target cells. The term "monoclonal antibody" refers to an antibody molecule consisting of a single molecular component that exhibits specific binding specificity and affinity for a specific epitope.

[0208] As used herein, the terms "regulation" and "regulation" generally encompass two different directions: upregulation and downregulation. In some cases, they can be understood as inhibition or enhancement; in others, as reduction or increase; and in still others, as decrease or increase, etc. Specific interpretations are not limited and should be understood and interpreted according to the actual application context. For example, in some implementations, "regulating" tumor cell growth can be understood as inhibiting or enhancing tumor cell growth.

[0209] As used herein, the terms “reduction” and “reduction” are used interchangeably and indicate any change less than the original. “Reduction” and “reduction” are relative terms and need to be compared between before and after measurement. “Reduction” and “reduction” include complete consumption; similarly, the terms “increase” and “enhancement” are interpreted in opposite ways. For the purposes of this invention, “identity” between two nucleotide or amino acid sequences refers to the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, which is purely statistical and the differences between the two sequences are randomly distributed and cover their full length.

[0210] As used herein, the terms "5H antibody" and "5H-ADC" refer to the humanized monoclonal antibody 5H targeting CD3L1 and its drug conjugates provided by this invention, unless otherwise specified. Similarly, as used herein, the terms "13B7 antibody" and "13B7-ADC", "18B12 antibody" and "18B12-ADC", "5B11 antibody" and "5B11-ADC", "43B2 antibody" and "43B2-ADC", "41 antibody" and "41-ADC", and "46 antibody" and "46-ADC" refer to the monoclonal antibody targeting CD3L1 and its drug conjugates provided by this invention, unless otherwise specified.

[0211] As used herein, the term "linker" refers to the portion of an antibody-drug conjugate that links the antibody to the drug, and it can be cleavable or non-cleavable. A cleavable linker can cleave within target cells, thereby releasing the drug. In some embodiments, the linkers of the present invention exhibit excellent stability, reducing drug release during delivery to the target and thus reducing side effects and toxicity. In some specific embodiments, the linkers of the present invention are selected from cleavable linkers, such as peptide linkers, disulfide-based linkers, and hydrazone linkers. In other specific embodiments, the linkers of the present invention are selected from non-cleavable linkers, such as thioether linkers.

[0212] As used in this disclosure, a variety of suitable linkers are known in the art. The linker may be cleavable (cleavable linker), for example, under physiological conditions, such as intracellular conditions, such that cleavage of the linker releases the drug in the intracellular environment. Alternatively, the linker may be cleavable under extracellular conditions, such as outside tumor cells or near tumor masses, such that cleavage of the linker releases the drug that preferentially penetrates into the tumor cells. In other embodiments, the linker is non-cleavable (non-cleavable linker) and releases the drug, for example, through antibody degradation.

[0213] In this paper, the term "acid-cleavable linker" refers to a linker that relies on an acidic environment to trigger cleavage. It is designed to utilize acidic environments in vitro and / or in vivo (e.g., endosome pH 5.5–6.2, lysosome pH 4.5–5.0) to precisely release drugs into target cells, thereby enhancing efficacy and reducing systemic toxicity, while remaining stable under physiological conditions of pH 7.4.

[0214] Linkers can bind to chemically reactive groups on the antibody moiety, such as free amino, imino, hydroxyl, thiol, or carboxyl groups (e.g., N- or C-terminal, ε-amino group of one or more lysine residues, free carboxylic acid group of one or more glutamic or aspartic acid residues, thiol group of one or more cysteine ​​residues, or hydroxyl group of one or more serine or threonine residues). The linker binding site can be a native residue in the amino acid sequence of the antibody moiety, or the site can be introduced into the antibody moiety, for example, through DNA recombination techniques (e.g., by introducing cysteine ​​or protease cleavage sites into the amino acid sequence) or through protein biochemistry (e.g., reduction, pH regulation, or proteolysis). Linker binding sites can also be non-natural amino acids. Linker binding sites can also be glycans on the antibody.

[0215] The term “therapeutic agent” as used herein refers to any substance or entity that can have a therapeutic effect (e.g., treat, prevent, alleviate or suppress any disease and / or condition), including but not limited to: chemotherapeutic agents, radiotherapy agents, immunotherapy agents, etc.

[0216] The terms “cytotoxic agent” and “cytotoxic molecule” are used interchangeably in this disclosure and refer to any molecule (chemical or biochemical) that is toxic to cells.

[0217] "Immunostimulants" refer to drugs or biological agents that promote anti-tumor immune responses by activating or enhancing the function of the immune system. They can activate immune cells (such as T cells and NK cells) or enhance antigen presentation, and also relieve immunosuppressive signals (such as PD-1 / PD-L1 blockade). Examples include, but are not limited to, cytokines such as IL-2 (aldeleukin) and IFN-α (interferon), immune checkpoint inhibitors such as pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4), and cancer vaccines.

[0218] Immunosuppressants suppress the activity of the immune system and are used to control excessive immune responses (such as in autoimmune diseases) or prevent transplant rejection. Examples include, but are not limited to, glucocorticoids (such as prednisone), calcineurin inhibitors (such as cyclosporine and tacrolimus), antimetabolites (such as mycophenolate mofetil), and monoclonal antibodies (such as balithiabab).

[0219] "Cell inhibitors" are drugs that inhibit the proliferation of tumor cells but do not directly kill them. They slow tumor growth by blocking the cell cycle or signaling pathways. Examples include, but are not limited to, CDK4 / 6 inhibitors (such as palbociclib) and tamoxifen.

[0220] "Chemotherapy agents" refer to traditional chemotherapy drugs that exert their anti-tumor effects by directly killing rapidly dividing cells (including tumor and normal cells). Examples include 5-fluorouracil, methotrexate, cyclophosphamide, cisplatin, and doxorubicin.

[0221] "Radiotherapy agents" refer to the use of ionizing radiation (such as X-rays and gamma rays) to destroy the DNA of tumor cells, leading to cell death.

[0222] Anti-angiogenic agents can inhibit tumor angiogenesis and cut off the tumor's nutrient supply. Examples include, but are not limited to, bevacizumab and sorafenib.

[0223] "Anti-metastatic agents" are drugs that prevent tumor cells from invading, migrating, or colonizing distant organs. Examples include, but are not limited to, silengiptide and praxavir.

[0224] "Targeted anticancer agents" refer to those that specifically act on key molecular targets (such as kinases and mutant proteins) of tumor cells, reducing the impact on normal cells. Examples include, but are not limited to, tyrosine kinase inhibitors (TKIs) such as imatinib (BCR-ABL) and osimertinib (EGFR T790M); and PARP inhibitors such as olaparib.

[0225] "Radioactive isotopes" refers to isotopes that emit ionizing radiation, which kill tumor cells through localized irradiation. Examples include, but are not limited to, PSMA-617, lutetium-177, iodine-131, and radium-223.

[0226] The term "tubulin inhibitor" refers to antimitotic agents that block tubulin polymerization and inhibit cell division, making microtubules unstable or altering microtubule dynamics.

[0227] "DNA damaging agents" are a class of anticancer drugs or treatments that directly or indirectly disrupt DNA structure or function, inducing DNA damage (such as breaks, cross-links, or base modifications), thereby interfering with cell replication or transcription processes and ultimately leading to cell death or apoptosis. These drugs are widely used in chemotherapy, radiotherapy, and targeted therapy, and are particularly effective against rapidly proliferating tumor cells. They include, but are not limited to, alkylating agents (e.g., cyclophosphamide, temozolomide), platinum-based drugs (cisplatin, oxaliplatin), carbamicins (e.g., calicamicinγ1), pamomycins (e.g., bleomycin, pepromycin), apromycin derivatives (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone, PBD), and / or camptothecin and its derivatives (e.g., camptothecin, topotecan, belotecone, SN-38, DxdDXd).

[0228] "Topoisomerases" are enzymes capable of altering the topological structure of DNA in eukaryotic cells. They are essential for cell function and cell proliferation. Generally, two classes of topoisomerases, type I and type II, exist in eukaryotic cells. Topoisomerase I is a monomeric enzyme with a molecular weight of approximately 100,000. This enzyme binds to DNA and introduces transient single-strand breaks, causing the double helix to unwind (or allowing it to unwind), and then reseals the breaks before dissociating from the DNA strand. The term "topoisomerase inhibitor" refers to any tumor cell growth inhibitory compound that is structurally related to camptothecin. According to some embodiments of this disclosure, the topoisomerase inhibitor is camptothecin or a camptothecin analogue. Camptothecin is a water-insoluble cytotoxic alkaloid produced by the native Chinese tree *Camptotheca accuminata* and the native Indian tree *Nothapodytes foetida*. Camptothecin exhibits tumor cell growth inhibitory activity against a variety of tumor cells. Camptothecin analogues are generally specific inhibitors of DNA topoisomerase I.

[0229] "DNA alkylating agents" are drugs that interfere with DNA replication and transcription by alkylating DNA bases (such as guanine) to form cross-links or single-strand breaks. Examples include, but are not limited to, cyclophosphamide, cisplatin, carmustine (BCNU), busulfan, and temozolomide.

[0230] "RNA polymerase inhibitors" refer to drugs that block RNA polymerase activity and inhibit transcription or viral replication. Examples include, but are not limited to, bacterial RNA polymerase inhibitors (such as rifampicin and rifapentine), eukaryotic RNA polymerase II inhibitors (α-Amanitin), and viral RNA polymerase inhibitors (Sofosbuvir).

[0231] "RNA splicing enzyme inhibitors" are drugs that interfere with the mRNA splicing process, leading to the accumulation of abnormal transcripts. Examples include, but are not limited to, ponatinib, E7107, and antisense oligonucleotides (such as nusinersen).

[0232] "Bcl-xL inhibitors" refer to drugs that inhibit the anti-apoptotic protein Bcl-xL, thereby restoring the apoptotic ability of tumor cells. Examples include, but are not limited to, Navitoclax (ABT-263), A-1331852, and Venetoclax.

[0233] "Antimitotic agents" can interfere with microtubule homeostasis or spindle formation, thereby blocking cell division. Examples include, but are not limited to, paclitaxel, vincristine, Monastrol, and Alisertib (MLN8237).

[0234] "Sodium-potassium ion pump ATPase inhibitors" can inhibit the sodium-potassium pump in cell membranes, affecting ion balance and signal transduction. Examples include, but are not limited to, digoxin and digitoxin.

[0235] "Matrix metalloproteinase inhibitors" can inhibit MMP enzyme activity, blocking tumor invasion and angiogenesis. Examples include, but are not limited to, marimastat, batimastat, and doxycycline.

[0236] "Immune agonists" can activate immune cells or signaling pathways, enhancing anti-tumor immune responses. Examples include, but are not limited to, imiquimod, ADU-S100 (MIW815), selicrelumab (CP-870,893), and aldesleukin (IL-2).

[0237] The term "sea hare toxin" used in this article refers to a polypeptide isolated from the truncated sea hare, a marine organism. Sea hare toxin peptides are mitotic inhibitors that exhibit strong anticancer activity, thus being considered as candidates for anticancer drugs. Researchers have further discovered and synthesized many derivatives of sea hare toxin peptides, including, but not limited to, Dolatatin 10, Dolatatin 15, MMAE, MMAF, Auristatin E, Auristatin F, PF-06380101, and Tubulysins.

[0238] MMAE is an olistatin derivative, also known as monomethyl olistatin E, with the following structural formula:

[0239] Specifically, the term "MMAE" refers to the compound (S)-N-((3R,4S,5S)-1-((S)2-((1R,2R)-3(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohepane-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butamido)butyramide. MMAE is actually demethylated olistatin E, meaning the N-terminal amino group has only one methyl substituent, instead of the two substituents found in olistatin E itself.

[0240] The term "camptothecin" used in this article refers to a cytotoxic quinoline alkaloid that inhibits DNA topoisomerase. Camptothecin has demonstrated significant anticancer properties and a high degree of adverse drug reaction in preliminary clinical trials. Researchers have further utilized these properties to develop a variety of camptothecin derivatives to enhance efficacy and safety, such as topotecan, irinotecan, and ethanotecan, as well as derivatives like DXd and SN-38.

[0241] DXd is a camptothecin derivative and an effective derivative of ethanotecan, CAS No.: 1599440-33-1, with the following structural formula:

[0242] SN-38 is 7-ethyl-10-hydroxycamptothecin, a camptothecin derivative. As a metabolite, SN38 is formed by the hydrolysis of irinotecan by a carboxylesterase. In some embodiments, SN38 has one of the following structures:

[0243] "Maytansin-like cytotoxic molecules" are well known in the art, and maytansin derivatives can be synthesized or isolated from natural sources using known techniques. According to this disclosure, particularly preferred maytansin alkaloids are thiol-containing derivatives of maytansin, such as DM1 and DM4. These thiol-containing derivatives of maytansin comprise compounds in which a methyl group bound to a carbonyl group is replaced by a group containing a free thiol group (e.g., the group -R-SH, where R represents an alkylene or other carbon-containing atom).

[0244] Mertansine DM1 is derived from maytansine, specifically referring to the compound N2'-deacetylated N2'-(3-mercapto-1-oxopropyl)-matansine, CAS No.: 139504-50-0, with the following structural formula:

[0245] Maytansin DM4 refers to the compound N2'-deacetylated-N2'-(4-methyl-4-mercapto-1-oxopentyl)-matansin.

[0246] The "drug / antibody ratio" or "drug-to-antigen binding protein ratio (DAR)" refers to the number of therapeutic agents attached to each antibody molecule, and it is an important factor affecting the efficacy and safety of antibody-drug conjugates (ADCs). ADCs are typically manufactured as mixtures with varying numbers of therapeutic agents attached. Therefore, the drug / antibody ratio of a mixture is usually characterized by an average number of therapeutic agents attached to each antibody molecule. Unless otherwise specified, the term "drug / antibody ratio" as used herein refers to an average value.

[0247] As used in this article, the terms "endocytosis," "endocytosis," or "internalization" refer to the process by which antibodies and their drug conjugates enter the cell. This can be specific, depending on the binding of the antibody or drug conjugate to cell surface antigens; or it can be non-specific, independent of the binding of the antibody or drug conjugate to cell surface antigens, such as phagocytosis and pinocytosis.

[0248] The term "object" as used in this article refers to mammals, such as humans, but can also be other animals, such as laboratory animals (monkeys, mice, rabbits, etc.).

[0249] As used in this article, "therapeutic effective dose" or "effective dose" refers to a dose sufficient to demonstrate its benefit to the recipient. The actual amount administered, as well as the rate and duration of administration, depends on the individual's condition and severity. The prescription of treatment (e.g., the determination of dosage) ultimately rests with the physician, typically taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0250] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug substance and optionally a pharmaceutically acceptable carrier or excipient, grouped together to achieve a particular purpose. In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided they can work together to achieve the objectives of the invention. For example, the components contained in the pharmaceutical composition may be applied to the subject holistically or separately; when the components contained in the pharmaceutical composition are applied to the subject separately, the components may be applied to the subject simultaneously or sequentially. The pharmaceutically acceptable carrier may be water, isotonic saline solution, glucose, mannitol, hyaluronic acid, triglycerides, etc.

[0251] The terms “pharmaceutical carrier” and “pharmaceuticalally acceptable carrier” are used interchangeably herein and may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or nontoxic excipients, combinations or more of various components known in the art.

[0252] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl arsenate. As disclosed in this disclosure, an antibody or its antigen-binding moiety containing a composition disclosed herein can be oxidized in a solvent containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding moiety. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this disclosure provides compositions comprising one or more antibodies or their antigen-binding moieties and one or more antioxidants such as methionine. This disclosure further provides various methods in which an antibody or its antigen-binding moieties are mixed with one or more antioxidants such as methionine. Thus, antibodies or their antigen-binding portions can be protected from oxidation to prolong their shelf life and / or increase their activity.

[0253] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's injection; non-aqueous media such as non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); isolating agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions containing phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride in multi-dose containers. Suitable excipients may include, for example, water, saline, dextran, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, or cyclodextrin.

[0254] The pharmaceutical compositions, vaccines, or pharmaceutical preparations according to the present invention may be administered via any suitable route, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous, and intrathecal administration, or by implantation or inhalation. The compositions disclosed herein may be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration may be selected according to the intended application and treatment regimen.

[0255] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers and their controlled-release formulations.

[0256] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Similarly, specific dosing regimens (including dose, time, and repetition) will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetic parameters (e.g., half-life, clearance, etc.).

[0257] The requirements for effective drug carriers for injectable formulations / compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, edited by Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)).

[0258] The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction in tumor cells, reducing tumor cell proliferation, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the therapeutic composition disclosed herein may be suitable.

[0259] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or adverse side effects. The chosen dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, timing of administration, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, the patient's sex, age, weight, condition, general health status, and previous medical history. The amount of compound and route of administration are ultimately determined by a physician, veterinarian, or clinician, but a dosage is typically chosen to achieve a local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

[0260] Generally, the antibody-drug conjugates or pharmaceutical compositions thereof disclosed herein can be administered in a variety of ranges. These include about 5 μg / kg body weight per dose to about 100 mg / kg body weight; about 50 μg / kg body weight per dose to about 5 mg / kg body weight per dose; and about 100 μg / kg body weight per dose to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight per dose to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight per dose to about 20 mg / kg body weight per dose. In some embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, and at least about 10 mg / kg body weight.

[0261] In any case, the antibody-drug conjugates or pharmaceutical compositions thereof disclosed herein are preferably administered to subjects in need as required. Those skilled in the art can determine the frequency of administration, for example, based on considerations by the attending physician regarding the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health condition of the subject being treated.

[0262] In some preferred embodiments, a treatment regimen involving the antibody-drug conjugate or pharmaceutical composition thereof of this disclosure will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody-drug conjugate or pharmaceutical composition thereof of this disclosure may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0263] The dosage and regimen of the disclosed therapeutic composition may also be determined empirically in individuals receiving one or more administrations. For example, an incremental dose of the therapeutic composition as described herein may be administered to an individual. In selected embodiments, the dosage may be gradually increased, decreased, or mitigated based on empirical determination or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, condition, or disease can be tracked as previously described. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein; reduction of pain or paralysis; improvement of tumor-related speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life or prolonged survival as measured by administered tests. Those skilled in the art will understand that the dosage will vary depending on the individual, the type of tumor, the stage of the tumor, whether the tumor has begun to metastasize to other sites within the individual, and past and concurrent treatments.

[0264] Compatible formulations intended for parenteral administration (e.g., intravenous injection) will comprise an antibody-drug conjugate or a pharmaceutical composition thereof as disclosed herein at a concentration of about 10 μg / mL to about 100 mg / mL. In some selected embodiments, the concentration of the antibody-drug conjugate or the pharmaceutical composition thereof will include 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, or 1 mg / mL. In other preferred embodiments, the ADC concentration will include 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL.

[0265] The antibody-drug conjugates, pharmaceutical compositions, and methods disclosed herein have numerous in vitro and in vivo uses, including, for example, detection of CD3L1 or enhancement of immune responses. These molecules can be administered, for example, in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects. Preferred subjects include mammals, such as individuals / patients in need. In the context of this disclosure, mammals include humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as zoo animals.

[0266] The antibody-drug conjugates or pharmaceutical compositions disclosed herein can be used alone as a monotherapy or in combination with chemotherapy or radiotherapy.

[0267] The antibody-drug conjugates or pharmaceutical compositions disclosed herein can be used in combination with immune checkpoint inhibitors (e.g., CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors), anticancer agents, antiproliferative agents, cytotoxic agents, chemotherapeutic agents, radiotherapy agents, antiangiogenic agents, and antimetastatic agents.

[0268] The disclosed antibody-drug conjugates or pharmaceutical compositions can be combined with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combined therapies using targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeted agents. Typically, radiotherapy is administered in a pulsed manner over a period of approximately 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for approximately 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0269] As used herein, a “kit” may have a single container containing the disclosed antibody-drug conjugate or pharmaceutical composition, with or without additional components, or they may have different containers for each desired reagent. In cases where a combination therapeutic agent for conjugation is provided, a single solution may be premixed in molar equivalents or in a manner where one component is more than another. Alternatively, the conjugate and any optional anticancer agent of the kit may be stored separately in different containers prior to administration to the patient. The kit may also contain a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0270] When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0271] As briefly described above, the kit may also contain tools for administering antibodies or their antigen-binding portions, antibody-drug conjugates, or pharmaceutical compositions and any optional components to a patient, such as one or more needles, IV bags (intravenous injection bags), or syringes, or even eye drops, pipettes, or other similar devices through which the preparation can be injected or introduced into an animal or applied to a diseased area of ​​the body. The kits disclosed herein typically also include devices for containing vials or the like, and other tightly sealed components for commercial sale, such as injection or blow-molded plastic containers, in which the desired vials and other devices are placed and held.

[0272] "CD3L1-expressing tumors" refers to tumor cells found to contain the transcriptional or translational product of CD3L1 (CD3 ligand 1) through gene or protein detection. "CD3L1-positive tumors" means tumor cells whose surface or intracellular CD3L1 protein expression reaches a specific threshold (e.g., ≥1% cell positivity or H-score criteria) confirmed by standardized clinical tests (e.g., immunohistochemistry / IHC, flow cytometry).

[0273] As used herein, the term "cancer" refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors. Examples include: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastrointestinal cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, cancers of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, vegetations / tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal cord axis Axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and combinations thereof.

[0274] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0275] This disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0276] Wherein: A is an antibody that specifically binds to CD3L1 or its antigen-binding fragment; U is a therapeutic agent, each U may be the same or different; L is a linker, each L may be the same or different; n is an integer greater than 1, preferably an integer selected from 1 to 8, for example 1, 2, 3, 4, 5, 6, 7 or 8.

[0277] According to some embodiments of this disclosure, the antibody or antigen-binding fragment specifically binding to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13. In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14.

[0278] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region including HCDR1, HCDR2 and HCDR3, HCDR1 including an amino acid sequence as shown in SEQ ID NO:5, HCDR2 including an amino acid sequence as shown in SEQ ID NO:6, and HCDR3 including an amino acid sequence as shown in SEQ ID NO:7.

[0279] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 includes a heavy chain variable region and a light chain variable region, the light chain variable region including LCDR1, LCDR2 and LCDR3, the LCDR1 containing an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, the LCDR2 containing an amino acid sequence as shown in SEQ ID NO:9, and the LCDR3 containing an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16.

[0280] According to some embodiments of this disclosure, the amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:7. In some embodiments, the amino acid sequence of LCDR1 is shown in SEQ ID NO:8 or SEQ ID NO:15, the amino acid sequence of LCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:10 or SEQ ID NO:16.

[0281] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 includes HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, HCDR3 as shown in SEQ ID NO:7, LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:10.

[0282] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 includes HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, HCDR3 as shown in SEQ ID NO:7, LCDR1 as shown in SEQ ID NO:15, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:16.

[0283] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence having greater than 80% identity with such sequences, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0284] According to some embodiments of this disclosure, the light chain variable region comprises an amino acid sequence selected from, for example, the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or an amino acid sequence having greater than 80% identity with them, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0285] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having greater than 80% identity with it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having greater than 80% identity with it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0286] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0287] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:11 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:12 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0288] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0289] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having greater than 80% identity with it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having greater than 80% identity with it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0290] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14.

[0291] According to some embodiments of the present disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:17 or an amino acid sequence having greater than 80% identity with it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:18 or an amino acid sequence having greater than 80% identity with it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0292] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:18.

[0293] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:19 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0294] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:20.

[0295] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:22 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0296] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:21, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:22.

[0297] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:23 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:24 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0298] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24.

[0299] According to some embodiments of this disclosure, the antibody or antigen-binding fragment specifically binding to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45. In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:38, or SEQ ID NO:45.

[0300] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, wherein HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48, and wherein HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49.

[0301] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 includes a heavy chain variable region and a light chain variable region, the light chain variable region including LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 contains an amino acid sequence as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52.

[0302] According to some embodiments of this disclosure, the amino acid sequence of HCDR1 is shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47; the amino acid sequence of HCDR2 is shown in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48; and the amino acid sequence of HCDR3 is shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49. In some embodiments, the amino acid sequence of LCDR1 is shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50; the amino acid sequence of LCDR2 is shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51; and the amino acid sequence of LCDR3 is shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52.

[0303] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 includes HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO:29, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:32.

[0304] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 includes HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:35, HCDR3 as shown in SEQ ID NO:36, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:37.

[0305] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:40, HDCR2 as shown in SEQ ID NO:41, HCDR3 as shown in SEQ ID NO:42, LCDR1 as shown in SEQ ID NO:43, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:44.

[0306] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 comprises HCRD1 as shown in SEQ ID NO:47, HDCR2 as shown in SEQ ID NO:48, HCDR3 as shown in SEQ ID NO:49, LCDR1 as shown in SEQ ID NO:50, LCDR2 as shown in SEQ ID NO:51, and LCDR3 as shown in SEQ ID NO:52.

[0307] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence selected from, for example, the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45, or an amino acid sequence having greater than 80% identity with such sequences, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0308] According to some embodiments of this disclosure, the light chain variable region comprises an amino acid sequence selected from, for example, the amino acid sequence shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46, or an amino acid sequence having greater than 80% identity with such sequences, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0309] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:25 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0310] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26.

[0311] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:33 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0312] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:34.

[0313] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:38 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:39 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0314] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:39.

[0315] According to some embodiments of this disclosure, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:45 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:46 or an amino acid sequence with greater than 80% identity to it, such as any amino acid sequence or an amino acid sequence composed thereof with at least 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0316] According to some embodiments of this disclosure, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:45, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:46.

[0317] According to some embodiments of this disclosure, the antibody that specifically binds to CD3L1 or its antigen-binding fragment is selected from chimeric antibodies, humanized antibodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant proteins or Fab fragments containing antigen-binding fragments, F(ab') fragments, F(ab')2 fragments, Fv fragments, single-chain antibodies (scFv), and diabody; in some embodiments, the antibody or its antigen-binding fragment further comprises a constant region of an immunoglobulin, wherein the immunoglobulin is selected from IgG1, IgG2, IgG3, and IgG4.

[0318] According to some preferred embodiments of this disclosure, the antibody that specifically binds to CD3L1 is a chimeric antibody or a humanized antibody. According to some preferred embodiments of this disclosure, the antibody that specifically binds to CD3L1 is a monoclonal antibody. In some more preferred embodiments, the antibody that specifically binds to CD3L1 or its antigen-binding fragment is a humanized monoclonal antibody containing the IgG1 constant region.

[0319] According to some embodiments of this disclosure, the linker can be linked to an antibody or its antigen-binding fragment in any manner known in the art. In some preferred embodiments, the linker is linked to an amino or thiol residue on the antibody or its antigen-binding fragment. In some more preferred embodiments, the linker of the present invention is covalently linked to a thiol residue formed after the interchain disulfide bond of the anti-CD3L1 antibody is opened.

[0320] According to some embodiments of this disclosure, the connector includes a shardable connector and a non-shardable connector.

[0321] According to some embodiments of this disclosure, the cleavable linker comprises a peptide unit containing 2-20 amino acids; in some preferred embodiments, the peptide unit is selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-arginine-(-Phe-Arg-), and combinations thereof; in some more preferred embodiments, the peptide unit is selected from -valine-citrulline-(-Val-Cit-) and -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-).

[0322] According to some embodiments of this disclosure, the cleavable linker comprises a structure selected from maleimide-hexanoyl (MC), maleimide-butyric acid (MB), or p-aminobenzyloxycarbonyl (PAB).

[0323] According to some embodiments of this disclosure, the cleavable linker is selected from maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleimide-hexanoyl-valine-alanine (MC-VA), maleimide-butyric acid-valine-citrulline (MB-VC), glycine-glycine-phenylalanine-glycine (GGFG), maleimide-hexanoyl-glycine-glycine-phenylalanine-glycine (MC-GGFG), or valine-citrulline-p-aminobenzyloxycarbonyl (VC-PAB).

[0324] According to some embodiments of this disclosure, the cleavable linker is a linker that can cleave under acidic conditions. According to some preferred embodiments of this disclosure, the cleavable linker is a linker that can cleave in endosomes or lysosomes.

[0325] According to some embodiments of this disclosure, the antibody or antigen-binding fragment that specifically binds to CD3L1 is capable of binding to the amino acid sequence in CD3L1 as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0326] According to some embodiments of this disclosure, the U is selected from the group consisting of cytotoxic molecules, immune enhancers, immunosuppressants, cell inhibitors, chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic agents, anti-metastatic agents, targeted anticancer agents, other anticancer agents, and radioisotopes.

[0327] According to some embodiments of this disclosure, the U is selected from microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, DNA alkylating agents, RNA polymerase inhibitors, RNA cleavage enzyme inhibitors, Bcl-xL inhibitors, antimitotic agents, sodium-potassium ion pump ATPase inhibitors, matrix metalloproteinase inhibitors, and / or immune agonists.

[0328] According to some embodiments of this disclosure, the microtubule inhibitor is selected from saurus toxin and olistatin cytotoxic molecules (e.g., including but not limited to MMAE, MMAF), and / or maytansine cytotoxic molecules (e.g., including but not limited to DM1, DM4).

[0329] According to some embodiments of this disclosure, the DNA damaging agent includes those selected from carbamicins (e.g., including but not limited to Caliceamicinγ1), pyromycins (e.g., including but not limited to bleomycin, pepromycin), atrazomycin derivatives (e.g., including but not limited to doxorubicin, epirubicin, idarubicin, mitoxantrone, PBD), and / or camptothecin and camptothecin derivatives (e.g., including but not limited to camptothecin, topotecan, belotecone, SN-38, DXd).

[0330] According to some embodiments of this disclosure, the U is selected from DXd and / or MMAE.

[0331] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0332] in:

[0333] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence with greater than 80% identity to the present; and / or, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence with greater than 80% identity to the present; and / or, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ The amino acid sequence shown in NO:24 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.The amino acid sequence is 9%; the U is a therapeutic agent, each U may be the same or different, and it is selected from MMAE or DXd; the L is a linker, each L may be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0334] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0335] in:

[0336] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:19 ... The amino acid sequence shown in SEQ ID NO:23 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it.The amino acid sequence is 9%; U is a therapeutic agent, and it is MMAE; L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0337] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0338] in:

[0339] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:19 ... The amino acid sequence shown in SEQ ID NO:23 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% with respect to it.The amino acid sequence is 9%; U is a therapeutic agent, and it is DXd; L is a linker, and it is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0340] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0341] in:

[0342] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28, SEQ ID NO:45, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:45, SEQ ID NO:39, SEQ ID NO:46 ... The amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48, and the HCDR3 contains an amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the light chain variable region including LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 contains an amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 contains an amino acid sequence shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region contains an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the heavy chain variable region contains an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49 ...36, SEQ ID NO:49, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49, SEQ ID The amino acid sequence shown in NO:45 or having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99% with respect to it.The amino acid sequence comprises 9%; and / or, the light chain variable region comprises an amino acid sequence selected from those shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with them; the U is a therapeutic agent, each U may be the same or different, and it is selected from MMAE or DXd; the L is a linker, each L may be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0343] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0344] in:

[0345] The A is an antibody or antigen-binding fragment specifically binding to CD3L1, wherein the antibody or antigen-binding fragment specifically binding to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment specifically binding to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28 or SEQ ID NO:45. The amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48, and the HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49 ...36 The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.9% of the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence selected from, as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; the U is a therapeutic agent, and it is MMAE; the L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0346] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0347] in:

[0348] The A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28, SEQ ID NO:45, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:45, SEQ ID NO:39, SEQ ID NO:46 ... The amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:48, and the HCDR3 contains an amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the light chain variable region including LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 contains an amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 contains an amino acid sequence shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region contains an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the heavy chain variable region contains an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49 ...36, SEQ ID NO:49, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:49, SEQ ID The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.9% of the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; the U is a therapeutic agent, and it is DXd; the L is a linker, and it is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0349] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0350] in:

[0351] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:5. HCDR3 as shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1 with an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 with an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; and / or, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:7; The amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, or those showing an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.5% with respect to them.The amino acid sequence is 9%; the U is a therapeutic agent, each U may be the same or different, and it is selected from MMAE or DXd; the L is a linker, each L may be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0352] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0353] in:

[0354] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:5. HCDR3 as shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1 with an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 with an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; and / or, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:7; The amino acid sequences shown in NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, or amino acid sequences with an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% compared to them; the U is a therapeutic agent, and it is MMAE; the L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0355] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0356] in:

[0357] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:5. HCDR3 as shown in SEQ ID NO:7; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprising LCDR1 with an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 with an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:16; and / or, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; and / or, the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:7; The amino acid sequences shown in NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24, or amino acid sequences with an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% compared to them; the U is a therapeutic agent and is DXd; the L is a linker and is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0358] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0359] in:

[0360] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in the amino acid sequence SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR1 in the amino acid sequence SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:46. HCDR2 as shown in NO:48, and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with amino acid sequences as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 with amino acid sequences as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 with amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region comprises HCDR3 selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49 ... The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.The amino acid sequence comprises 9%; and / or, the light chain variable region comprises an amino acid sequence selected from those shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with them; the U is a therapeutic agent, each U may be the same or different, and it is selected from MMAE or DXd; the L is a linker, each L may be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0361] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0362] in:

[0363] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR1 in SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:46. HCDR2 as shown in NO:48, and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with amino acid sequences as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 with amino acid sequences as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 with amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region comprises HCDR3 selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49 ... The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.9% of the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence selected from, as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; the U is a therapeutic agent, and it is MMAE; the L is a linker, and it is Mc-Val-Cit-PAB; and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0364] According to some preferred embodiments of this disclosure, this disclosure provides an antibody-drug conjugate with the general structural formula: A-(LU). n

[0365] in:

[0366] A is an antibody or antigen-binding fragment that specifically binds to CD3L1, wherein the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:46; and / or, the antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 as shown in the amino acid sequence SEQ ID NO:27, SEQ ID NO:40, or SEQ ID NO:47, and HCDR1 in the amino acid sequence SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:41, or SEQ ID NO:46. HCDR2 as shown in NO:48, and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:36, SEQ ID NO:42, or SEQ ID NO:49; and / or, the antibody specifically binding to CD3L1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises LCDR1 with amino acid sequences as shown in SEQ ID NO:30, SEQ ID NO:43, or SEQ ID NO:50, LCDR2 with amino acid sequences as shown in SEQ ID NO:31, SEQ ID NO:9, or SEQ ID NO:51, and LCDR3 with amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:44, or SEQ ID NO:52; and / or, the heavy chain variable region comprises HCDR3 selected from SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:49 ... The amino acid sequence shown in NO:45 or its identity with it is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99%.9% of the amino acid sequence; and / or, the light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence with greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with such sequences; the U is a therapeutic agent, and it is DXd; the L is a linker, and it is Gly-Gly-Phe-Gly (GGFG); and n is an integer greater than 1, preferably an integer selected from 1 to 8.

[0367] According to some preferred embodiments of this disclosure, the antibody that specifically binds to CD3L1 involved in this invention is a humanized monoclonal antibody 5H. The 5H monoclonal antibody is linked to MMAE or DXd via a Mc-Val-Cit-PAB or GGFG linker to form two antibody-drug conjugates, 5H-MMAE or 5H-DXd.

[0368] According to some preferred embodiments of this disclosure, the antibody that specifically binds to CD3L1 involved in this invention is a monoclonal antibody 13B7, 18B12, 5B11, 43B2, 41, or 46. The above-mentioned monoclonal antibodies are linked to MMAE or DXd via Mc-Val-Cit-PAB or GGFG linkers to form antibody-drug conjugates such as 13B7-MMAE, 18B12-MMAE, 5B11-MMAE, 43B2-MMAE, 41-MMAE, 46-MMAE, 13B7-DXd, 18B12-DXd, 5B11-DXd, 43B2-DXd, 41-DXd, or 46-DXd.

[0369] According to some embodiments of this disclosure, the drug fraction to antigen-binding protein ratio (DAR) is in the range of 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the DAR is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, the DAR is about 2, about 2.5, about 3, about 4, about 5, or about 6, about 7, or about 8. In some embodiments, the DAR is in the range of about 2 to about 4. In some embodiments, the DAR is in the range of about 6 to about 8. The DAR can be characterized by conventional methods, such as mass spectrometry, UV / Vis spectroscopy, ELISA assay, and / or HPLC.

[0370] According to some preferred embodiments of this disclosure, each antibody molecule is conjugated to an average of 3 to 4 MMAEs (DAR is 3 to 4).

[0371] According to some preferred embodiments of this disclosure, each antibody molecule is conjugated to an average of 6 to 8 DXd (DAR is 6 to 8).

[0372] According to some preferred embodiments of this disclosure, the antigen-binding power of the anti-CD3L1 antibody drug conjugate provided by the present invention is comparable to that of the anti-CD3L1 antibody that forms part of it, and both can bind specifically to the extracellular domain of CD3L1 with high affinity.

[0373] According to some preferred embodiments of this disclosure, the CD3L1-targeting antibody-drug conjugates provided by this invention have highly efficient endocytosis into tumor cells and drug release in lysosomes, with endocytosis efficiency significantly superior to anti-HER2 antibody-drug conjugates produced under the same preparation conditions. This highly efficient endocytosis capability facilitates its rapid entry into tumor cells after binding to them to exert its cytotoxic activity, which is something that those skilled in the art could not have anticipated.

[0374] In certain specific embodiments, the anti-CD3L1 antibody drug conjugate provided by the present invention has shown the ability to inhibit the proliferation of CD3L1-positive tumor cells under both in vivo and in vitro experimental conditions, and its tumor-suppressing effect is significantly better than that of anti-CD3L1 antibody and anti-HER2 antibody drug conjugate produced under the same preparation conditions, which is something that those skilled in the art could not have expected.

[0375] According to some embodiments of this disclosure, the antibody-drug conjugate can be efficiently internalized into target cells and release a therapeutic agent within the cells.

[0376] This disclosure also provides a method for preparing the above-mentioned antibody-drug conjugate, wherein the method is capable of linking the antibody or its antigen-binding fragment to the therapeutic agent and achieving a stable drug / antibody ratio.

[0377] This disclosure also provides a pharmaceutical composition having the aforementioned antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0378] This disclosure also provides the use of the above-mentioned antibody-drug conjugates, or antibody-drug conjugates or pharmaceutical compositions prepared by the aforementioned methods, in the preparation of antitumor drugs.

[0379] According to some preferred embodiments of this disclosure, the tumor is a tumor that does not respond well to treatment with antibody-drug conjugates targeting HER2.

[0380] This disclosure also provides a kit comprising a container, a formulation placed in the container, and optionally instructions for use; wherein the formulation comprises the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition.

[0381] According to some embodiments of this disclosure, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody-drug conjugate, with or without one or more other reagents. In other embodiments, such a unit dose is supplied in a single-use, pre-filled syringe. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; a buffer solution, such as phosphate; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., sterile water or a saline solution). In some preferred embodiments, the composition contains one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the packaged conjugate or composition is intended for the treatment of selected oncological conditions.

[0382] This disclosure also provides the use of the aforementioned antibody-drug conjugates, or antibody-drug conjugates or pharmaceutical compositions prepared by the aforementioned methods, in the preparation of products for the treatment or prevention of tumors.

[0383] According to some embodiments of this disclosure, the tumor is a solid tumor or hematologic tumor expressing CD3L1.

[0384] According to some embodiments of this disclosure, the tumor is a CD3L1 positive tumor, including melanoma, sarcoma, lung cancer, breast cancer, thyroid cancer, nervous system tumors, kidney cancer, urothelial carcinoma, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, head and neck cancer, leukemia, lymphoma, and other CD3L1 positive related tumors.

[0385] This disclosure also provides the use of the aforementioned antibody-drug conjugates, or antibody-drug conjugates prepared by the aforementioned methods, or the aforementioned pharmaceutical compositions in the preparation of medicaments for treating diseases related to CD3L1 expression.

[0386] This disclosure provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative disorders, as well as methods for screening cells from patients to identify tumor cells, including tumorigenic cells. Such methods include identifying individuals for treating cancer or monitoring cancer progression, including contacting a patient or a sample obtained from a patient (in vivo or in vitro) with an antibody described herein and detecting the presence or absence of the antibody in the sample, or the binding level, of a bound or free target molecule. In some embodiments, the antibody will comprise a detectable marker or reported molecule as described herein. Preferred subjects include mammals, such as human patients in need.

[0387] The present application will be further described below with reference to the embodiments. It should be understood that these embodiments are only for illustrative purposes. The present application can be embodied in many different forms of embodiments, and the protection scope of the present application is not limited to the embodiments mentioned in the text.

[0388] Example

[0389] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0390] Example 1: Drug distribution and metabolism of CD3L1 humanized antibody 5H in tumor-bearing nude mice

[0391] CD3L1 humanized antibody 5H was labeled using the Cy7 antibody labeling kit (Multi-Fluorescence Biotechnology, China). Refer to the kit's instructions for specific procedures. A375 cells (2 × 10⁶) were then labeled with the humanized antibody 5H. 6 (One per mouse) was injected under the armpit of BALB / c nude mice (Balb / C Nude, Jicui Yaokang). When the tumor grew to 200 mm... 3 The labeled antibody was intraperitoneally injected into tumor-bearing mice (n=3) at a dose of 10 mg / kg. At different time points after injection, the mice were treated with the anesthetic isoflurane (Reward, R510-22) and placed on an observation board (simultaneously anesthetized). The distribution and metabolism of antibody fluorescence in the tumor-bearing nude mice were monitored using a PerkinElmer small animal in vivo imaging system (model IVIS Spectrum CT). The results are shown in Figure 1. The experiment showed that the 5H antibody could rapidly target the xenograft site in nude mice, with strong intratumoral fluorescence enrichment observed within 24 hours, and the fluorescence intensity could be maintained for 4 days, while fluorescence enrichment was rarely observed in other non-tumor sites.

[0392] Example 2: Preparation of antibody-drug conjugates

[0393] Reagent preparation: Dissolve 25 mM Na2B4O7 (Sigma, USA), 25 mM NaCl (Sangon Biotech, China), and 1 mM DPTA (Sigma, USA) in pure water and adjust the pH to 7.4 as the coupling solution; prepare 1 mM and 10 mM TCEP (Sigma, USA) solutions in pure water; prepare 100 mM cysteine ​​(Sigma, USA) solution in pure water; dissolve 50 mM potassium phosphate, 50 mM NaCl, and 2 mM EDTA in pure water and adjust the pH to 6.5 as buffer A; prepare 1×PBS at pH 6.5 as buffer B; prepare 10 mM mc-vc-PAB-MMAE (MCE, USA), 10 mM GGFG-DXd (MCE, USA), and 20 mM SMCC solutions in dimethyl sulfoxide (DMSO); prepare 10 mM DM1 solution in dimethylacetamide (DMA).

[0394] 2.1 Preparation of antibody-drug conjugates with MMAE as toxicity load

[0395] Anti-CD3L1 antibody and anti-HER2 antibody were transferred from PBS to the conjugation buffer using ultrafiltration. After ultrafiltration, the antibody concentration was adjusted to 2.5 mg / mL. An appropriate volume of 1 mM TCEP aqueous solution was added to the antibody solution to achieve a TCEP:antibody molar ratio of 3:1, and the mixture was incubated at room temperature for 2 hours. Then, an appropriate volume of 10 mM mc-vc-PAB-MMAE solution was added to achieve a mc-vc-PAB-MMAE:antibody molar ratio of 7:1, and the mixture was incubated at room temperature for 2 hours. After the reaction was complete, the product was transferred to PBS using ultrafiltration, removing unreacted mc-vc-PAB-MMAE. After ultrafiltration, the drug / antibody ratio (DAR) of CD3L1 humanized antibody-drug conjugates (5H-MMAE, 13B7-MMAE, 18B12-MMAE, 5B11-MMAE, 43B2-MMAE, 41-MMAE, 46-MMAE, etc.) was analyzed by hydrophobic high performance liquid chromatography. Some results are shown in Figure 2(A), with an average value of 3.81.

[0396] In addition, this embodiment also prepared conjugates of human IgG1 (Biobio, China, SEQ ID NO: 53-60) and mc-vc-PAB-MMAE (MCE, USA, HY-15575-G) (i.e., control IgG1-MMAE) and conjugates of anti-HER2 antibody (Biobio, China, BTAT008) and mc-vc-PAB-MMAE (i.e., anti-HER2-MMAE) using the same method. As shown in Figure 2(A), the average DAR values ​​of the two were 4.05 and 3.87, respectively.

[0397] The aggregation rate analysis of 5H-MMAE, control IgG1-MMAE and anti-HER2-MMAE is shown in Figure 2(B). The monomer molecule proportion of all three is greater than 95%.

[0398] 2.2 Preparation of antibody-drug conjugates with DXd as toxicity load

[0399] The CD3L1 humanized antibody 5H was transferred from PBS to the conjugation buffer using ultrafiltration. After ultrafiltration, the antibody concentration was adjusted to 10 mg / mL. An appropriate volume of 10 mM TCEP aqueous solution was added to the antibody solution, resulting in a TCEP:antibody molar ratio of 5.5:1, and the mixture was incubated at 37°C for 2 hours. Next, an appropriate volume of 10 mM GGFG-DXd solution was added, resulting in a GGFG-DXd:antibody molar ratio of 12:1, and the mixture was incubated at 25°C for 2 hours. Subsequently, an appropriate volume of 100 mM cysteine ​​aqueous solution was added to the reaction system, resulting in a cysteine:antibody molar ratio of 4:1, and the reaction was terminated by stirring at room temperature for 20 minutes. The product (5H-DXd) was then transferred to PBS using ultrafiltration. In addition, this example also prepared a conjugate of anti-HER2 antibody (Baiquan Biotechnology, China, BTAT008) and GGFG-DXd (i.e., anti-HER2-DXd) using the same method. The UV absorbance of the obtained antibody-drug conjugates at two wavelengths, 280 nm and 370 nm, was measured to calculate the average DAR value of 5H-DXd and anti-HER2-DXd. The calculation formula is: C D / C A C D and C A The value can be obtained from the following two equations: A 280 =ε D,280 C D +ε A,280 C A (Formula I); A 370 =ε D,370 C D +ε A,370 C A (Formula II).

[0400] Here, A 280 A represents the absorbance of the antibody-drug conjugate solution at 280 nm. 370 ε represents the absorbance of the antibody-drug conjugate solution at 370 nm. A,280 ε represents the molar absorptivity of the antibody at 280 nm. A,370 ε represents the molar absorptivity of the antibody at 370 nm. D,280 ε represents the molar absorptivity of the drug at 280 nm. D,370 C represents the molar absorptivity of the drug at 370 nm.A C represents the antibody concentration in the antibody-drug conjugate. D This indicates the drug concentration in the antibody-drug conjugate.

[0401] Analysis showed that the average DAR values ​​of 5H-DXd and anti-HER2-DXd were 6.7 and 6.3, respectively.

[0402] The preparation methods for other antibody-DXd conjugates are similar to those for 5H-DXd.

[0403] 2.3 Preparation of antibody-drug conjugates with DM1 as the toxicity load

[0404] Anti-CD3L1 antibody and anti-HER2 antibody (Baiquan Biotechnology, China, BTAT008) were exchanged from PBS into buffer A. Antibodies were modified with SMCC (succinimide-maleimide crosslinking agent) at a concentration of 20 mg / mL and an excess of 7.5 moles. The reaction was carried out in 5% DMSO (v / v) buffer A with stirring at room temperature for 2 h. A 1.5 × 4.9 cm Sephadex G25 resin (Sigma, USA, G25150) pre-packed column was equilibrated in buffer A. The anti-CD3L1 antibody-SMCC reaction mixture was then passed through the packed column for gel filtration, and the concentration of the modified antibody solution was analyzed using spectrophotometry / extinction coefficient method. Antibodies were dissolved in 6% DMA (v / v) buffer A. At a concentration of 10 mg / mL antibody, DM1 was added in an excess of 1.7 times the amount of linkers (assuming 5 linkers per antibody). The reaction was carried out with stirring at room temperature for 16.5 h. A 1.5 × 4.9 cm Sephadex G25 resin (Sigma, USA, G25150) pre-packed column was equilibrated in buffer B. The ligation reaction mixture was passed through the packed column and gel filtered. The absorbance of the eluent was measured at 252 nm and 280 nm to determine the number of DM1 molecules ligated per mole of antibody. Analysis showed that the average DAR values ​​for 5H-DM1 and anti-HER2-DM1 were 3.55 and 3.64, respectively.

[0405] The preparation methods for other antibody-DM1 conjugates are similar to those for 5H-DM1.

[0406] Example 3: Comparison of antigen-binding affinity between CD3L1 humanized antibody 5H and its drug conjugate

[0407] 3.1 Enzyme-linked immunosorbent assay (ELISA) to compare the binding of 5H antibody and 5H-MMAE to CD3L1.

[0408] To verify the binding affinity of 5H and 5H-MMAE to CD3L1, ELISA was used to confirm their direct binding to the CD3L1 protein. A dedicated ELISA plate (Costar, USA) was used. First, the CD3L1-his recombinant protein was diluted to 0.2 μg / mL with ELISA coating buffer (Solepro, China), and 100 μL of this buffer was applied to each well. For the negative control, 100 μL of coating buffer without CD3L1-his recombinant protein was used. Coating was performed overnight at 4°C. After washing with PBST, 100 μL of 5% BSA (VWR, USA) dissolved in PBS was added for blocking, and the plate was incubated at 37°C for 90 minutes. After washing with PBST, serially diluted 5H antibody, 5H-MMAE, and control IgG1-MMAE were added, and the plate was incubated at 37°C for 60 minutes. The serial dilutions were performed as follows: eight 1.5 mL centrifuge tubes were taken and numbered 1-8, and 300 μL of PBS was added to each tube as diluent. Add a certain amount of 5H antibody stock solution to the first centrifuge tube to a final concentration of 8 μg / mL, and mix thoroughly by tapping with a pipette. Then, take 100 μL of the mixture from the first centrifuge tube and add it to the second centrifuge tube, mixing thoroughly by pipetting. Repeat the above steps, taking 100 μL of the diluent from the previous centrifuge tube and adding it to the next, until the eighth centrifuge tube has been diluted. The procedures for 5H-MMAE and control IgG1-MMAE are the same. After washing with PBST, incubate with goat anti-human IgG Fcγ antibody (JIR, USA, 109-035-098) diluted in PBS at 37°C for 30 minutes. After washing with PBST, add 100 μL of chromogenic reagent (Solepro, China) to each well, incubate for 15 minutes, then add stop solution and read the value at 450 nm using a microplate reader. Figure 3(A) shows the ELISA results of the binding of 5H antibody, 5H-MMAE, and control IgG1-MMAE to CD3L1 protein. The experiment shows that both 5H and 5H-MMAE have a strong affinity for the CD3L1-his recombinant protein, and their EC50 values ​​are high. 50 The concentrations were 0.02262 and 0.02924 μg / mL, respectively. The antibody-drug conjugates produced under this conjugation method did not exhibit reduced affinity for their antigen-binding sites. Furthermore, no binding was observed between the control IgG1-MMAE and the CD3L1-his recombinant protein, indicating that the binding of 5H and 5H-MMAE to the CD3L1-his recombinant protein is specific.

[0409] 3.2 Flow cytometry comparison of the binding of 5H antibody and 5H-MMAE to CD3L1

[0410] RD cells washed with PBS (1×10⁻⁶) 6Cells / tubes were fixed with 4% paraformaldehyde at room temperature for 15 minutes. After cell fixation, the cells were washed twice with PBS. Primary antibodies 5H and 5H-MMAE were serially diluted 4-fold from 40 μg / mL to 0.0098 μg / mL using cell staining buffer. Cells were resuspended in 100 μL of each concentration solution. The blank control group was directly resuspended in staining buffer. After incubation on ice for 60 minutes, the cells were washed twice. 100 μL of FITC-labeled goat anti-human IgG fluorescent secondary antibody (Invitrogen, USA, 31529) diluted 1:200 with staining buffer was added to each tube to resuspend the cells. The cells were incubated on ice for 30 minutes, followed by washing and flow cytometry analysis. As shown in Figure 3(B), the relative geometric mean fluorescence intensity represents the percentage of the highest geometric mean fluorescence intensity at each primary antibody concentration. The results showed that both 5H and 5H-MMAE strongly bound to CD3L1-positive RD tumor cells, EC... 50 The values ​​were 0.4619 and 0.9352 micrograms per milliliter, respectively. The antibody-drug conjugates produced under this conjugation method did not significantly affect their affinity for cell surface antigens.

[0411] Example 4: Flow cytometry detection of CD3L1 expression in different cell lines

[0412] Take different types of tumor cells (1×10) 6 Cells / tube), after washing with PBS, were diluted with cell staining buffer to 20 μg / mL with primary antibody 5H-MMAE and control IgG1-MMAE. 100 μL of 5H-MMAE dilution was used to resuspend cells in each tube of the experimental group, and 100 μL of control IgG1-MMAE dilution was used in each tube of the negative control group. After incubation on ice for 60 minutes, the cells were washed twice. 100 μL of FITC-labeled goat anti-human IgG fluorescent secondary antibody (Invitrogen, USA) diluted 1:200 with staining buffer was added to each tube. The cells were resuspended and incubated on ice for 30 minutes, followed by washing and flow cytometry analysis. Figure 4 shows the fluorescence intensity of each cell line bound to 5H-MMAE and control IgG1-MMAE. The results showed that in the 5H-MMAE experimental group, except for the HCT116 CD3L1 knockout cells which did not show obvious CD3L1 expression, the other cell lines all had high levels of CD3L1 expression; in the control IgG1-MMAE group, the expression of CD3L1 was lower than that in the 5H-MMAE experimental group.

[0413] Example 5: Cell endocytosis experiment of CD3L1 humanized antibody 5H and its drug conjugate

[0414] 5.1 Intracellular endocytosis assay of CD3L1 humanized antibody 5H

[0415] The pH-sensitive dye (Promega, USA, G9841) fluoresces strongly in the acidic pH environment of endosomes and lysosomes. 5H antibody and control IgG1 antibody were labeled with the pH-sensitive dye according to the product instructions. RD cells were cultured at 5 × 10⁶ cells per well. 4 Cells were seeded in 20 mm confocal culture dishes (Nex, China). After cell adhesion, 10 μg / mL of 5H antibody conjugated with a pH-sensitive dye and control IgG1 antibody were added to the experimental and control wells, respectively, and the cells were incubated in a cell culture incubator for 24 hours. After endocytosis, the cells were washed twice with PBS and photographed under a confocal microscope; the results are shown in Figure 5. The experiment shows that the CD3L1 humanized antibody 5H provided by this invention can be endocytosed into endosomes and lysosomes in large quantities, while the control IgG1 antibody only shows a small amount of weak fluorescence due to nonspecific endocytosis.

[0416] 5.2 Intracellular endocytosis assay of CD3L1 humanized antibody-drug conjugate 5H-ADC

[0417] RD cells were placed at a density of 1 × 10⁶ cells per well. 5Cells were seeded in 20 mm confocal culture dishes. After cell adhesion, 10 μg / mL 5H-MMAE was added to each well, and the cells were incubated on ice for 1 hour. After washing with PBS, complete culture medium was added, and the cells were placed in a cell culture incubator. At different time points, the cells were removed from the incubator and placed on ice to terminate endocytosis. After washing twice with PBS, 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 30 minutes. After thorough washing with PBS, permeabilization blocking buffer containing 0.3% Triton and 3% BSA was added to each well, and the cells were incubated at room temperature for 1 hour. Subsequently, the corresponding organelle-labeled antibodies (rabbit EEA1 antibody, CST, 3288T, labeled early endosomes; rabbit RAB7 antibody, CST, 9367T, labeled late endosomes; rabbit LAMP1 antibody, CST, 9091T, labeled lysosomes; rabbit RAB11 antibody, CST, 5589T, labeled circulating endosomes) diluted with permeabilization blocking buffer were added, and the cells were incubated overnight at 4°C. After washing with PBS, different fluorescently labeled secondary antibodies (goat anti-human antibody labeled Alexa Fluor 488, Invitrogen, A-11013; goat anti-rabbit antibody labeled Alexa Fluor 594, Invitrogen, A-11012) were added to each well and incubated at room temperature for 2 hours. Finally, DAPI staining was performed for 5 minutes. After washing, images were taken under a confocal microscope, and the results are shown in Figure 6. In the figure, green fluorescence indicates the location of 5H-MMAE, and red fluorescence indicates the location of different organelle marker antibodies. The figure shows that before endocytosis (0 minutes), 5H-MMAE binds to the RD cell membrane. As the incubation time at 37°C increased, 5H-MMAE began to translocate between organelles. To dynamically quantify the colocalization of 5H-MMAE with different organelle marker antibodies, we analyzed the tM1 (Mander's colocalization coefficients, representing the ratio of green to red fluorescence colocalization) of multiple regions of interest (ROIs) and plotted their changes over time. The trends shown in the curves indicate that 5H-MMAE, after being endocytosed by cells, is first transported to early endosomes, exhibiting the strongest colocalization with the early endosome marker EEA1 antibody at 30 minutes (mean tM1 0.84). As shown in the image, most of the green and red fluorescence in the field of view merges into yellow fluorescence. Subsequently, 5H-MMAE gradually concentrates in late endosomes and lysosomes. Significant colocalization of 5H-MMAE with the late endosome marker RAB7 antibody was observed at 120 minutes (mean tM1 0.79), and the degree of colocalization between 5H-MMAE and lysosomes continuously increases with incubation time, reaching significant colocalization at 240 minutes (mean tM1 0.76). Additionally, we also observed colocalization of 5H-MMAE with the circulating endosome marker RAB11 antibody.This indicates that after 5H-MMAE is endocytosed into the cell, it can be rapidly delivered to lysosomes, and some of it is also transported to circulating endosomal tissues.

[0418] 5.3 Comparison of endocytosis efficiency between CD3L1 humanized antibody-drug conjugate 5H-ADC and anti-HER2-ADC

[0419] After digesting RD cells and A375 cells, dispense 1×10-1 cells into each tube. 6 Cells were incubated on ice for 1 hour with saturated concentrations of 5H-MMAE and anti-HER2-MMAE, respectively. After washing twice with PBS, the cells were resuspended in complete culture medium and transferred to an incubator for endocytosis. Cells were harvested at a series of time points, then centrifuged, washed, and incubated with a 1:200 dilution of FITC-labeled goat anti-human IgG fluorescent secondary antibody (Invitrogen, USA, 31529) to bind any residual primary antibody on the cell surface. For the control group, cells were treated directly with the secondary antibody after primary antibody incubation without being placed in an incubator. As shown in Figure 7, RD cells showed a significant decrease in fluorescence intensity compared to the control group after 30 minutes of co-incubation with 5H-MMAE at 37°C, indicating that most of the 5H-MMAE had been internalized during this period. However, A375 cells did not show a significant decrease in fluorescence intensity compared to the control group after 4 hours of co-incubation with anti-HER2-MMAE at 37°C, indicating that the anti-HER2-MMAE had not yet been significantly internalized. This demonstrates that the endocytosis efficiency of 5H-ADC is significantly better than that of anti-HER2-ADC prepared under the same conditions.

[0420] Example 6: In vitro cytotoxic activity of CD3L1 humanized antibody-drug conjugate.

[0421] 6.1 Comparison of cytotoxicity between CD3L1 humanized antibody-drug conjugate 5H-ADC, control IgG1-ADC, and CD3L1 humanized antibody 5H

[0422] CD3L1 knockout cell lines with different CD3L1 expression levels (RD, RKO, A375, NCIH520, MCF7, HCT116, and HCT116) were seeded in 96-well plates and incubated overnight. The following day, 5H antibody, 5H-MMAE, and control IgG1-MMAE were added to the microplates at 2-fold serial dilutions, starting at the same concentration. Blank control wells were also included. After 5 days of incubation, CCK8 assay solution (APExBIO, USA) was added, and absorbance at 450 nm was measured using a microplate reader. Cell viability (%) = (OD...) 药物 -OD 空白 / OD 对照 -OD 空白The half-maximal inhibitory concentration (MCI) was calculated using Prism software with cell viability as the y-value and drug concentration as the x-value for four-parameter curve fitting. The results are shown in Figure 8. The experiment showed that within the set concentration range, the 5H antibody had no killing effect on cells at any expression level. 5H-MMAE exhibited significant killing activity against CD3L1-expressing cell lines, killing cells even at low concentrations. However, it only showed weak killing activity against CD3L1 knockout cells at high concentrations. Its MCI for A375, RKO, RD, HCT116, NCIH520, and MCF7 cell lines was 1.406, 0.436, 1.780, 0.942, 6.488, and 9.800 μg / mL, respectively. Cells treated with the control IgG1-MMAE were only partially killed at high drug concentrations, and the killing efficiency at these concentrations was much weaker than that of the 5H-MMAE-treated group.

[0423] Although existing technologies have shown that anti-CD3L1 antibodies have good anti-tumor effects, anti-CD3L1-ADCs still exhibit significantly better tumor cell killing effects than anti-CD3L1 antibodies.

[0424] 6.2 Comparison of tumor cell killing activity between anti-CD3L1 antibody-drug conjugates and anti-HER2 antibody-drug conjugates

[0425] Multiple tumor cell lines were seeded at 1000-3000 cells / well / 50 μL in 96-well plates and incubated overnight. The next day, 50 μL of different concentrations of anti-CD3L1-ADC or anti-HER2-ADC were added to each well. Control wells and blank wells without drugs and cells were also included. After incubation for 4-5 days, CCK8 (APExBIO, USA) assay solution was added, and the reaction was carried out at 37°C for 2-3 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = (OD0.05) / (Cell Viability (%)) 药物 -OD 空白 / OD 对照 -OD 空白 The above data were analyzed using Prism software, and the IC50 of each antibody-drug conjugate in different cell lines was calculated. 50 Values. The VH and VL sequences of the CD3L1 antibody used in this embodiment include, but are not limited to, the amino acid sequences shown in SEQ ID NO:3-4, 11-14, and 17-24.

[0426] Figure 9 shows the inhibition curves of different sequences of anti-CD3L1-MMAE and anti-HER2-MMAE drug conjugates on the growth of NCI-H520, UMUC3, RD, A2780, A375, and Raji tumor cells. The table below shows their corresponding IC50 values. 50 Different sequence anti-CD3L1-MMAE drug conjugates exhibited good cytotoxic activity in various types of tumor cells. Among them, 5H-MMAE showed significantly better cytotoxic activity against all tumor cells than anti-HER2-MMAE. Other sequence anti-CD3L1-MMAE (e.g., 5B11-MMAE, 43B2-MMAE, 13B7-MMAE, etc.) showed better tumor cell killing activity than anti-HER2-MMAE.

[0427] This embodiment tested the cytotoxic effects of different sequence anti-CD3L1 antibodies conjugates with DXd in different types of solid tumor and hematological malignancy cell lines, and compared their cytotoxic effects with anti-HER2-DXd. The results showed that, under the same treatment conditions, different sequence anti-CD3L1-DXd exhibited superior cytotoxic effects compared to anti-HER2-DXd in the tested tumor cell lines. The cytotoxic activity of 5H-DXd was stronger than that of anti-HER2-DXd at the same concentration. 5H-DXd inhibited the growth of A2780, RD, A375, Raji, U87, NCI-H520, UMUC3, and Jurkat tumor cells by an IC50 value. 50 The values ​​were all lower than those of anti-HER2-DXd (Figure 10-11); 5B11-DXd, 41-DXd, and 46-DXd inhibited the growth of A2780, A375, RD, and Jurkat tumor cells. 50 The values ​​were also lower than those for anti-HER2-DXd (Figure 12).

[0428] This embodiment tested the killing effects of different sequence anti-CD3L1 antibodies conjugates with DM1 in different types of solid tumors and hematological malignancies, and compared them with the killing effects of anti-HER2-DM1. The results showed that, under the same treatment conditions, the tumor cell killing effects of different sequence anti-CD3L1-DM1 in the tested tumor cell lines were weaker than those of anti-HER2-DM1. Furthermore, the killing activity of anti-HER2-DM1 against the tumor cells shown in the figure was slightly better than that of 5H-DM1, with an IC50 value of [missing information]. 50 The values ​​were all lower than those of 5H-DM1 (Figure 13); the killing activity of 5B11-DXd and 46-DXd against tumor cells was comparable to that of 5H-DM1 (Figure 14).

[0429] In addition, the same method was used to verify the cytotoxicity of antibody-drug conjugates using other therapeutic agents such as MMAF, DM4, cacimycin, pyromycin, PBD, and SN-38.

[0430] Example 7: Pharmacodynamic assay of CD3L1 humanized antibody-drug conjugate 5H-ADC in different subcutaneous xenograft tumor models.

[0431] 7.1 Efficacy of 5H-MMAE in the treatment of human colorectal cancer cell line RKO and human rhabdomyosarcoma cell line RD in subcutaneous xenografts in BALB / cNude mice

[0432] RKO / RD cells (5 × 10⁻⁶) were subcutaneously injected into BALB / c Nude mice. 6 (each individual), until the tumor grows to 50-150 mm. 3 Tumors were randomly assigned to two groups based on tumor size: a control IgG1-MMAE treatment group and a 5H-MMAE treatment group. The 5H-MMAE was administered twice weekly at a dose of 5 mg / kg intraperitoneally for three weeks. Tumor length and short diameter were measured simultaneously with administration, and tumor volume was calculated as length × short diameter / 2. After administration, the tumors were dissected and weighed; the results are shown in Figures 15(A) and 15(B). The experiment demonstrated that, compared to the control IgG1-MMAE treatment group, the same dose of 5H-MMAE significantly inhibited the growth of RKO / RD xenografts. This indicates that the efficacy of ADCs depends on their binding to CD3L1.

[0433] 7.2 Efficacy of 5H-MMAE in the treatment of human melanoma cell line A375 in subcutaneous xenografts in BALB / c Nude mice

[0434] BALB / c Nude mice were subcutaneously inoculated with A375 cells (5 × 10⁻⁶). 6 (each individual), until the tumor grows to 50-150 mm. 3 At that time, patients were randomly assigned to three groups according to tumor size: a PBS group, a 5H antibody treatment group, and a 5H-MMAE treatment group. The drugs were administered twice weekly at a dose of 5 mg / kg intraperitoneally for three weeks. The long and short diameters of the tumor were measured simultaneously with drug administration. Tumor volume = long diameter × short diameter. 2 / 2. After administration, the tumors were dissected and weighed, and the results are shown in Figure 15(C). The experiment showed that in immunodeficient mice, 5H antibody did not have a significant tumor-suppressing effect compared to PBS, while the same dose of 5H-MMAE had a significant inhibitory effect on A375 xenografts, suggesting that 5H antibody coupled with a toxic load greatly enhanced its anti-tumor effect.

[0435] Example 8: Comparison of the efficacy of anti-CD3L1-ADC and anti-HER2-ADC in an A375 nude mouse subcutaneous xenograft model

[0436] BALB / c Nude mice were subcutaneously inoculated with A375 cells (5 × 10⁻⁶). 6 (each individual), until the tumor grows to 50-150mm 3 At approximately 10:00 AM, patients were randomly assigned to two groups based on tumor size: a 5H-DXd treatment group and an anti-HER2-DXd treatment group. The 5H-DXd treatment group received the drug 2 mg / kg every three days via intraperitoneal administration for a total of three administrations. The long and short diameters of the tumor were measured simultaneously with each administration. Tumor volume = long diameter × short diameter. 2 / 2. After the administration was completed, the tumor was dissected and photographed. The results are shown in Figure 16. The experiment showed that the same dose of 5H-DXd had a stronger inhibitory effect on A375 melanoma compared with anti-HER2-DXd.

[0437] Example 9: Comparison of the efficacy of anti-CD3L1-ADC, anti-HER2-ADC, and Isotype mAb-ADC in LoVo, SW480, and SW1116 subcutaneous xenograft tumor models of colorectal cancer in nude mice.

[0438] BALB / c Nude mice were subcutaneously inoculated with LoVo / SW480 / SW1116 cells (5 × 10⁻⁶). 6 (each individual), wait until the tumor grows to 50-150mm. 3 Tumors were randomly assigned to four groups based on tumor size: a PBS solvent control group, an Isotype-DXd treatment group, an anti-HER2-DXd treatment group, and a 5H-DXd treatment group. The tumors were administered 5 mg / kg intraperitoneally every three days for 2-3 weeks. The long and short diameters of the tumors were measured simultaneously with administration. Tumor volume = long diameter × short diameter. 2 / 2. After the administration was completed, the tumors were dissected and weighed. The results are shown in Figure 17. The experiment showed that the same dose of anti-CD3L1-DXd had a stronger inhibitory effect on colorectal cancer tumors such as LoVo, SW480, and SW1116 compared with anti-HER2-DXd.

[0439] Example 10: Immunohistochemical staining for detecting apoptosis markers in tumors

[0440] Caspase-3 is a key executor of apoptosis, responsible for the hydrolysis and cleavage of many crucial proteins. Activation of caspase-3 requires the hydrolysis of its inactive proenzyme form into activated p17 and p12 fragments. By staining tumor tissue for activated caspase-3 fragments, the degree of tumor cell apoptosis can be determined. After dissecting an A375 xenograft tumor, paraffin sections were prepared, and histochemical staining was performed using a specific antibody against the caspase-3 cleavage variant. The specific procedure is as follows:

[0441] (1) Dewaxing / hydration:

[0442] a. Incubate the slices in xylene solution three times, for 5 minutes each time.

[0443] b. Incubate the slices in 100% ethanol twice, for 10 minutes each time.

[0444] c. Incubate the slices in 95% ethanol twice, 10 minutes each time.

[0445] d. Rinse the slides twice with deionized water for 5 minutes each time.

[0446] (2) Antigen retrieval:

[0447] Immerse the sections in 1× citrate retrieval solution, then microwave until boiling; continue to maintain a sub-boiling temperature for 10 minutes (95℃-98℃). Cool the sections for 30 minutes.

[0448] (3) Staining:

[0449] a. Wash the slides three times with deionized water, 5 minutes each time.

[0450] b. Place the slices in a 3% hydrogen peroxide aqueous solution and incubate for 10 minutes.

[0451] c. Wash the slides twice with deionized water, 5 minutes each time.

[0452] d. Wash the slides with washing buffer (1×Tris buffer containing Tween 20) for 5 minutes.

[0453] e. Use 200 μL of blocking solution (CST, USA) to block each slide at room temperature for 1 hour.

[0454] f. Remove the blocking solution and add 200 μL of diluted primary antibody to each slice, incubate overnight at 4°C.

[0455] g. Remove antibody solution and wash the slides three times with washing buffer for 5 minutes each time.

[0456] h. Place 2 drops on the slice Boost Detection Reagent (CST, USA)

[0457] Incubate at room temperature in a humidifier for 30 minutes.

[0458] i. Wash the slides three times with washing buffer, five minutes each time.

[0459] j. Add 200 microliters to each slice DAB (CST, USA), incubate for 10 minutes.

[0460] k. Immerse the slices in deionized water.

[0461] (4) Dehydration:

[0462] a. Incubate the slices in 95% ethanol twice, for 10 seconds each time.

[0463] b. Incubate the slices in 100% ethanol twice, for 10 seconds each time.

[0464] c. Incubate the slices in xylene solution twice, 10 seconds each time.

[0465] (5) Use coverslips and mounting medium (CST, USA) to mount the slide.

[0466] The results are shown in Figure 18. The experiment showed that, compared with the 5H antibody control group, the tumor tissue samples of the 5H-MMAE treatment group had a wider range of caspase 3 splice positive staining cells, indicating that 5H-MMAE effectively penetrated into the tumor tissue and induced apoptosis of tumor cells.

[0467] The experimental methods listed in the above embodiments are equally applicable to experiments on other antibodies and antibody-drug conjugates of this application. The above embodiments are merely illustrative of the technical solutions of this application and are not intended to limit the scope of this application. Other anti-CD3L1 antibodies and other antibody-drug conjugates intended to be protected in this application also possess the corresponding effects.

[0468] Table A. Sequence Information

[0469] By incorporating references

[0470] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0471] Equivalence

[0472] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. An antibody-drug conjugate having the general structural formula: A-(LU) n in: A is an antibody or its antigen-binding fragment that specifically binds to CD3L1; The U is a therapeutic agent, and each U may be the same or different; The L is a connector, and each L can be the same or different; n is an integer greater than 1, preferably an integer selected from 1 to 8.

2. The antibody-drug conjugate according to claim 1, wherein, The U is selected from the group consisting of cytotoxic molecules, immune enhancers, immunosuppressants, cell inhibitors, chemotherapeutic agents, radiotherapy agents, anti-angiogenic agents, anti-metastatic agents, targeted anticancer agents, other anticancer agents, and radioisotopes.

3. The antibody-drug conjugate according to claim 1 or 2, wherein, The U is selected from microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, DNA alkylating agents, RNA polymerase inhibitors, RNA cleavage enzyme inhibitors, Bcl-xL inhibitors, antimitotic agents, sodium-potassium ion pump ATPase inhibitors, matrix metalloproteinase inhibitors, and / or immune agonists.

4. The antibody-drug conjugate according to claim 3, wherein, The microtubule inhibitor is selected from sarsaparilla toxin and olistatin cytotoxic molecules (e.g., MMAE, MMAF), and / or maytansine cytotoxic molecules (e.g., DM1, DM4); and / or The DNA damaging agents include those selected from chachiin, pyromycin, atrazomycin derivatives (e.g., PBD), and / or camptothecin and camptothecin derivatives (e.g., SN-38, Dxd).

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein, The U is selected from DXd and / or MMAE.

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein, The linker is covalently linked to an amino or thiol residue on the antibody or its antigen-binding fragment.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein, The connectors include spliable connectors and non-spliable connectors.

8. The antibody-drug conjugate according to claim 7, wherein, The cleavable linker comprises a peptide unit containing 2-20 amino acids, preferably selected from any one or a combination of -valine-citrulline-, -glycine-glycine-phenylalanine-glycine-, -valine-alanine-, -valine-lysine-, -valine-arginine-, -phenylalanine-citrulline-, -phenylalanine-lysine-, and -phenylalanine-arginine-.

9. The antibody-drug conjugate according to claim 7, wherein, The cleavable linker comprises a structure selected from maleimide-hexanoyl (MC), maleimide-butyric acid (MB), or p-aminobenzyloxycarbonyl (PAB).

10. The antibody-drug conjugate according to claim 7, wherein, The cleavable linker is selected from maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleimide-hexanoyl-valine-alanine (MC-VA), maleimide-butyric acid-valine-citrulline (MB-VC), glycine-glycine-phenylalanine-glycine (GGFG, SEQ ID NO:61), maleimide-hexanoyl-glycine-glycine-phenylalanine-glycine (MC-GGFG, SEQ ID NO:62) or valine-citrulline-p-aminobenzyloxycarbonyl (VC-PAB).

11. The antibody-drug conjugate according to claim 7, wherein, The cleavable linker is a linker that can be cleaved under acidic conditions, preferably a linker that cleaves in endosomes or lysosomes.

12. The antibody-drug conjugate according to any one of claims 1 to 11, wherein, The antibody or its antigen-binding fragment that specifically binds to CD3L1 can bind to the amino acid sequence in CD3L1, such as SEQ ID NO:1 or SEQ ID NO:

2.

13. The antibody-drug conjugate according to any one of claims 1 to 12, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 contains the amino acid sequence shown in SEQ ID NO:5, HCDR2 contains the amino acid sequence shown in SEQ ID NO:6, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:

7.

14. The antibody-drug conjugate according to any one of claims 1 to 13, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:9, and LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:10 or SEQ ID NO:

16.

15. The antibody-drug conjugate according to any one of claims 1 to 12, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:

13. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:

14.

16. The antibody-drug conjugate according to any one of claims 13 to 15, wherein, The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to the amino acid sequence shown therein; and / or The light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to them.

17. The antibody-drug conjugate according to any one of claims 13 to 16, wherein, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

4.

18. The antibody-drug conjugate according to any one of claims 1 to 12, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 contains the amino acid sequence shown in SEQ ID NO:27, HCDR2 contains the amino acid sequence shown in SEQ ID NO:28 or SEQ ID NO:35, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:

36.

19. The antibody-drug conjugate according to any one of claims 1 to 12 and 18, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:30, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:31 and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32 or SEQ ID NO:

37.

20. The antibody-drug conjugate according to any one of claims 1 to 12, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25 or SEQ ID NO:

33. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26 or SEQ ID NO:

34.

21. The antibody-drug conjugate according to any one of claims 18 to 20, wherein, The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:25, SEQ ID NO:33, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to them; and / or The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26, SEQ ID NO:34, or an amino acid sequence with greater than 80%, 85%, 90%, 95%, or 99% identity with it.

22. The antibody-drug conjugate according to any one of claims 18 to 21, wherein, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:25, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

26.

23. The antibody-drug conjugate according to any one of claims 18 to 21, wherein, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:33, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

34.

24. The antibody-drug conjugate according to any one of claims 1 to 12, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 contains an amino acid sequence as shown in SEQ ID NO:40 or SEQ ID NO:47, HCDR2 contains an amino acid sequence as shown in SEQ ID NO:41 or SEQ ID NO:48, and HCDR3 contains an amino acid sequence as shown in SEQ ID NO:42 or SEQ ID NO:

49.

25. The antibody-drug conjugate according to any one of claims 1 to 12 and 24, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:43 or SEQ ID NO:50, LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:51, and LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:44 or SEQ ID NO:

52.

26. The antibody-drug conjugate according to any one of claims 1 to 12, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:38 or SEQ ID NO:

45. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:39 or SEQ ID NO:

46.

27. The antibody-drug conjugate according to any one of claims 24 to 26, wherein, The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:38, SEQ ID NO:45, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to such sequences; and / or The light chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having greater than 80%, 85%, 90%, 95%, or 99% identity with them.

28. The antibody-drug conjugate according to any one of claims 24 to 27, wherein, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

39.

29. The antibody-drug conjugate according to any one of claims 24 to 27, wherein, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:45, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

46.

30. The antibody-drug conjugate according to any one of claims 1 to 29, wherein, The antibody that specifically binds to CD3L1 is a monoclonal antibody.

31. The antibody-drug conjugate according to any one of claims 1 to 30, wherein, The antibody that specifically binds to CD3L1 is a chimeric antibody or a humanized antibody.

32. An antibody-drug conjugate with the general structural formula: A-(LU) n in: A is an antibody or its antigen-binding fragment that specifically binds to CD3L1, wherein, The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:3, SEQ ID NO:11, or SEQ ID NO:13; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:14; and / or The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:7; and / or The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:15, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:16; and / or The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to the amino acid sequence shown therein; and / or The light chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to them. The U is a therapeutic agent, and each U may be the same or different, and it is selected from MMAE or DXd; The L is a connector, and each L can be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and n is an integer selected from 1 to 8.

33. An antibody-drug conjugate with the general structural formula: A-(LU) n in: A is an antibody or its antigen-binding fragment that specifically binds to CD3L1, wherein, The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25 or SEQ ID NO:33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26 or SEQ ID NO:34; and / or The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28 or SEQ ID NO:35, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:36; and / or The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:30, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:31, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:32 or SEQ ID NO:37; and / or The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:25, SEQ ID NO:33, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% compared to it; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:26, SEQ ID NO:34, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% compared to it; The U is a therapeutic agent, and each U may be the same or different, and it is selected from MMAE or DXd; The L is a connector, and each L can be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and n is an integer selected from 1 to 8.

34. An antibody-drug conjugate with the general structural formula: A-(LU) n in: A is an antibody or its antigen-binding fragment that specifically binds to CD3L1, wherein, The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:38 or SEQ ID NO:45; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:39 or SEQ ID NO:46; and / or The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:40 or SEQ ID NO:47, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:48, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:42 or SEQ ID NO:49; and / or The antibody or antigen-binding fragment that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:43 or SEQ ID NO:50, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:51, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:44 or SEQ ID NO:52; and / or The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:38, SEQ ID NO:45, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to such sequences; and / or The light chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to them. The U is a therapeutic agent, and each U may be the same or different, and it is selected from MMAE or DXd; The L is a connector, and each L can be the same or different, and it is selected from Mc-Val-Cit-PAB or Gly-Gly-Phe-Gly (GGFG); and n is an integer selected from 1 to 8.

35. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 34, and a pharmaceutically acceptable carrier.

36. A kit comprising a container, a formulation disposed in the container, and optionally instructions for use; wherein the formulation comprises an antibody-drug conjugate according to any one of claims 1 to 34 or a pharmaceutical composition according to claim 35.

37. Use of the antibody-drug conjugate according to any one of claims 1 to 34 or the pharmaceutical composition according to claim 35 in the preparation of an antitumor drug, preferably, the tumor is a tumor that has not responded well to treatment with antibody-drug conjugates targeting HER2.

38. Use of the antibody-drug conjugate of any one of claims 1 to 34 or the pharmaceutical composition of claim 35 in the preparation of a product for the treatment or prevention of tumors.

39. The use according to claim 37 or 38, wherein the tumor is a solid tumor or hematologic malignancy expressing CD3L1.

40. The use according to claim 37 or 38, wherein the tumor is a CD3L1 positive tumor, including melanoma, sarcoma, lung cancer, breast cancer, thyroid cancer, nervous system tumors, kidney cancer, urothelial carcinoma, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, head and neck cancer, leukemia, lymphoma, and other CD3L1 positive associated tumors.

41. Use of the antibody-drug conjugate of any one of claims 1 to 34 or the pharmaceutical composition of claim 35 in the preparation of a medicament for treating diseases related to CD3L1 expression.

42. An antibody or antigen-binding fragment thereof that specifically binds to CD3L1, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 contains the amino acid sequence shown in SEQ ID NO:27, HCDR2 contains the amino acid sequence shown in SEQ ID NO:28 or SEQ ID NO:35, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:

36. The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains the amino acid sequence shown in SEQ ID NO:30, LCDR2 contains the amino acid sequence shown in SEQ ID NO:31 and LCDR3 contains the amino acid sequence shown in SEQ ID NO:32 or SEQ ID NO:

37.

43. The antibody or antigen-binding fragment thereof that specifically binds to CD3L1 according to claim 42, wherein, The antibody that specifically binds to CD3L1 comprises: (1) HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO:29, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:32; or (2) HCRD1 as shown in SEQ ID NO:27, HDCR2 as shown in SEQ ID NO:35, HCDR3 as shown in SEQ ID NO:36, LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:

37.

44. An antibody or antigen-binding fragment thereof that specifically binds to CD3L1, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:25 or SEQ ID NO:

33. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:26 or SEQ ID NO:

34.

45. The antibody or antigen-binding fragment thereof that specifically binds to CD3L1 according to any one of claims 42 to 44, wherein, The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:25, SEQ ID NO:33, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to them; and / or The light chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:34, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to them.

46. ​​The antibody or antigen-binding fragment thereof that specifically binds to CD3L1 according to any one of claims 42 to 45, wherein, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:25, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26; or The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:33, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

34.

47. An antibody or antigen-binding fragment thereof that specifically binds to CD3L1, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. HCDR1 contains an amino acid sequence as shown in SEQ ID NO:40 or SEQ ID NO:47, HCDR2 contains an amino acid sequence as shown in SEQ ID NO:41 or SEQ ID NO:48, and HCDR3 contains an amino acid sequence as shown in SEQ ID NO:42 or SEQ ID NO:

49. The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:43 or SEQ ID NO:50, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:9 or SEQ ID NO:51, and LCDR3 contains an amino acid sequence as shown in SEQ ID NO:44 or SEQ ID NO:

52.

48. The antibody or antigen-binding fragment thereof that specifically binds to CD3L1 according to claim 47, wherein, The antibody that specifically binds to CD3L1 comprises: HCRD1 as shown in SEQ ID NO:40, HDCR2 as shown in SEQ ID NO:41, HCDR3 as shown in SEQ ID NO:42, LCDR1 as shown in SEQ ID NO:43, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:44; or HCRD1 as shown in SEQ ID NO:47, HDCR2 as shown in SEQ ID NO:48, HCDR3 as shown in SEQ ID NO:49, LCDR1 as shown in SEQ ID NO:50, LCDR2 as shown in SEQ ID NO:51, and LCDR3 as shown in SEQ ID NO:

52.

49. An antibody or antigen-binding fragment thereof that specifically binds to CD3L1, wherein, The antibody that specifically binds to CD3L1 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region VH as shown in SEQ ID NO:38 or SEQ ID NO:

45. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region VL as shown in SEQ ID NO:39 or SEQ ID NO:

46.

50. The antibody or antigen-binding fragment thereof that specifically binds to CD3L1 according to any one of claims 47 to 49, wherein, The heavy chain variable region comprises an amino acid sequence selected from, for example, SEQ ID NO:38, SEQ ID NO:45, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to such sequences; and / or The light chain variable region comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:46, or an amino acid sequence having an identity greater than 80%, 85%, 90%, 95%, or 99% with respect to them.

51. The antibody or antigen-binding fragment thereof that specifically binds to CD3L1 according to any one of claims 47 to 50, wherein, The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39; or The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:45, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:46.

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