Antibodies and uses thereof

Abstract

Relates to single domain antibodies or antigen binding fragments thereof targeting MSLN humanization or PD-L1. The invention further relates to a multispecific antibody, and particularly provides the multispecific antibody targeting CD3, tumor-associated antigens and immune checkpoints, and the multispecific antibody has good medication safety and an anti-tumor effect. The invention also relates to application of the single-domain antibody or the multispecific antibody in disease treatment.

Description

Antibodies and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202211646964.4 filed on December 21, 2022 and Chinese patent application No. 202310771156.9 filed on June 27, 2023, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to single-domain antibodies targeting MSLN or PD-L1. The present invention also relates to multispecific antibodies, specifically providing multispecific antibodies targeting CD3, tumor-associated antigens, and immune checkpoints. The present invention also relates to the use of such single-domain antibodies or multispecific antibodies in the treatment of diseases. Background Art

[0004] Mesothelin (MSLN) is a cell surface glycoprotein encoded by the MSLN gene. Compared to normal tissues, MSLN is highly expressed in a variety of cancers, including mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, bile duct cancer, endometrial cancer, thymic cancer, colon cancer, and breast cancer. Its abnormal expression plays an important role in tumor cell proliferation, adhesion, and drug resistance. PD-L1, also known as B7-H1 or CD274, is the first characterized functional ligand for the coinhibitory programmed death receptor 1 (PD-1). Studies have found that PD-L1 is highly expressed in different types of tumors, including melanoma, ovarian cancer, lung cancer, and renal cancer. PD-L1 plays a key role in preventing autoimmunity in normal tissues and maintaining peripheral tolerance. Tumor cells can exploit the PD-1 / PD-L1 signaling pathway to evade antitumor immune responses and ultimately spread and metastasize. Therefore, blocking the PD-1 / PD-L1 signaling pathway can activate the endogenous anti-tumor immune response, thereby exerting a therapeutic effect on tumors.

[0005] Currently, most MSLN or PD-L1 antibodies in the field are traditional IgG heavy-light chain antibodies. Nanobodies, also known as single-domain antibodies (sdAbs), are composed solely of heavy chains and have the characteristics of small molecular weight, good stability, and strong penetrability. However, there is currently a lack of satisfactory nanobodies targeting MSLN or PD-L1 in the field. Therefore, there is a need to develop new and effective nanobodies specifically targeting MSLN or PD-L1. In addition, T cell-activating CD3 bispecific antibody (T Cell Engage, TCE) drugs for the treatment of solid tumors have attracted widespread attention. TCEs contain two domains: one that binds to CD3 on T cells and the other that targets and binds to cancer cells. Their molecular design is designed to help T cells recognize cancer cells and fully activate them, releasing cytokines such as TNFα, perforin, granzyme B, and IFN-γ to kill cancer cells. MSLN, as a TAA for solid tumors, is still under investigation for related multi-antibody drugs, among which HPN536 has made rapid clinical progress. However, in clinical applications, TCE is not very effective in treating solid tumors, and there are risks of CRS or T cell dysfunction caused by excessive T cell activation. Therefore, there is a need to develop T cell activating drugs with better performance in this field.

[0006] Summary of the Invention

[0007] To address the above issues, the present invention provides nanobodies targeting MSLN or PD-L1 with excellent properties, as well as multispecific antibodies targeting CD3, tumor-associated antigens (TAAs), and immune checkpoints. The CD3-targeting portion can moderately activate T cells to improve drug safety, and further combining portions targeting tumor-associated antigens (TAAs) and immune checkpoints can significantly enhance anti-tumor efficacy. This provides the following aspects.

[0008] CD3 Antibody

[0009] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3, which has a moderate CD3 affinity, a moderate ability to activate T cells, and reduced cytokine release, thereby improving drug safety. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0010] The VH comprises HCDR1 shown in SEQ ID NO: 55, HCDR2 shown in SEQ ID NO: 56, and HCDR3 shown in any one of SEQ ID NOs: 57-59, 65; and / or,

[0011] The VL comprises LCDR1 shown in SEQ ID NO:60, LCDR2 shown in SEQ ID NO:61, and LCDR3 shown in SEQ ID NO:62.

[0012] In certain embodiments, the VH comprises the sequence shown in any one of SEQ ID NOs: 40-42, 64.

[0013] In certain embodiments, the VL comprises the sequence shown in SEQ ID NO:43.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises the VH shown in SEQ ID NO:40 and the VL shown in SEQ ID NO:43.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises the VH shown in SEQ ID NO:41 and the VL shown in SEQ ID NO:43.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises the VH shown in SEQ ID NO:42 and the VL shown in SEQ ID NO:43.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises the VH shown in SEQ ID NO:64 and the VL shown in SEQ ID NO:43.

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

[0019] MSLN single domain antibody

[0020] In a second aspect, the present invention relates to a humanized single domain antibody or an antigen binding fragment thereof that can specifically bind to MSLN. A single domain antibody is typically composed of four framework regions (FRs) and three complementary determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, and its antigen binding fragment comprises at least a portion of the single domain antibody, which portion is sufficient to confer upon the fragment the ability to specifically bind to an antigen (e.g., MSLN). The single domain antibody can be truncated at the N-terminus or C-terminus so that it comprises only a portion of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. The single domain antibody can be humanized to form a humanized single domain antibody, wherein one or more framework regions in its VHH have been substantially replaced by human framework regions. In certain embodiments, the humanized single-domain antibody or antigen-binding fragment thereof comprises a camelid CDR region and a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), wherein the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to camelid residues.

[0021] In certain embodiments, the humanized single-domain antibody or antigen-binding fragment thereof of the present invention comprises: a VHH sequence as shown in any one of SEQ ID NOs: 1-8, or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions; preferably, the substitutions are conservative substitutions.

[0022] In certain embodiments, the humanized single-domain antibody or antigen-binding fragment thereof of the present invention comprises a CDR1 as set forth in SEQ ID NO:52, a CDR2 as set forth in SEQ ID NO:53, and a CDR3 as set forth in SEQ ID NO:54, as defined by the IMGT numbering system.

[0023] In certain embodiments, the single domain antibody or antigen-binding fragment thereof specifically binds to human and / or cynomolgus monkey MSLN.

[0024] PD-L1 single domain antibody

[0025] In a third aspect, the present invention relates to a single domain antibody or an antigen-binding fragment thereof that can specifically bind to PD-L1.

[0026] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the IMGT numbering system: (1A) a CDR1 comprising the sequence shown in SEQ ID NO: 11 or 63 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 12 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 13 or a variant thereof; or (1B) a CDR1 comprising the sequence shown in SEQ ID NO: 68 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 69 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 70 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the IMGT numbering system: (1A) a CDR1 comprising the sequence shown in SEQ ID NO: 11 or 63, a CDR2 comprising the sequence shown in SEQ ID NO: 12, and a CDR3 comprising the sequence shown in SEQ ID NO: 13; or (1B) a CDR1 comprising the sequence shown in SEQ ID NO: 68, a CDR2 comprising the sequence shown in SEQ ID NO: 69, and a CDR3 comprising the sequence shown in SEQ ID NO: 70.

[0027] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Kabat numbering system: (2A) a CDR1 comprising the sequence shown in SEQ ID NO: 14 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 15 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 16 or a variant thereof; or (2B) a CDR1 comprising the sequence shown in SEQ ID NO: 71 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 72 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 73 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Kabat numbering system: a CDR1 comprising the sequence shown in SEQ ID NO: 14, a CDR2 comprising the sequence shown in SEQ ID NO: 15, and a CDR3 comprising the sequence shown in SEQ ID NO: 16; or (2B) a CDR1 comprising the sequence shown in SEQ ID NO: 71, a CDR2 comprising the sequence shown in SEQ ID NO: 72, and a CDR3 comprising the sequence shown in SEQ ID NO: 73.

[0028] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the AbM numbering system: (3A) a CDR1 comprising the sequence shown in SEQ ID NO: 17 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 18 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 16 or a variant thereof; or (3B) a CDR1 comprising the sequence shown in SEQ ID NO: 74 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 75 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 73 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the AbM numbering system: a CDR1 comprising the sequence shown in SEQ ID NO: 17, a CDR2 comprising the sequence shown in SEQ ID NO: 18, and a CDR3 comprising the sequence shown in SEQ ID NO: 16; or (3B) a CDR1 comprising the sequence shown in SEQ ID NO: 74, a CDR2 comprising the sequence shown in SEQ ID NO: 75, and a CDR3 comprising the sequence shown in SEQ ID NO: 73.

[0029] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Chothia numbering system: (4A) a CDR1 comprising the sequence shown in SEQ ID NO: 19 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 20 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 16 or a variant thereof; or (4B) a CDR1 comprising the sequence shown in SEQ ID NO: 76 or a variant thereof, a CDR2 comprising the sequence shown in SEQ ID NO: 77 or a variant thereof, and a CDR3 comprising the sequence shown in SEQ ID NO: 73 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Chothia numbering system: a CDR1 comprising the sequence shown in SEQ ID NO: 19, a CDR2 comprising the sequence shown in SEQ ID NO: 20, and a CDR3 comprising the sequence shown in SEQ ID NO: 16; or (4B) a CDR1 comprising the sequence shown in SEQ ID NO: 76, a CDR2 comprising the sequence shown in SEQ ID NO: 77, and a CDR3 comprising the sequence shown in SEQ ID NO: 73.

[0030] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Contact numbering system: (5A) a CDR1 comprising the sequence set forth in SEQ ID NO: 21 or a variant thereof, a CDR2 comprising the sequence set forth in SEQ ID NO: 22 or a variant thereof, and a CDR3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof; or (5B) a CDR1 comprising the sequence set forth in SEQ ID NO: 78 or a variant thereof, a CDR2 comprising the sequence set forth in SEQ ID NO: 79 or a variant thereof, and a CDR3 comprising the sequence set forth in SEQ ID NO: 80 or a variant thereof. The variant comprises one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Contact numbering system: a CDR1 comprising the sequence shown in SEQ ID NO:21, a CDR2 comprising the sequence shown in SEQ ID NO:22, and a CDR3 comprising the sequence shown in SEQ ID NO:23; or (5B) a CDR1 comprising the sequence shown in SEQ ID NO:78, a CDR2 comprising the sequence shown in SEQ ID NO:79, and a CDR3 comprising the sequence shown in SEQ ID NO:80.

[0031] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises the CDR1, CDR2, and CDR3 comprised in the VHH set forth in any one of SEQ ID NOs: 10, 24-39, 67, 81-88. In certain embodiments, the CDRs are identified by the IMGT, Kabat, AbM, Chothia, or Contact numbering systems.

[0032] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises a VHH sequence as shown in SEQ ID NO: 10, 67 or a variant thereof, wherein the variant has a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared thereto; preferably, the substitutions are conservative substitutions.

[0033] In certain embodiments, the single domain antibody or antigen binding fragment thereof is humanized, wherein one or more framework regions in its VHH have been substantially replaced by human framework regions. In certain embodiments, the humanized single domain antibody or antigen binding fragment thereof comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), the heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to camelid residues.

[0034] In certain embodiments, the humanized single-domain antibody or antigen-binding fragment thereof comprises a VHH sequence as shown in any one of SEQ ID NOs: 24-39, 81-88, or a variant thereof, wherein the variant has a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared thereto; preferably, the substitutions are conservative substitutions.

[0035] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof specifically binds to human PD-L1, murine PD-L1, and / or cynomolgus monkey PD-L1, for example, the single-domain antibody is as defined in (1A) to (5A) in the above embodiments. In certain embodiments, the single-domain antibody or antigen-binding fragment thereof specifically binds to human PD-L1 and / or cynomolgus monkey PD-L1, for example, the single-domain antibody is as defined in (1B) to (5B) in the above embodiments.

[0036] polypeptide constructs

[0037] In a fourth aspect, the present invention relates to a polypeptide construct comprising the single domain antibody or antigen-binding fragment thereof according to any one of the above aspects, and an immunoglobulin Fc domain.

[0038] In certain embodiments, the polypeptide construct comprises the single-domain antibody that specifically binds to MSLN as described in the second aspect and an immunoglobulin Fc domain.

[0039] In certain embodiments, the polypeptide construct comprises the single-domain antibody that specifically binds to PD-L1 as described in the third aspect and an immunoglobulin Fc domain.

[0040] As used herein, the Fc domain, also referred to as the Fc region, refers to the portion of the heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc domain comprises a hinge, CH2, and CH3. When the Fc domain comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc domain can be of any antibody heavy chain constant region isotype. In some embodiments, the Fc domain is IgG1, IgG2, IgG3, or IgG4.

[0041] In certain embodiments, the Fc domain included in the polypeptide construct of the present invention is a native Fc region, which comprises an amino acid sequence consistent with the amino acid sequence of the Fc region found in nature. For example, the Fc domain can be a native sequence human IgG1 Fc region, a native sequence human IgG2 Fc region, a native sequence human IgG3 Fc region, or a native sequence human IgG4 Fc region. A native Fc region may have effector functions. Exemplary "effector functions" include binding to Fc receptors; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. Functional changes can be produced by replacing at least one amino acid residue in the native Fc region with a different residue or chemical modification, for example, changing the affinity of the antibody for effector ligands (such as FcR or complement C1q), thereby changing the effector function (e.g., reducing or enhancing).

[0042] In certain embodiments, the Fc domain contained in the polypeptide construct of the present invention may also be a variant Fc region, which may contain one or more (e.g., 1-10, such as 1-5) amino acid mutations or chemical modifications compared to the native Fc region to alter one or more of the following properties of the antibody of the present invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function or complement function, etc.

[0043] In certain embodiments, the immunoglobulin Fc domain is optionally linked to the N-terminus and / or C-terminus (eg, C-terminus) of the single-domain antibody or antigen-binding fragment thereof via a peptide linker.

[0044] In certain embodiments, the immunoglobulin Fc domain is linked to the C-terminus of the single domain antibody or antigen-binding fragment thereof, optionally via a peptide linker.

[0045] In certain embodiments, the immunoglobulin Fc domain is the Fc domain of IgG (eg, the Fc domain of IgG1).

[0046] In certain embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 48, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) thereto.

[0047] In certain embodiments, the peptide linker is a peptide linker comprising one or more glycine and / or one or more serine. In certain embodiments, the peptide linker is (G4S)n, where n is 1, 2, 3 or 4. In certain embodiments, the peptide linker comprises the sequence shown in SEQ ID NO:50.

[0048] Multispecific antibodies

[0049] In the fifth aspect, the present invention relates to a multispecific antibody comprising the humanized single-domain antibody or antigen-binding fragment thereof targeting MSLN according to the second aspect and / or the single-domain antibody or antigen-binding fragment thereof targeting PD-L1 according to the third aspect.

[0050] In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0051] In certain embodiments, the multispecific antibody comprises the humanized single-domain antibody or antigen-binding fragment thereof targeting MSLN as described in the second aspect as an antigen-binding domain targeting MSLN, and comprises at least one additional antigen-binding domain that binds to a different target from the antigen-binding domain.

[0052] In certain embodiments, the multispecific antibody comprises the single-domain antibody or antigen-binding fragment thereof targeting PD-L1 as described in the third aspect as an antigen-binding domain targeting PD-L1, and comprises at least one additional antigen-binding domain that binds to a different target from the antigen-binding domain.

[0053] In certain embodiments, the multispecific antibody comprises the humanized single-domain antibody or antigen-binding fragment thereof targeting MSLN described in the second aspect as an antigen-binding domain targeting MSLN, comprises the single-domain antibody or antigen-binding fragment thereof targeting PD-L1 described in the third aspect as an antigen-binding domain targeting PD-L1, and further comprises at least one additional antigen-binding domain that binds to a different target from the above-mentioned antigen-binding domain.

[0054] In a sixth aspect, the present invention relates to a multispecific antibody comprising: a first antigen-binding domain targeting CD3, a second antigen-binding domain targeting MSLN, and a third antigen-binding domain targeting PD-L1, wherein:

[0055] (i) the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to the first aspect;

[0056] (ii) the second antigen-binding domain comprises a VHH, wherein the VHH comprises the CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NOs: 1-9; preferably, the VHH comprises the CDR1 shown in SEQ ID NO: 52, the CDR2 shown in SEQ ID NO: 53, and the CDR3 shown in SEQ ID NO: 54; preferably, the second antigen-binding domain comprises the humanized single-domain antibody or antigen-binding fragment thereof that specifically binds to MSLN according to the second aspect, or comprises the VHH sequence shown in SEQ ID NO: 9;

[0057] and / or,

[0058] (iii) The third antigen-binding domain comprises the single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 as described in the third aspect.

[0059] In certain embodiments, in (i), the first antigen-binding domain comprises VH and VL, wherein the VH comprises HCDR1 shown in SEQ ID NO:55, HCDR2 shown in SEQ ID NO:56, and HCDR3 shown in any one of SEQ ID NOs:57-59, 65; and / or the VL comprises LCDR1 shown in SEQ ID NO:60, LCDR2 shown in SEQ ID NO:61, and LCDR3 shown in SEQ ID NO:62; preferably, the VH comprises the sequence shown in any one of SEQ ID NOs:40-42, 64; preferably, the VL comprises the sequence shown in SEQ ID NO:43.

[0060] In certain embodiments, the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in the first aspect; the second antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to MSLN, such as a full-length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, scFv or VHH; the third antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to PD-L1, such as a full-length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, scFv or VHH.

[0061] In certain embodiments, the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to CD3, such as a full-length antibody, an Fv fragment, a Fab fragment, a F(ab')2 fragment, a scFv, or a VHH; the second antigen-binding domain comprises a humanized single-domain antibody or an antigen-binding fragment thereof that specifically binds to MSLN as described in the second aspect; the third antigen-binding domain comprises an antibody or an antigen-binding fragment thereof that specifically binds to PD-L1, such as a full-length antibody, an Fv fragment, a Fab fragment, a F(ab')2 fragment, a scFv, or a VHH.

[0062] In certain embodiments, the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to CD3, such as a full-length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, scFv or VHH; the second antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to MSLN, such as a full-length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, scFv or VHH; the third antigen-binding domain comprises the single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 as described in the third aspect.

[0063] In certain embodiments, the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in the first aspect; the second antigen-binding domain comprises the humanized single-domain antibody or antigen-binding fragment thereof that specifically binds to MSLN as described in the second aspect; the third antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to PD-L1, such as a full-length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, scFv or VHH.

[0064] In certain embodiments, the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in the first aspect; the second antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to MSLN, such as a full-length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, scFv or VHH; the third antigen-binding domain comprises the single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 as described in the third aspect.

[0065] In certain embodiments, the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to CD3, such as a full-length antibody, an Fv fragment, a Fab fragment, a F(ab')2 fragment, a scFv, or a VHH; the second antigen-binding domain comprises the humanized single-domain antibody or antigen-binding fragment thereof that specifically binds to MSLN as described in the second aspect; and the third antigen-binding domain comprises the single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 as described in the third aspect.

[0066] In certain embodiments, the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in the first aspect; the second antigen-binding domain comprises the humanized single-domain antibody or antigen-binding fragment thereof that specifically binds to MSLN as described in the second aspect; and the third antigen-binding domain comprises the single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 as described in the third aspect.

[0067] In the seventh aspect, the present invention relates to a multispecific antibody comprising: a first antigen binding domain targeting CD3, a second antigen binding domain targeting a tumor-associated antigen (TAA), and a third antigen binding domain targeting an immune checkpoint, wherein the first antigen binding domain is Fab, and the second and third antigen binding domains are VHH.

[0068] In certain embodiments, the multispecific antibody further comprises an Fc domain comprising a first Fc monomer and a second Fc monomer; wherein:

[0069] The N-terminus of the first Fc monomer is optionally connected to the first antigen-binding domain (e.g., the heavy chain CH1 domain thereof) via a linker, and the C-terminus thereof is optionally connected to one of the second antigen-binding domains via a linker;

[0070] The N-terminus of the second Fc monomer is optionally connected to another second antigen-binding domain via a linker, and the C-terminus thereof is optionally connected to the third antigen-binding domain via a linker.

[0071] In certain embodiments, the tumor-associated antigen is selected from CD19, BCMA, EGFR, HER2, HER3, HER4, PSMA, EpCAM, EphA2, CD33, CD123, CD38, CLDN18, MSLN, TROP2, Mucin1, AFP, CD79b, GUCY2C, LRRC15, gp100, STEAP1, ROR1, 5T4, CEA, DLL3, CD20, CD7, PRAME, CDH19, CDH17, GPA33, HLA-A2, CD34, FAP, GPRC5D, GPC3, B7-H3, CLL-1, CLDN6, Flt 3, NY-ESO-1, PSCA, NECTIN-4, ENPP3, IGFR-1, TSA1, Melan-A, MUC16 (CA125), MUC17, SSTR2, c-Met, B7-H6, CSPG4, CAIX, MCSP, BIRC5, BIRC7, BRCA1, BORIS, CCR5, GD2, GD3, GloboH, GM3, hTERT, LMP2, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, Ras, SART-3, TRP-1, TRP-2, CD22, CD30, FOLR1, or any combination thereof.

[0072] In certain embodiments, the tumor-associated antigen is MSLN.

[0073] In certain embodiments, the immune checkpoint is selected from PD-1, PD-L1, PD-L2 CTLA-4, TIM-3, Lag-3, TIGIT, CD73, VISTA, B7-H3, NKG2D, NKG2A, OX40, OX40L, CD40, CD47, LIGHT, ICOS, HVEM, BTLA, B7-H4, 4-1BB, 4-1BBL, or any combination thereof.

[0074] In certain embodiments, the immune checkpoint is selected from PD1, PD-L1, TIGIT, LAG3.

[0075] In certain embodiments, the immune checkpoint is selected from PD1, PD-L1, or a combination thereof. In certain embodiments, the immune checkpoint is PD-L1.

[0076] In certain embodiments, the Fc domain of the multispecific antibody comprises a modification to promote dimerization of the first Fc monomer and the second Fc monomer, thereby (hetero)dimerization occurs between the polypeptide comprising the first Fc monomer and the polypeptide comprising the second Fc monomer to form a complex.

[0077] Such modifications are known to those skilled in the art and can include separately modifying each of the two Fc subunits (i.e., the first and second monomers of Fc) that are desired to be combined, wherein the modifications are complementary to each other, thereby promoting the combination of the two Fc subunits. For example, the modification that promotes the combination can change the structure or charge of one or both Fc subunits, thereby promoting their combination in three dimensions or electrostatically. For example, the modification that promotes the combination comprises an amino acid mutation (e.g., an amino acid replacement) in Fc. In certain embodiments, the modification is in the CH3 domain of Fc.

[0078] In certain embodiments, the CH3 domains of both monomers of the Fc domain comprise amino acid substitutions.

[0079] In certain embodiments, the modification comprises a "knob" modification in one of the two monomers of the Fc domain and a "hole" modification in the other of the two monomers of the Fc domain to form a "knob-into-hole" modification. Typically, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide such that the protuberance can be placed in the cavity to promote heterodimer formation and hinder homodimer formation. The protuberance is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0080] In certain exemplary embodiments, one amino acid in the CH3 domain of the first Fc monomer is substituted with an amino acid residue having a larger side chain volume, thereby forming a protuberance in the CH3 domain of the first Fc monomer, and one amino acid in the CH3 domain of the second Fc monomer is substituted with an amino acid residue having a smaller side chain volume, thereby forming a complementary cavity having the same or similar size as the protuberance in the CH3 domain of the second Fc monomer; or one amino acid in the CH3 domain of the second Fc monomer is substituted with an amino acid residue having a larger side chain volume, thereby forming a protuberance in the CH3 domain of the second Fc monomer, and one amino acid in the CH3 domain of the first Fc monomer is substituted with an amino acid residue having a smaller side chain volume, thereby forming a complementary cavity having the same or similar size as the protuberance in the CH3 domain of the first Fc monomer.

[0081] In certain embodiments, the amino acid residue with a larger side chain volume is selected from tryptophan (W), arginine (R), phenylalanine (P), and tyrosine (Y).

[0082] In certain embodiments, the amino acid residue with a smaller side chain volume is selected from valine (V), alanine (A), serine (S), and threonine (T).

[0083] In certain exemplary embodiments, the first Fc monomer and the second Fc monomer of the Fc domain comprise the amino acid sequences shown in SEQ ID NOs: 46 and 47, respectively.

[0084] In certain embodiments, the linker is selected from a peptide linker comprising one or more glycines and / or one or more serines, such as (G4S)n, where n is 1, 2, 3 or 4, such as a sequence as shown in any one of SEQ ID NOs: 49-51.

[0085] In certain embodiments, the multispecific antibody comprises:

[0086] (i) a first peptide chain comprising the VL and light chain constant region (CL) of the first antigen-binding domain; preferably, the CL is a kappa light chain constant region;

[0087] (ii) a second peptide chain comprising the VH, CH1 and first Fc monomer of the first antigen-binding domain and the second antigen-binding domain; preferably, the first Fc monomer is IgG, such as IgG1 or IgG4; preferably, the first Fc monomer comprises a hinge region, CH2 and CH3; preferably, the second antigen-binding domain is connected to the C-terminus of the first Fc monomer via a linker (e.g., a flexible peptide comprising (G4S)n);

[0088] and

[0089] (iii) a third peptide chain comprising the second antigen-binding domain, a second Fc monomer and a third antigen-binding domain; preferably, the second Fc monomer is IgG, such as IgG1 or IgG4; preferably, the second Fc monomer comprises a hinge region, CH2 and CH3; preferably, the second antigen-binding domain is connected to the N-terminus of the second Fc monomer via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the third antigen-binding domain is connected to the C-terminus of the second Fc monomer via a linker (e.g., a flexible peptide comprising (G4S)n).

[0090] In certain embodiments, the second Fc monomer of the third peptide chain is capable of forming a dimer with the first Fc monomer of the second peptide chain.

[0091] In certain embodiments, the first Fc monomer of the second peptide chain and the second Fc monomer of the third peptide chain comprise modifications to promote dimerization. Preferably, the modifications comprise amino acid replacements in the CH3 domain of the Fc domain. Preferably, the modifications comprise a "knob" modification in one of the two Fc monomers and a "hole" modification in the other of the two Fc monomers to form a "knob-into-hole" modification.

[0092] In certain embodiments, the two Fc monomers comprise the amino acid sequences set forth in SEQ ID NOs: 46 and 47, respectively. In certain embodiments, the first Fc monomer of the second peptide chain comprises the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the second peptide chain comprises the heavy chain constant region sequence set forth in SEQ ID NO: 45. In certain embodiments, the second Fc monomer of the third peptide chain comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0093] In certain embodiments, the first antigen binding domain comprises VH and VL, wherein the VH comprises HCDR1 shown in SEQ ID NO: 55, HCDR2 shown in SEQ ID NO: 56, and HCDR3 shown in any one of SEQ ID NOs: 57-59, 65; and / or the VL comprises LCDR1 shown in SEQ ID NO: 60, LCDR2 shown in SEQ ID NO: 61, and LCDR3 shown in SEQ ID NO: 62.

[0094] In certain embodiments, the VH comprises the sequence shown in any one of SEQ ID NOs: 40-42, 64.

[0095] In certain embodiments, the VL comprises the sequence shown in SEQ ID NO:43.

[0096] In certain embodiments, the second antigen-binding domain comprises a VHH, wherein the VHH comprises the CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NOs: 1-9; preferably, the VHH comprises the CDR1 shown in SEQ ID NO: 52, the CDR2 shown in SEQ ID NO: 53, and the CDR3 shown in SEQ ID NO: 54.

[0097] In certain embodiments, the second antigen-binding domain comprises a VHH as shown in any one of SEQ ID NOs: 1-9 or a variant thereof, which has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared thereto; preferably, the substitutions are conservative substitutions.

[0098] In certain embodiments, the third antigen-binding domain comprises the single-domain antibody or antigen-binding fragment thereof capable of specifically binding to PD-L1 as described in the third aspect.

[0099] In certain embodiments, the multispecific antibody comprises:

[0100] (i) a first peptide chain having the structure [VL]-[CL],

[0101] (ii) a second peptide chain having the structure [VH]-[CH]-[L1]-[VHH1], and

[0102] (iii) a third peptide chain having the structure [VHH1]-[L2]-[Fc monomer]-[L3]-[VHH2];

[0103] One of the following:

[0104] (1) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 81; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0105] (2) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 29; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0106] (3) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 29; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 and L3 are the same, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0107] (4) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 29; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 and L3 are the same, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0108] (5) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 30; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 and L3 are the same, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0109] (6) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 30; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0110] (7) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 30; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 and L3 are the same, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0111] (8) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 38; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0112] (9) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 38; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0113] (10) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 38; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0114] (11) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 31; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0115] (12) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 31; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0116] (13) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 31; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0117] (14) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 39; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0118] (15) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 39; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0119] (16) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 39; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51;

[0120] (17) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 64, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 9; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 10; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; or,

[0121] (18) The VL comprises the sequence shown in SEQ ID NO:43, the CL comprises the sequence shown in SEQ ID NO:44; the VH comprises the sequence shown in SEQ ID NO:40, the CH comprises the sequence shown in SEQ ID NO:45, and the VHH1 comprises the sequence shown in SEQ ID NO:9; the Fc monomer comprises the sequence shown in SEQ ID NO:47, and the VHH2 comprises the sequence shown in SEQ ID NO:10; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (for example, a peptide linker shown in (G4S)n), preferably L1 is the same as L3, preferably, L1 is SEQ ID NO:51, L2 is SEQ ID NO:49, and L3 is SEQ ID NO:51.

[0122] Antibody preparation

[0123] The antibodies, single domain antibodies, polypeptide constructs or multispecific antibodies described in any of the above aspects can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology. For example, DNA molecules encoding them are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies, single domain antibodies, polypeptide constructs or multispecific antibodies of the present invention.

[0124] In another aspect, the present invention provides isolated nucleic acid molecules encoding:

[0125] - the antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof according to the first aspect;

[0126] - the humanized single-domain antibody or antigen-binding fragment thereof according to the second aspect;

[0127] - the single domain antibody or antigen-binding fragment thereof according to the third aspect;

[0128] - the polypeptide construct according to the fourth aspect;

[0129] - The multispecific antibody according to the fifth, sixth or seventh aspect, or a polypeptide chain thereof.

[0130] In another aspect, the present invention provides a vector comprising the nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0131] In certain embodiments, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the present invention, and the nucleotide sequence encoding each peptide chain is present on the same or different vectors.

[0132] On the other hand, the invention provides host cells comprising nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells etc.).

[0133] On the other hand, the present invention provides a method for preparing the antibody, single domain antibody, polypeptide construct or multispecific antibody described in any of the above aspects, which comprises culturing the host cell described above under conditions that allow protein expression, and collecting the antibody, single domain antibody, polypeptide construct or multispecific antibody from the cultured host cell culture.

[0134] Conjugate

[0135] As one of the derivatives of the antibody, the present invention provides a conjugate comprising the antibody, single domain antibody, polypeptide construct or multispecific antibody described in any one of the above aspects and a coupling portion.

[0136] In certain embodiments, the conjugated moiety is selected from a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0137] In certain embodiments, the conjugated moiety is selected from a therapeutic agent. In certain embodiments, the therapeutic agent is preferably an anti-tumor agent, such as a cytotoxic agent, a cytokine, a toxin, a radionuclide, an immunostimulant, an immunosuppressant, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis.

[0138] Pharmaceutical composition

[0139] The antibodies, single domain antibodies, polypeptide constructs, multispecific antibodies or conjugates disclosed herein (also referred to as active ingredients) can be incorporated into pharmaceutical compositions suitable for administration.

[0140] On the other hand, the present invention relates to a pharmaceutical composition comprising the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, or conjugate described in any one of the above aspects, and a pharmaceutically acceptable carrier and / or excipient.

[0141] In certain embodiments, the pharmaceutical composition of the present invention comprises the humanized single-domain antibody or antigen-binding fragment thereof according to the second aspect, or a nucleic acid molecule or vector encoding the same.

[0142] In certain embodiments, the pharmaceutical composition of the present invention comprises the single-domain antibody or antigen-binding fragment thereof according to the third aspect, or a nucleic acid molecule or vector encoding the same.

[0143] In certain embodiments, the pharmaceutical composition of the present invention comprises the polypeptide constructs described in the fourth aspect or nucleic acid molecules or vectors encoding them.

[0144] In certain embodiments, the pharmaceutical composition of the present invention comprises the multispecific antibodies described in the fifth aspect or nucleic acid molecules or vectors encoding the same.

[0145] In certain embodiments, the pharmaceutical composition of the present invention comprises the multispecific antibodies described in the sixth aspect or nucleic acid molecules or vectors encoding the same.

[0146] In certain embodiments, the pharmaceutical composition of the present invention comprises the multispecific antibodies described in the seventh aspect or nucleic acid molecules or vectors encoding the same.

[0147] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent, such as an anti-tumor agent. In certain embodiments, the additional pharmaceutically active agent is provided separately from the antibody, single domain antibody, polypeptide construct, multispecific antibody or conjugate, isolated nucleic acid molecule, vector or host cell of the present invention, or is provided as a component of the same composition.

[0148] Therapeutic uses

[0149] On the other hand, the present invention relates to a method for preventing and / or treating and / or neoadjuvant treatment and / or adjuvant treatment of a disease, comprising administering to a subject in need thereof an antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition as described in any of the above aspects. The present invention also relates to the use of the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition as described in any of the above aspects for preventing and / or treating and / or neoadjuvant treatment and / or adjuvant treatment of a disease, or for the preparation of a medicament for preventing and / or treating and / or neoadjuvant treatment and / or adjuvant treatment of a disease.

[0150] In certain embodiments, the disease is a tumor. In certain embodiments, the tumor is a solid tumor or a hematologic tumor. In certain embodiments, the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer; and the hematologic tumor comprises acute myeloid leukemia.

[0151] On the other hand, the present invention relates to a method for preventing and / or treating and / or neoadjuvant therapy and / or adjuvant therapy of a disease, comprising administering to a subject in need thereof the humanized single-domain antibodies, polypeptide constructs comprising them, multispecific antibodies or conjugates, nucleic acid molecules, vectors or host cells encoding them, or pharmaceutical compositions comprising any of the foregoing. The present invention also relates to the humanized single-domain antibodies, polypeptide constructs comprising them, multispecific antibodies or conjugates, nucleic acid molecules, vectors or host cells encoding them, or pharmaceutical compositions comprising any of the foregoing, as described in the second aspect, for use in preventing and / or treating and / or neoadjuvant therapy and / or adjuvant therapy of a disease, or in the preparation of a medicament for preventing and / or treating and / or neoadjuvant therapy and / or adjuvant therapy of a disease.

[0152] In certain embodiments, the disease is a tumor.

[0153] In certain embodiments, the tumor is an MSLN-positive tumor.

[0154] In certain embodiments, the tumor is a solid tumor or a hematological tumor, such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, colorectal cancer, head and neck squamous cell carcinoma, pancreatic cancer, bile duct cancer, colorectal cancer, fallopian tube cancer, malignant melanoma, soft tissue cancer, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.

[0155] In certain embodiments, the tumor is selected from a solid tumor, such as mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer.

[0156] In certain embodiments, the tumor is selected from a hematological tumor, such as acute myeloid leukemia.

[0157] On the other hand, the present invention relates to a method for preventing and / or treating and / or neoadjuvant therapy and / or adjuvant therapy of a disease, comprising administering to a subject in need thereof the single domain antibodies, polypeptide constructs comprising them, multispecific antibodies or conjugates, nucleic acid molecules, vectors or host cells encoding them, or pharmaceutical compositions comprising any of the foregoing. The present invention also relates to the single domain antibodies, polypeptide constructs comprising them, multispecific antibodies or conjugates, nucleic acid molecules, vectors or host cells encoding them, or pharmaceutical compositions comprising any of the foregoing, for use in preventing and / or treating a disease, or in the preparation of a medicament for preventing and / or treating and / or neoadjuvant therapy and / or adjuvant therapy of a disease.

[0158] In certain embodiments, the disease is a disease associated with PD-L1. In certain embodiments, the disease associated with PD-L1 is a tumor.

[0159] In certain embodiments, the tumor is a PD-L1 positive tumor.

[0160] In certain embodiments, the tumor is a solid tumor or a hematological tumor. In certain embodiments, the tumor is selected from the group consisting of: gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, melanoma, head and neck cancer, esophageal cancer, and skin cancer.

[0161] On the other hand, the present invention relates to a method for preventing and / or treating and / or neoadjuvant treatment and / or adjuvant treatment of a disease, comprising administering to a subject in need thereof the multispecific antibodies of the fifth, sixth or seventh aspects, nucleic acid molecules, vectors or host cells encoding the same, or pharmaceutical compositions comprising any of the foregoing. The present invention also relates to the use of the multispecific antibodies of the fifth, sixth or seventh aspects, nucleic acid molecules, vectors or host cells encoding the same, or pharmaceutical compositions comprising any of the foregoing, for preventing and / or treating a disease, or for preparing a medicament for preventing and / or treating and / or neoadjuvant treatment and / or adjuvant treatment of a disease.

[0162] The multispecific antibodies described in the fifth, sixth or seventh aspects of the present invention can be used in the treatment of any disease, as long as the effector mechanism of cytotoxic T cells is required in the treatment of the disease. For example, when the multispecific antibodies of the present invention include a CD3 binding arm for T cell recruitment and a tumor targeting arm specific for a tumor-associated antigen (TAA), the T cells are brought into close contact with the target tumor cells, locally activating the T cells, and then the perforins and granzymes released by the T cell toxic granules destroy the target cells. When further including a targeting arm specific for an immune checkpoint molecule, it activates the T cells in the tumor microenvironment, subsequently killing tumor cells and enhancing the anti-tumor effect.

[0163] In certain embodiments, the disease is a tumor.

[0164] In certain embodiments, the tumor is a solid tumor or a hematological tumor, such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, colorectal cancer, head and neck squamous cell carcinoma, pancreatic cancer, bile duct cancer, colorectal cancer, fallopian tube cancer, malignant melanoma, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.

[0165] In certain embodiments, the treatment of the disease involves the effector mechanism of T cells (e.g., cytotoxic T cells). In certain embodiments, the multispecific antibody is used to recruit T cells to the vicinity of target cells, activate T cells, and induce T cell-mediated cell killing (TDCC), thereby effectively killing target cells to treat or prevent the disease.

[0166] In certain embodiments, the tumor is an MSLN-positive tumor, such as a solid tumor, such as mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, cervical cancer, bile duct cancer, prostate cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer; for example, a blood tumor, such as acute myeloid leukemia.

[0167] In certain embodiments, the tumor is a PD-L1 positive tumor, such as a solid tumor, such as gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, melanoma, head and neck tumor, esophageal cancer, skin cancer.

[0168] In any of the above aspects, the single domain antibody, polypeptide construct, multispecific antibody or pharmaceutical composition comprising the same of the present invention can be formulated into any dosage form known in the medical field.

[0169] In any of the above aspects, the single domain antibodies, polypeptide constructs, multispecific antibodies or pharmaceutical compositions comprising the same of the present invention can be administered by any suitable method known in the art.

[0170] In any of the above aspects, the single domain antibodies, polypeptide constructs, multispecific antibodies or pharmaceutical compositions comprising the same of the present invention can be formulated in dosage unit form for ease of administration.

[0171] In any of the above aspects, the single domain antibodies, polypeptide constructs, multispecific antibodies or pharmaceutical compositions comprising the same of the present invention can be administered alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor agent) or another therapy (e.g., an anti-tumor therapy).

[0172] In any of the above aspects, the subject may be a mammal, such as a human.

[0173] Detection Application

[0174] In another aspect, the present invention provides a kit comprising the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition described in any one of the above aspects.

[0175] In certain embodiments, the kit comprises instructions for use, such as instructions for using the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition to detect the presence or level of PD-L1, MSLN and / or CD3 in a sample and / or diagnose or differentially diagnose a disease associated with PD-L1, MSLN and / or CD3.

[0176] In certain embodiments, the kit comprises: an antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in the first aspect. The antibody or antigen-binding fragment thereof is optionally detectably labeled. The kit is used to detect the presence or level of CD3 in a sample and / or to diagnose or differentially diagnose a disease associated with CD3.

[0177] In certain embodiments, the kit comprises: a humanized single-domain antibody, antigen-binding fragment thereof, or polypeptide construct thereof that specifically binds to MSLN as described in the second aspect. The humanized single-domain antibody, antigen-binding fragment thereof, or polypeptide construct thereof is optionally detectably labeled. The kit is used to detect the presence or level of MSLN in a sample and / or to diagnose or differentially diagnose a disease associated with MSLN.

[0178] In certain embodiments, the kit comprises: a single-domain antibody, antigen-binding fragment thereof, or polypeptide construct thereof that specifically binds to PD-L1 as described in the third aspect. The single-domain antibody, antigen-binding fragment thereof, or polypeptide construct thereof is optionally detectably labeled. The kit is used to detect the presence or level of PD-L1 in a sample and / or to diagnose or differentially diagnose a disease associated with PD-L1.

[0179] In certain embodiments, the kit comprises: the multispecific antibody of the fifth, sixth, or seventh aspect. The multispecific antibody is optionally detectably labeled. The kit is used to detect the presence or level of PD-L1, MSLN, and / or CD3 in a sample and / or diagnose or differentially diagnose a disease associated with PD-L1, MSLN, and / or CD3.

[0180] In this article, the detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Particularly preferably, such label can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).

[0181] In another aspect, the present invention provides a method for detecting the presence or level of PD-L1, MSLN, and / or CD3 in a sample, the method comprising contacting the sample with the antibody, single-domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition described in any one of the above aspects under conditions that allow the formation of an antibody-antigen immune complex; and detecting the formation of the complex. The formation of the complex indicates the presence of a target antigen or a cell expressing the target antigen. The target antigen refers to the antigen targeted by the above-mentioned detection reagent used. The method can be used for diagnostic purposes or non-diagnostic purposes.

[0182] In certain embodiments, the methods are performed in vitro.

[0183] In certain embodiments, the method is an immunological assay (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).

[0184] In certain embodiments, the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is further detectably labeled.

[0185] In certain embodiments, the method uses the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to the first aspect, and is used to detect the presence or level of CD3 in a sample and / or diagnose or differentially diagnose a disease associated with CD3.

[0186] In certain embodiments, the method uses the humanized single-domain antibody or its antigen-binding fragment or its polypeptide construct that specifically binds to MSLN as described in the second aspect, and is used to detect the presence or level of MSLN in a sample and / or diagnose or differentially diagnose diseases related to MSLN.

[0187] In certain embodiments, the method uses the single-domain antibody or antigen-binding fragment thereof or polypeptide construct that specifically binds to PD-L1 as described in the third aspect, and is used to detect the presence or level of PD-L1 in a sample and / or diagnose or differentially diagnose a disease associated with PD-L1.

[0188] In certain embodiments, the method uses the multispecific antibody of the fifth, sixth or seventh aspect and is used to detect the presence or level of PD-L1, MSLN and / or CD3 in a sample and / or diagnose or differentially diagnose a disease associated with PD-L1, MSLN and / or CD3.

[0189] In another aspect, the present invention provides a method for diagnosing or differentially diagnosing a disease associated with PD-L1, MSLN and / or CD3, the method comprising using the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition described in any one of the above aspects.

[0190] In certain embodiments, the method diagnoses or differentially diagnoses a disease by detecting the presence or level of PD-L1, MSLN and / or CD3 in a sample from a subject using the detection methods described above.

[0191] In certain embodiments, the disease is associated with a target antigen, for example, characterized by expression or abnormal expression of a target antigen. The target antigen refers to the antigen targeted by the detection reagent used.

[0192] In certain embodiments, the method may further comprise the step of comparing the detected value of the target antigen in the obtained sample with a reference value. The reference value may be the level of the target antigen in a sample from a healthy control (also referred to as a "negative reference value"). For example, if the amount of the target antigen in the sample from the subject is elevated relative to the negative reference value, it indicates that the subject suffers from a disease associated with the target antigen.

[0193] In certain embodiments, the disease is a tumor. In certain embodiments, the tumor is a solid tumor or a hematological tumor; more preferably, the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, cervical cancer, bile duct cancer, prostate cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer; and the hematological tumor includes acute myeloid leukemia.

[0194] On the other hand, provided is a use of the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, conjugate, pharmaceutical composition, or kit described in any of the above aspects in the preparation of a detection reagent for detecting the presence or level of PD-L1, MSLN and / or CD3 in a sample and / or diagnosing or differentially diagnosing a disease associated with PD-L1, MSLN and / or CD3.

[0195] Definition of terms

[0196] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0197] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0198] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0199] The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site.

[0200] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA86:9268–9272) or the Contact numbering system (MacCallum, RM, Martin, ACR, & Thornton, JM (1996). Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5), 732-745.). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art.

[0201] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, AbM, or Contact numbering systems. In certain embodiments, the CDRs contained in the anti-CD3 antibodies or antigen-binding fragments thereof involved in the present invention are determined by the Kabat numbering system. In certain embodiments, the CDRs contained in the anti-MSLN or PD-L1 single domain antibodies or antigen-binding fragments thereof involved in the present invention are determined by the Kabat, Chothia, IMGT, AbM, or Contact numbering systems.

[0202] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0203] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0204] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises multiple antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain comprised by a multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a scFv). In some cases, each antigen-binding domain is connected by a peptide linker.

[0205] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion thereof". Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementary determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0206] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that, in the direction from N-terminus to C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the direction from N-terminus to C-terminus. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. A full-length antibody contains two antigen-binding sites formed by a VH and VL pair, respectively, and these two antigen-binding sites specifically recognize / bind to the same antigen.

[0207] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody may be truncated at the N-terminus or C-terminus so that it contains only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Single domain antibodies are also referred to as nanobodies, and the two are used interchangeably. As used herein, the term "antigen-binding fragment" of a single domain antibody refers to a polypeptide comprising a fragment of a single domain antibody that retains the ability to specifically bind to the same antigen to which the single domain antibody binds, and / or competes with the single domain antibody for specific binding to the antigen. Antigen-binding fragments of the single-domain antibodies of the present invention can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of the single-domain antibodies of the present invention. In some embodiments, the "antigen-binding fragment" of the single-domain antibody can be truncated at the N-terminus or C-terminus compared to the full-length single-domain antibody so that it only contains part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity.

[0208] As used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0209] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0210] As used herein, the term "Fc domain" or "Fc region" means a portion of the heavy chain constant region comprising CH2 and CH3. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding. The "effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, among others. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge regulates the dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0211] The Fc domain may include both a native Fc region and a variant Fc region. A native Fc region comprises an amino acid sequence that is consistent with the amino acid sequence of an Fc region found in nature, for example, a native sequence human Fc region includes a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may have altered effector functions (e.g., Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.

[0212] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)2 may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0213] As used herein, the term "diabodies" means antibodies whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites.

[0214] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0215] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0216] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0217] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0218] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived. In certain embodiments, the variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived.

[0219] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0220] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore, bioluminescence interferometry, or Kinexa.

[0221] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an origin of replication.

[0222] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0223] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Furthermore, amino acid residues can be further divided into categories defined by optional physical and functional properties, e.g., alcohol-containing residues (S and T), aliphatic residues (I, L, V, and M), cycloalkenyl-related residues (F, H, W, and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W, and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S, and T), positively charged residues (H, K, and R), small residues (A, C, D, G, N, P, S, T, and V), very small residues (A, G, and S), residues involved in turn formation (A, C, D, E, G, H, K, N, Q, R, S, P, and T), and flexible residues (Q, T, K, S, G, P, D, E, and R). Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0224] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0225] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives.

[0226] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment. In this article, treatment can include neoadjuvant therapy and / or adjuvant therapy. "Neoadjuvant therapy" refers to a therapy administered to a patient before a planned surgery for the treatment of the disease. "Adjuvant therapy" refers to a therapy administered to a patient after surgery for the treatment of the disease.

[0227] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from a tumor, an inflammatory disease, or an autoimmune disease, or is at risk of suffering from the above diseases.

[0228] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0229] Advantageous Effects of the Invention

[0230] The nanoantibodies targeting PD-L1 of the present invention have good binding affinity and specificity to PD-L1, can effectively promote the secretion of IL2 and IFNγ, and can be used to prevent and / or treat diseases or conditions related to PD-L1. The humanized antibodies of the nanoantibodies retain the functions and properties of the parent camel-derived antibodies and have a high degree of humanization, thereby having the potential to be administered to human subjects. The humanized nanoantibodies targeting MSLN of the present invention have a high degree of humanization and are therefore less likely to induce immune side effects. At the same time, they have good binding affinity and specificity for MSLN, thereby achieving efficient targeting of MSLN-expressing tumors. In addition, compared to known multispecific T cell activation structures, the multispecific antibodies targeting CD3, tumor-associated antigens, and immune checkpoints further provided by the present invention have obvious advantages. The multispecific antibodies of the present invention can moderately activate T cells, have improved drug safety, and at the same time, the combined targeting of tumor-associated antigens and immune checkpoints significantly increases the anti-tumor effect. In particular, the present invention specifically provides a trispecific antibody targeting CD3, MSLN and PD-L1, which can reduce cytokine release and ensure safety while having good anti-tumor effects.

[0231] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0232] Figures 1A-1C show the blocking activity of the 4A7 Nanobody in Example 2 on the binding of human PD-L1 to human PD-1 (A), the binding of mouse PD-L1 to mouse PD-1 (B), and the binding of monkey PD-L1 to monkey PD-1 (C).

[0233] FIG2 shows the binding activity detection of Benchmark-ATE, 7B11 and 7B11_huGS_HM4 to human PD-L1 cells in Example 2.

[0234] FIG3 shows the binding activity detection of Benchmark-ATE, 7B11 and 7B11_huGS_HM4 to monkey PD-L1 cells in Example 2.

[0235] FIG4 shows the blocking activity detection of Benchmark-ATE, 7B11 and 7B11_huGS_HM4 against human PD-L1 and human PD-1, respectively, in Example 2.

[0236] FIG5 shows the activity detection of Benchmark-ATE, 7B11 and 7B11_huGS_HM4 in blocking the PD-1 / PD-L1 signaling pathway of T cells in Example 2.

[0237] Figure 6 shows the detection results of cytokine production by T cells activated by Benchmark-ATE, 7B11, and 7B11_huGS_HM4 in Example 2; Figure 6a shows the detection results of IL2 production by T cells activated by Benchmark-ATE, 7B11, and 7B11_huGS_HM4; Figure 6b shows the detection results of IFNγ production by T cells activated by Benchmark-ATE, 7B11, and 7B11_huGS_HM4.

[0238] FIG7 shows a schematic structural diagram of the CD3-PDL1-MSLN trispecific antibody constructed in Example 3.

[0239] 8A-8D respectively show the binding activity of the CD3-PDL1-MSLN trispecific antibody in Example 4 to human MC38 / MSLN cells.

[0240] Figures 9A-9C respectively show the binding activity of the CD3-PDL1-MSLN trispecific antibody in Example 4 to CHO-hPDL1 cells.

[0241] Figures 10A-10C respectively show the blocking activity of the CD3-PDL1-MSLN trispecific antibody in Example 5 on the binding of human PD1 to human PD-L1.

[0242] Figures 11A-11C respectively show the activation of Jurkat-NFAT-luc cells by the CD3-PDL1-MSLN trispecific antibody in Example 7 in the tumor group.

[0243] Figures 12A-12C respectively show the activation of Jurkat-NFAT-luc cells by the CD3-PDL1-MSLN trispecific antibody in Example 7 in the group without tumor addition.

[0244] Figures 13A-13C respectively show the TDCC activity of the CD3-PDL1-MSLN trispecific antibody in Example 8 against MSLN-expressing human tumor cells HCC1806.

[0245] Figures 14A-14F respectively show the results of the detection of IL-2 release levels mediated by the CD3-PDL1-MSLN trispecific antibody in Example 9.

[0246] Figures 15A-15F respectively show the results of the detection of INF-γ release levels mediated by the CD3-PDL1-MSLN trispecific antibody in Example 9.

[0247] Figures 16A-16B respectively show the in vivo killing activity and safety of the 5Y3-16 antibody in Example 10.

[0248] Figures 17A-17B respectively show the schematic structural diagrams of the CD3-PDL1-MSLN trispecific antibody constructed in Example 11.

[0249] Figures 18A-18E respectively show the TDCC activity of the CD3-PDL1-MSLN trispecific antibodies with different structures in Example 11 against tumor cells.

[0250] Sequence information

[0251] A description of the sequences involved in this application is provided in the table below.

[0252] Table 1: Sequence information

[0253] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0254] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0255] Example 1: Humanization of MSLN Antibody Molecule, Expression of Humanized Antibody and Dynamic Affinity Detection

[0256] Alpacas were immunized with hFc-tagged human MSLN recombinant protein (huMSLN-hFc, Kaixia Biotechnology, MSL-HM280) as the immunizing antigen. The camelid-derived nanobody 3C6, which exhibited excellent cross-binding activity to humans, monkeys, and mice, was screened. Its VHH sequence is shown in SEQ ID NO:9, and its IMGT-defined CDR1-CDR3 are shown in SEQ ID NOs:52-54, respectively. A humanized version of 3C6 was further constructed.

[0257] 1.1 Humanized modification of MSLN antibody molecules

[0258] The anti-MSLN nanoantibody 3C6 was humanized. The 3C6 sequence was compared with the IMGT offline database to obtain the germline gene sequence with the highest homology. Using the germline gene sequence as a template, the CDR of the target sequence was transplanted. After the transplantation was completed, the Vernier region residues in the framework region of the target sequence were back mutated to ensure that the antibody affinity was not affected after the humanization. The 3D structure of the humanized sequence was constructed using the Modeller program, and the energy contribution of each amino acid residue to the stability of the protein conformation was calculated using the Chimera software. The high-energy amino acid residues were back mutated to improve the thermal stability of the humanized sequence. After completing the above steps, 8 sequences with different humanization depths were obtained.

[0259] 1.2 Expression of humanized MSLN antibody

[0260] The eight humanized sequences, each with varying degrees of humanization, were constructed into the N-terminus of human IgG1 Fc (SEQ ID NO: 48) using a linker (SEQ ID NO: 50) and subsequently transferred into the PTT5 expression vector (Ubao Bio, lot: VT2202). Transient expression was performed using the Expicho-s expression system (Gibco) to obtain VHH-Fc antibodies. The supernatant of the expressing cells was collected and purified via a protein A column to obtain multiple humanized VHH-Fc antibodies. The humanized VHH-Fc antibodies were designated 3C6hu-1, 3C6hu-2, 3C6hu-3, 3C6hu-4, 3C6hu-5, 3C6hu-6, 3C6hu-7, and 3C6hu-8. The variable regions of the humanized anti-MSLN nanobodies are shown in SEQ ID NOs: 1-8.

[0261] 1.3 Dynamic affinity testing of MSLN humanized antibody molecules

[0262] The anti-DYKDDDDK antibody was bound to an HC30M chip via amino conjugation. Then, 2 μg / ml of the MSLN single-domain antibody was captured. After the baseline stabilized, a gradient of huMSLN-Fc (Kaixia Biotechnology, MSL-HM280) and cynoMSLN-Fc (Kaixia Biotechnology, MSL-CM280) was flowed over the chip (starting at 100 nM and followed by a gradient dilution) from low to high concentrations. The association time was 8 minutes and the dissociation time was 20 minutes. Kinetic constants were fitted using Carteria software. The dynamic affinity of the MSLN single-domain antibody for the MSLN protein is shown in Table 2 below. The results demonstrate that the humanized MSLN single-domain antibody has good binding activity to both human and monkey MSLN.

[0263] Table 2. Dynamic affinity of humanized MSLN single domain antibodies to MSLN protein

[0264] Example 2: Preparation of PD-L1 Antibody

[0265] 2.1 PD-L1 immunity in alpacas

[0266] In order to produce a humoral immune response against PD-L1 in alpacas, the first immunization was performed by mixing Freund's complete adjuvant (CFA) with 0.5 mg of PD-L1-Fc (Kaixia Bio, PDL-HM210) protein and injecting it subcutaneously. Then, Freund's incomplete adjuvant (IFA) was mixed with 0.25 mg of PD-L1-Fc protein and injected subcutaneously. Immunization was performed once every 3 weeks for a total of 4 times, so that the alpacas could produce antigen-specific nanoantibodies.

[0267] Starting from the second immunization, alpaca serum was collected every 7 days for serum titer monitoring. 2 μg / ml antigen was coated overnight with CBS (carbonate buffered saline). After washing with PBST (phosphate buffered saline), 5% skim milk was added and blocked at 37°C for 1 hour. After washing with PBST, serum diluent (starting at a serial dilution of 1:2000) was added and blocked at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-conjugated goat anti-Alpaca secondary antibody (alpaca S001H, diluted 1:1 with PBS) was added and incubated at 37°C for 45 minutes. After washing with PBST, TMB colorimetric solution (Thermo, 34029) was added for color development at 37°C for 5 minutes. The reaction was terminated with stop solution, and serum titer was measured by optical density at 450 nm.

[0268] 2.2 Establishment of PD-L1 Antibody Phage Library

[0269] After the three and four immunizations, 50 mL of peripheral blood was collected, and PBMCs were isolated and total RNA was extracted according to the instructions for sample density separation solution (Haoyang Biotechnology, HY2015). Total RNA from the three and four immunizations was extracted using RNAiso Plus reagent (Takara, 9109). cDNA was synthesized and the VHH sequence was amplified using slot-type PCR. The vector pComb3XSS (EdiGene, 63890) and the target fragment were digested with SfiI and recovered. The ligation products were electroporated into TG1 competent cells, and a PD-L1 nanobody library was constructed and the library capacity was determined. The library capacity was 2.15×10 9 CFM. 48 clones were randomly selected from the plate for testing the number of transformants in the library for identification. The results showed that the insertion rate was 100%. The library was packaged using helper phage M13KO7 (NEB, N0315S), and the phage library titer was 1.02×10 13 cfu / mL.

[0270] 2.3 Anti-PD-L1 Nanobody Screening

[0271] 2.3.1 Phage panning

[0272] Using the protein magnetic bead selection method, streptavidin magnetic beads (Dynabeads TM M-280 Streptavidin) was added to 3% skim milk powder and rotated at room temperature for 1 hour. 5 μg of biotinylated PD-L1 (ACRO, PD1-H82E5) antigen was added and incubated at room temperature for 1 hour to form a magnetic bead protein complex. 60 μl of VHH phage library was then added to the magnetic bead protein complex and incubated at room temperature for 1 hour. Non-specifically bound phages were washed 10 times with 0.05% PBST to remove the phages. TG1 bacteria were then infected at 37°C for 1 hour and then coated with 2YT medium plates overnight. The same screening operation was repeated for two rounds to complete the enrichment of positive phages.

[0273] 2.3.2 Specific clone screening

[0274] (1) Preparation of periplasmic proteins

[0275] Ninety-six individual colonies were selected from the phage-containing bacterial culture medium after two rounds of panning and cultured in 2YT medium. IPTG (Sangon, B541007-0001) was added to a final concentration of 1 mM and incubated overnight at 30°C to induce periplasmic protein expression. Chicken lysozyme was used to break down the bacterial wall and extract the periplasmic protein for specific clone screening.

[0276] (2) Enzyme-linked immunosorbent assay (ELISA) screening of positive monoclonal

[0277] 2 μg / ml human PD-L1 protein (ACRO, PD1-H5258), monkey PD-L1 protein (Kaika Bio, PDL-CM210), and / or mouse PD-L1 protein (ACRO, PD1-M5251) were coated with CBS coating solution, and 50 μl of the periplasmic protein prepared above was added and incubated for 1 hour. Unbound nonspecific clones were washed away with 0.05% PBST, and anti-mouse HA-HRP secondary antibody (GenScript, A01296) was added and incubated for 1 hour. Unbound nonspecific clones were washed away with 0.05% PBST, and TMB colorimetric solution was added and incubated for 1 hour. The readings were read at a wavelength of 450 nm using a microplate reader.

[0278] (3) T cell signaling pathway blocking experiment to screen positive monoclonal

[0279] One day in advance, GS-C2 cells (cells overexpressing human PD-L1, GenScript, M00613) were plated at 4×10 4 Cells were plated in a white 96-well flat-bottom plate (corning) at 37°C with 5% CO2 overnight. 5 μl / well of the above periplasmic protein was taken and diluted in 45 μl / well 1640 culture medium. The plate on which GS-C2 cells were plated one day earlier was removed, the upper layer of culture medium was discarded, and the diluted periplasmic protein was added to the cell plate. Jurket-NFAT-PD1 cells (GenScript, M00613) were then plated at 8×10 cells per well. 4 Cells were suspended in 5% 1640 medium, added to a white cell plate, mixed, and incubated at 37°C, 5% CO2 for 6 hours. Following incubation, 100 μl of detection reagent was added to each well using the Bio-Lite Luciferase Assay System (Novozymes, DD1201) according to the instructions. The plates were incubated at room temperature for 3 minutes before fluorescence signal analysis (BMG). Two positive monoclonal clones were obtained, designated 4A7 and 260-7-B11.

[0280] 2.4 Anti-PD-L1 Nanobody Expression

[0281] Positive clones 4A7 and 260-7-B11 obtained from the above screening were cloned into prokaryotic expression vectors, and the nanobodies (VHH antibodies) were expressed by IPTG induction. The nanobodies were then purified to obtain the nanobodies. DNA sequencing of the nanobodies was then performed. The sequencing results were analyzed, and the CDR regions were defined. The variable region sequences of the anti-PD-L1 nanobodies obtained are shown in SEQ ID NOs: 10 and 67, and the CDR sequences are shown in the table below.

[0282] Table 3. CDR sequences of PD-L1 nanobodies 4A7 and 260-7-B11

[0283] 2.5 Dynamic affinity detection of anti-PD-L1 nanobody

[0284] 2.5.1 Dynamic Affinity Testing of 4A7 Nanobody

[0285] The anti-DYKDDDDK antibody was bound to an HC30M chip via amino conjugation, and then captured at 2 μg / ml of the 4A7 nanobody. After baseline stabilization, gradient dilutions of human PD-L1 protein (ACRO, PD1-H5258), monkey PD-L1 protein (Kaika Bio, PDL-CM210), and mouse PD-L1 protein (ACRO, PD1-M5251) (starting at 100 nM) were passed over the chip from low to high concentrations. The association time was 8 minutes and the dissociation time was 20 minutes. Kinetic constants were calculated using Carteria software. The dynamic affinity of the 4A7 nanobody for human, mouse, and monkey PD-L1 proteins is shown in the table below.

[0286] Table 4. Dynamic affinity of 4A7 nanobody to human, mouse, and monkey PD-L1 proteins

[0287] 2.5.2 Dynamic affinity testing of 260-7-B11 nanobody

[0288] The affinity and kinetic data of the 260-7-B11 nanobody and the human PD-L1 protein (ACRO, PD1-H5258) were measured using surface plasma resonance (SPR) technology on the Carterra LSA platform. The measurement used a capture kinetics process. The antibody to be tested was immobilized on the surface of the NiHC200M chip via a His-Tag. After several rounds of blank buffer injection and stabilization of the detection baseline, the human PD-L1 protein was injected as the analyte. Each round of injection consisted of a 2-minute baseline, 8 minutes of binding, and 20 minutes of dissociation. After deducting the reference point and zero concentration signal from the original measurement data (Double Reference), the binding curve was fitted by the Carterra LSA Kinetics software, and the kinetic parameters were calculated. The results of the dynamic affinity of the 260-7-B11 nanobody and the human PD-L1 protein are shown in the table below.

[0289] Table 5. Dynamic affinity of 260-7-B11 nanobody to human PD-L1 protein

[0290] The results in Tables 4 and 5 show that the 4A7 nanobody can bind to human, mouse, and monkey PD-L1 proteins; the 260-7-B11 nanobody can bind to human PD-L1.

[0291] 2.6 Detection of cell binding ability of anti-PD-L1 nanoantibodies

[0292] GS-C2 cells (GenScript, M00613) were washed with PBS and resuspended in 1% BSA. 1×10 cells were added to each well. 5 Cells were added to a 96-well V-bottom plate, and then the serially diluted 260-7-B11 nanoantibody (starting at 33.33 nM) was added. The cells were incubated on ice for 1 hour, washed with PBS, and then PE-labeled goat anti-Flag fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes, washed with PBS, and detected by flow cytometry. The experimental data were fitted with EC by GraphPad Prism. 50 The binding affinity results of 260-7-B11 nanobody to GS-C2 cells expressing human PD-L1 are shown in the table below.

[0293] Table 6. Binding affinity of 260-7-B11 nanobody to GS-C2 cells overexpressing human PD-L1

[0294] From the results in Table 6, it can be seen that the 260-7-B11 nanobody can bind to GS-C2 cells overexpressing human PD-L1.

[0295] 2.7 Detection of the ability of anti-PD-L1 nanoantibodies to block the binding of PD-L1 to PD1

[0296] 2.7.1 Detection of the ability of 4A7 nanobody to block the binding of PD-L1 to PD1

[0297] The huPDL1 sequences (NCBI: Accession#NP_054862.1), mPDL1 (NCBI: Accession#NP_068693), and cynoPDL1 (NCBI: Accession#F6VEW6-1) were cloned into lentiviral vectors (plvx-IRES-puro, psPAX2, pMD2G). The constructed lentiviral plasmids and packaging plasmids were co-transfected into HEK293T (thermo K1649B) cells. After 48 hours, the cell supernatants were collected and added to CHO cells (Thremo, R80007). After 24 hours, 4 μg / ml puromycin was added for selection. Single cells expressing huPDL1, mPDL1, and cynoPDL1 were sorted using a cell sorter (Sony, LESH800SBP) to obtain stable CHO-huPDL1, CHO-mPDL1, and CHO-cynoPDL1 cells.

[0298] CHO-huPDL1, CHO-mPDL1, and CHO-cynoPDL1 cells were taken, washed with PBS, and resuspended in 1% BSA. 1×105 Cells were added to a 96-well V-bottom plate, and then gradiently diluted anti-PD-L1 nanoantibodies (starting at 33.33nM) and a constant concentration of 1.25μg / ml of human PD-1-hFc (ACRO, PD1-H5257), mouse PD-1-hFc (ACRO, PD1-M5251), and monkey PD-1-hFc (ACRO, PD1-C5254) protein premix were added. The cells were incubated on ice for 1 hour, washed with PBS, and then PE anti-human Fc (Biolegend, 410708) was added. The cells were incubated on ice for 30 minutes, washed with PBS, and then detected by flow cytometry. The experimental data were fitted with IC by GraphPad Prism. 50 The activity of 4A7 nanobody in blocking the binding of PD-L1 to PD-1 is shown in Figures 1A-1C and the table below.

[0299] Table 7. Activity of 4A7 Nanobody in Blocking the Binding of PD-L1 to PD-1

[0300] The results show that the 4A7 nanoantibody can block the binding of human PD-L1 to human PD-1, block the binding of mouse PD-L1 to mouse PD-1, and block the binding of monkey PD-L1 to monkey PD-1.

[0301] 2.7.2 Detection of the ability of 260-7-B11 nanobody to block the binding of PD-L1 to PD1

[0302] The monkey PD-L1 (Cyno-PD-L1) sequence (NCBI: Accession # F6VEW6-1) was cloned into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). The constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells. The cell supernatant was collected 48 hours later and added to 293F cells (Nanjing Kebai, CBP60437). 4 μg / ml puromycin was added for selection at 24 hours. Single cells expressing Cyno-PD-L1 were sorted using a cell sorter (Sony, LESH800SBP) to obtain 293F-cyno PD-L1 cells that stably overexpressed monkey PD-L1.

[0303] GS-C2 cells (GenScript, M00613) and 293F-cyno PD-L1 cells were taken, washed with PBS, and resuspended in 1% BSA. 1×10 5Cells were added to a 96-well V-bottom plate, and then gradiently diluted anti-PD-L1 nanoantibodies (starting at 33.33nM) and a constant concentration of 1.25μg / ml of human PD-1-mFc (ACRO, PD1-H5255) and monkey PD-1-hFc (ACRO, PD1-C5254) protein premix were added, incubated on ice for 1 hour, washed with PBS, and then PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes, washed with PBS, and detected by flow cytometry. The experimental data were fitted with IC by GraphPad Prism. 50 The activity of 260-7-B11 nanobody in blocking the binding of PD-L1 and PD-1 is shown in the following table.

[0304] Table 8. Activity of 260-7-B11 Nanobody in Blocking the Binding of PD-L1 to PD-1

[0305] From the results in Table 8, it can be seen that the 260-7-B11 nanobody can block the binding of human PD-L1 to human PD-1 and can block the binding of monkey PD-L1 to monkey PD-1.

[0306] 2.8 Screening and Assay for T Cell PD-1 / PD-L1 Signaling Pathway Blockade Activity

[0307] One day in advance, GS-C2 cells (GenScript, M00613) were plated at 4×10 4 Cells were plated in a white 96-well flat-bottom plate (corning) and cultured overnight at 37°C with 5% CO2. The plate with GS-C2 cells plated one day earlier was removed, the upper layer of culture medium was discarded, and the anti-PD-L1 nanobody after serial dilution (starting at 266.67 nM) was added. Then Jurket-NFAT-PD1 cells (GenScript, M00613) were plated at 8×10 per well. 4 Each cell was suspended in 5% 1640 medium, added to a white cell plate, mixed, and incubated at 37°C, 5% CO2 for 6 hours. Following incubation, 100 μl of detection reagent was added to each well using the Bio-Lite Luciferase Assay System (Novozymes, DD1201) according to the instructions. The plate was incubated at room temperature for 3 minutes, and then the fluorescence signal (BMG) was measured. The results of the 260-7-B11 nanoantibody blocking the T cell PD-1 / PD-L1 signaling pathway are shown in the table below.

[0308] Table 9. Activity of 260-7-B11 Nanobody in Blocking T Cell PD-1 / PD-L1 Signaling Pathway

[0309] From the results in Table 9, it can be seen that the 260-7-B11 nanobody can block the activity of the PD-1 / PD-L1 signaling pathway of T cells.

[0310] 2.9 Humanization of anti-PD-L1 nanobody

[0311] The anti-PD-L1 nanobodies 4A7 and 260-7-B11 were humanized. The sequences of 4A7 and 260-7-B11 were aligned with the IMGT offline database to identify the germline sequence with the highest homology. Using this germline sequence as a template, CDR grafting of the target sequence was performed. Following grafting, Vernier region residues in the framework region of the target sequence were backmutated to ensure that the affinity of the antibody was not affected by humanization. The 3D structure of the humanized sequence was constructed using the Modeller program, and the energy contribution of each amino acid residue to protein conformational stability was calculated using Chimera software. High-energy amino acid residues were backmutated to improve the thermal stability of the humanized sequence. After completing these steps, several humanized sequences with varying depths were obtained. Sixteen sequences with varying depths of humanization were obtained for 4A7, and eight sequences with varying depths of humanization were obtained for 260-7-B11.

[0312] 2.10 Expression of humanized PDL1 antibody

[0313] The 16 humanized sequences of 4A7 with varying depths were constructed and linked to the N-terminus of human IgG1 Fc (SEQ ID NO: 48) using a linker (SEQ ID NO: 50) and then transferred into the PTT5 expression vector (Ubao Bio, lot: VT2202). The eight humanized sequences of 260-7-B11 with varying depths were constructed and linked to the N-terminus of human IgG1 Fc (SEQ ID NO: 48) using a linker (SEQ ID NO: 50) and then transferred into the PTT5 expression vector (Ubao Bio, lot: VT2202). VHH-Fc antibodies were transiently expressed using the Expicho-S expression system (Gibco). The supernatant of the expressing cells was collected and purified using a protein A column to obtain multiple humanized VHH-Fc antibodies. Among them, 16 4A7 humanized VHH-Fc antibodies were named 4A7hu-1, 4A7hu-2, 4A7hu-3, 4A7hu-4, 4A7hu-5, 4A7hu-6, 4A7hu-7, 4A7hu-8, 4A7hu-9, 4A7hu-10, 4A7hu-11, 4A7hu-12, 4A7hu-13, 4A7hu-14, 4A7hu-15, and 4A7hu-16, and their variable region (VHH) sequences are shown in SEQ ID NOs: 24-39. Eight 260-7-B11 humanized VHH-Fc antibodies were designated 7B11_huGS_HM4, 7B11_huBSM1, 7B11_huBSM_HM1, 7B11_huBSM_HM2, 7B11_huGS1, 7B11_huGS_HM1, 7B11_huGS_HM2, and 7B11_huGS_HM3, and their variable region (VHH) sequences are shown in SEQ ID NOs: 81 to 88. 7B11 represents a VHH-Fc antibody fused to human IgG1 Fc (SEQ ID NO: 48) using the method of this example.

[0314] 2.11 Dynamic affinity testing of humanized PD-L1 nanobody

[0315] 2.11.1 Dynamic affinity testing of 4A7 humanized nanobody

[0316] The anti-DYKDDDDK antibody was bound to an HC30M chip via amino conjugation, and then captured at 2 μg / ml of a humanized PD-L1 single-domain antibody. After baseline stabilization, gradient dilutions of human PD-L1 protein (ACRO, PD1-H5258), monkey PD-L1 protein (Kaika Biotechnology, PDL-CM210), and mouse PD-L1 protein (ACRO, PD1-M5251) (starting at 100 nM) were passed over the chip from low to high concentrations. The association time was 8 minutes and the dissociation time was 20 minutes. Kinetic constants were obtained using Carteria software. The results are shown in the table below. These humanized single-domain antibodies exhibited good cross-reactivity with human, mouse, and monkey PD-L1 proteins.

[0317] Table 10. Dynamic affinity of humanized single domain antibodies to human, mouse and monkey PD-L1 proteins

[0318] 2.11.2 Dynamic affinity testing of 260-7-B11 humanized nanobody

[0319] Anti-DYKDDDDK antibodies were bound to an HC30M chip via amino conjugation, and then captured at 2 μg / ml of a humanized PD-L1 single-domain antibody. After baseline stabilization, gradient dilutions of human PD-L1 protein (ACRO, PD1-H5258) and monkey PD-L1 protein (Kaika Bio, PDL-CM210) (starting at 100 nM) were passed over the chip from low to high concentrations. The association time was 8 minutes and the dissociation time was 20 minutes. Kinetic constants were fitted using Carteria software. The results are shown in the table below. These humanized single-domain antibodies exhibited good cross-reactivity with both human and monkey PD-L1 proteins.

[0320] Table 11. Dynamic affinity detection of humanized anti-PD-L1 nanobodies

[0321] As can be seen from Table 11, the humanized single domain antibodies in the table can bind well to human and monkey PD-L1 proteins.

[0322] 2.12 Cell Binding Activity Assay of Humanized Anti-PD-L1 Nanobodies

[0323] 2.12.1 Binding activity to human PD-L1 cells

[0324] GS-C2 cells (GenScript, M00613) were washed with PBS and resuspended in 1% BSA. 1×10 cells were added to each well. 5Cells were plated into a 96-well V-bottom plate. A serial dilution series (starting at 16.67 nM) of 7B11_huGS_HM4, 7B11, and the control antibody Benchmark-ATE (atezolizumab) were then added. The cells were incubated on ice for 1 hour. After washing with PBS, PE-conjugated goat anti-human IgG Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes. After washing with PBS, the cells were detected using a flow cytometer. Data were fitted using GraphPad Prism, as shown in Figure 2.

[0325] 2.12.2 Cross-binding activity with monkey PD-L1 cells

[0326] 293F-cyno PD-L1 cells (293F, overexpressing monkey PD-L1) were taken, washed with PBS and resuspended in 1% BSA, and 1×10 5 Cells were plated into a 96-well V-bottom plate, followed by serial dilutions of 7B11_huGS_HM4, 7B11, and Benchmark-ATE (starting at 16.67 nM). The cells were incubated on ice for 1 hour, washed with PBS, and then a PE-labeled goat anti-human IgG Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes, washed with PBS, and analyzed using a flow cytometer. Data were fitted using GraphPad Prism, and the results are shown in Figure 3. The cell-binding activity of the 7B11_huGS_HM4 and 7B11 nanobodies is shown in Table 12 and Figures 2-3.

[0327] Table 12. Humanized anti-PD-L1 nanobody cell binding activity

[0328] As shown in Figures 2-3 and Table 12, 7B11 and 7B11_huGS_HM4 have excellent binding activity to cells overexpressing human and monkey PD-L1.

[0329] 2.13 Blocking activity of humanized anti-PD-L1 nanoantibodies against human PD-L1 and PD-1

[0330] GS-C2 cells (GenScript, M00613) were washed with PBS and resuspended in 1% BSA. 1×10 cells were added to each well. 5Cells were plated into a 96-well V-bottom plate. A serial dilution of 7B11_huGS_HM4, 7B11, and Benchmark-ATE (starting at 22.22 nM) was added, along with a constant concentration of 1.25 μg / ml human PD-1-mFc protein premix. The cells were incubated on ice for 1 hour. After washing with PBS, PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes. After washing with PBS, the cells were detected using a flow cytometer. Data were fitted using GraphPad Prism, and the results are shown in Figure 4 and Table 13.

[0331] Table 13. Blocking activity of humanized anti-PD-L1 nanobody

[0332] As shown in Figure 4 and Table 13, 7B11 and 7B11_huGS_HM4 can effectively block the binding of PD-L1 and PD-1.

[0333] 2.14 Detection of the blocking activity of humanized anti-PD-L1 nanobody on the PD-1 / PD-L1 signaling pathway in T cells

[0334] One day in advance, GS-C2 cells (GenScript, M00613) were plated at 4×10 4 Cells were plated in a white 96-well flat-bottom plate (corning) and cultured overnight at 37°C in 5% CO2. The plate with GS-C2 cells plated one day earlier was removed, the upper layer of culture medium was discarded, and serial dilutions of 7B11_huGS_HM4, 7B11, and Benchmark-ATE (starting at 20 nM) were added. Then, Jurket-NFAT-PD1 cells (GenScript, M00613) were plated at 8×10 cells per well. 4 Cells were suspended in 5% 1640 medium, added to a white cell plate, mixed, and incubated at 37°C, 5% CO2 for 6 hours. After incubation, 100 μl of detection reagent was added to each well according to the Bio-Lite Luciferase Assay System (Novozymes) instructions. The plates were incubated at room temperature for 3 minutes, and then the fluorescence signal (BMG) was measured. The results are shown in Table 14 and Figure 5.

[0335] Table 14. Activity of humanized anti-PD-L1 nanobodies in blocking the PD-1 / PD-L1 signaling pathway in T cells

[0336] The results in Table 14 and Figure 5 show that 7B11_huGS_HM4 and 7B11 can effectively block the PD-1 / PD-L1 signaling pathway of T cells.

[0337] 2.15 Effects of humanized anti-PD-L1 nanobody on T cell activation in lymphocyte responses

[0338] Resuscitation of DC cells (Aoneng Biotech, FPB-DC002F-C) and PBMC cells (Aoneng Biotech, FPB004F-C-MLR), DC cells were 1×10 per well. 4 PBMC cells per well: 1×10 5 The mixture was mixed, and 200 μl was added to each well of a 96-well flat-bottom plate. The 96-well plate was divided into three groups, each of which was added with Benchmark-ATE, 7B11, and 7B11_huGS_HM4. In the Benchmark-ATE group, different concentrations of Benchmark-ATE (111.1 nM, 44.44 nM, 4.444 nM, and 0 nM) were added to each well; in the 7B11 group, different concentrations of 7B11 (66.7 nM, 6.67 nM, 0.667 nM, and 0 nM) were added to each well; and in the 7B11_huGS_HM4 group, different concentrations of 7B11_huGS_HM4 (66.7 nM, 6.67 nM, 0.667 nM, and 0 nM) were added to each well. The 96-well plate was incubated at 37°C under 5% CO2 for 5 days, and the concentrations of IL2 and IFNγ were detected using HUMAN IL2 KITS (Cisbio Bioassays, 62HIL02PEG) and HUMAN IFNγ KITS (Cisbio Bioassays, 62HIFNGPEG).

[0339] The results are shown in Figures 6a and 6b. Figure 6a shows the results of IL-2 production by Benchmark-ATE, 7B11, and 7B11_huGS_HM4 on T cell activation; Figure 6b shows the results of IFNγ production by Benchmark-ATE, 7B11, and 7B11_huGS_HM4 on T cell activation. These results demonstrate that 7B11 and 7B11_huGS_HM4 can stimulate cytokine production in donors, demonstrating that 7B11 and 7B11_huGS_HM4 effectively activate T cells.

[0340] Example 3: Preparation of CD3-PDL1-MSLN trispecific antibody

[0341] 3.1 Construction of CD3-PDL1-MSLN trispecific antibody

[0342] The structure of the CD3-PDL1-MSLN trispecific antibody of this embodiment is shown in Figure 7, and is composed of three chains: 1. a light chain, consisting of a CD3 antibody light chain variable region and a light chain CL1 constant region (SEQ ID NO: 44); 2. a heavy chain 1, consisting of a humanized CD3 heavy chain variable region, an IgG1 mutant 1 (SEQ ID NO: 45) at the C-terminus connected to a humanized MSLN single domain antibody via (GGGGS)3; 3. a heavy chain 2, consisting of a humanized MSLN single domain antibody connected to the N-terminus of an IgG1 mutant 2 (SEQ ID NO: 47) via (GGGGS)1, and a PD-L1 single domain antibody at the C-terminus of an IgG1 mutant 2 via (GGGGS)3; wherein the CD3 heavy chain variable region is selected from VH1 (SEQ ID NO: 40), VH2 (SEQ ID NO: 41), and VH3 (SEQ ID NO: 42); and the CD3 light chain variable region is VL (SEQ ID NO: 43). Specifically, a total of 16 triple antibodies (5Y3-1 to 5Y3-15, 5Y3-16) were obtained, and their structures are shown in the table below; the nucleic acid sequences encoding the above 16 polypeptide chains were constructed into the PTT5 plasmid vector (Ubao Bio, lot: VT2202), and sufficient plasmids were extracted for use.

[0343] Table 15. Trispecific antibody structures

[0344] 3.2 Expression and purification of CD3-PDL1-MSLN trispecific antibody

[0345] One day before transfection (D1), the cell density was diluted to 2×10 6 On the day of transfection (D0), count the cells (cell viability should be ≥95%) and adjust the cell density to 4.0×10 6 The antibody fragment plasmids were mixed at a ratio of heavy chain 1:heavy chain 2:light chain = 1:1:1, mixed with PEI, and co-transfected into CHO-S cells (Gibco A29127). After transfection, the cells were transferred to a 37°C, 120 rpm, 8% CO2 incubator for culture. On the first day after transfection (D1), preheated CHO gro (Mirus6200A) complete medium was added at 1 / 5 of the expression volume. The culture was cooled to 32°C and continued to be cultured. On days 2, 4, and 6 after transfection, Advanced CHO Feed 1 (Sigma 24367C) was supplemented at 8%, 5%, and 5%, respectively. Cell viability was monitored daily starting on D7 and cells were harvested when the viability was <80%. The supernatant was collected by centrifugation and purified using a protein A column for later use.

[0346] Example 4: Verification of binding activity of CD3-PDL1-MSLN trispecific antibody

[0347] 4.1 CD3-PDL1-MSLN trispecific antibody binding to human Jurkat cells

[0348] (1) Jurkat cells (Company: ATCC; Catalog No.: TIB-152) were adjusted to 1×10 7 cells / ml, prepare each sample and add 50 μl of cell suspension.

[0349] (2) For 5Y3-1 to 5Y3-15, adjust the final concentration to 2 μg / ml with PBS, then perform a 3-fold serial dilution, and perform 8 steps. For 5Y3-16, adjust the final concentration to 2000 nM with PBS, then perform a 3-fold serial dilution, and perform 7 steps. 50 μl of antibody was added to each well.

[0350] (3) Take 50 μl of cell suspension, the total cell volume is 5×10 5 For 5Y3-1 to 5Y3-15, the final antibody concentration was a starting concentration of 2 μg / ml. Three-fold serial dilutions were performed in 8 wells, and the cells were incubated at 4°C for 1 hour. For 5Y3-16, the final antibody concentration was a starting concentration of 2000 nM. Three-fold serial dilutions were performed in 7 wells, and the cells were incubated at 4°C for 1 hour.

[0351] (4) 500g, 5 min, gently remove the supernatant, add 200μl PBS and wash twice.

[0352] (5) Add secondary antibody PE anti-human Fc (Biolegend 410708): Add fluorescent secondary antibody diluted in PBS (1:100) and incubate at 4°C for 30 min.

[0353] (6) Wash the fluorescent secondary antibody at 500g for 5 min, gently remove the supernatant, add 200μl PBS to wash once, and then detect by upflow cytometry.

[0354] The results are shown in the table below, and the constructed trispecific antibody is able to bind to Jurkat cells expressing CD3.

[0355] Table 16. Binding results of trispecific antibodies to human Jurkat cells

[0356] 4.2 CD3-PDL1-MSLN trispecific antibody binding to human MC38 / MSLN cells

[0357] Human MC38 / MSLN cell construction: The full-length human MSLN (huMSLN) cDNA (purchased from Sino Biological, HG13128-UT) was cloned into a lentiviral vector. The constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells. Cell supernatants were collected 48 and 72 hours later and added to MC38 cells (Nanjing Kebai, Cat. No. CBP60825). 24 hours later, 4 μg / ml puromycin was added for selection. Single cells overexpressing huMSLN were sorted using a cell sorter (Sony, LE-SH800SBP) to obtain stable MC38 / MSLN monoclonal cells expressing high huMSLN expression.

[0358] Take 50 μl of MC38 / MSLN cells (2×10 5 Cells were added to a 96-well V-shaped plate, and 50 μl of serially diluted antibodies (antibodies were serially diluted from 100 nM) were added to each well and incubated on ice for 1 hour. After washing away the primary antibody, PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added and incubated on ice for 1 hour. After washing, each well was resuspended in 200 μl PBS and detected by flow cytometry. EC were counted using Graphpad Prism software. 50 8A-8D . The results show that the constructed trispecific antibody can bind to cells expressing MSLN.

[0359] 4.3 CD3-PDL1-MSLN trispecific antibody binding to CHO-hPDL1 cells

[0360] CHO-huPDL1 cells were washed with PBS and resuspended in 1% BSA at a rate of 1×10 cells per well. 5 Cells were added to a 96-well V-bottom plate, and then a gradient dilution of anti-CD3-PDL1-MSLN trispecific antibody was added (for 5Y3-1 to 5Y3-15, starting at 1500nM, 3-fold gradient dilution, a total of 8 gradients; for 5Y3-16, starting at 100nM, 3-fold gradient dilution, a total of 11 gradients), incubated on ice for 1 hour, washed with PBS, and then PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes, washed with PBS, and detected using a flow cytometer. The experimental data were fitted with EC by GraphPad Prism. 50 9A-9C . The results show that the constructed trispecific antibody can bind to cells expressing PD-L1.

[0361] Example 5: CD3-PDL1-MSLN trispecific antibody blocks binding of human PD1 to human PD-L1

[0362] CHO-huPDL1 cells were washed with PBS and resuspended in 1% BSA at a rate of 1×10 cells per well. 5 Cells were added to a 96-well V-bottom plate, and then a gradient dilution of the CD3-PDL1-MSLN trispecific antibody was added (for 5Y3-1 to 5Y3-15, starting at 1500nM, 3-fold gradient dilution, a total of 8 gradients; for 5Y3-16, starting at 50nM and gradient dilution) and a constant concentration of 1.25ug / ml human PD-1-mFc (ACRO, PD1-H5255) protein premix were added. The cells were incubated on ice for 1 hour, washed with PBS, and then PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes, washed with PBS, and detected by flow cytometry. The experimental data were fitted with EC by GraphPad Prism. 50 .

[0363] The results are shown in Figures 10A-10C , and the constructed trispecific antibody can effectively block the binding of human PD1 to human PD-L1.

[0364] Example 6: Dynamic affinity detection of CD3-PDL1-MSLN trispecific antibody

[0365] Antibodies at three concentrations, 4, 2, and 1 μg / mL, were immobilized on an HC200M chip containing an anti-human IgG Fc secondary antibody, controlling the antibody binding capacity to approximately 1000 RU. After baseline stabilization, a series of dilutions (eight steps of three-fold dilutions starting from 592.5 nM) of human CD3εγ (Acrobiosystems, Catalog No. CDG-H52W5), human MSLN-Fc (Kaixia Biotechnology, MSL-HM280), and human PD-L1 protein (ACRO, PD1-H5258) were flowed over the chip at a flow rate of 1000 μL / min. The binding time was 8 minutes and the dissociation time was 20 minutes. Kinetic constants were calculated using a 1:1 binding model fitted using Carterra's Kinetics software. The results, as shown in the table below, show that the constructed trispecific antibody exhibits good affinity for human CD3εγ, human MSLN, and human PD-L1.

[0366] Table 17. Affinity determination of trispecific antibodies to human CD3εγ, human MSLN and human PD-L1

[0367] Example 7: Activation of T cell activation signaling pathways by CD3-PDL1-MSLN trispecific antibodies

[0368] (1) No tumor group

[0369] For 5Y3-1 to 5Y3-15, dilute the antibody to 2 μg / ml with 1640+10% FBS, and add 50 μl of diluted antibody to each well; take Jurkat-NFAT-luc cells (Nanjing Kebai, catalog number CBP74020) in the logarithmic growth phase and adjust the volume to 6×10 5 cells / ml, 50 μl of cells (3×10 4 After gently mixing, the cells were incubated at 37°C and 5% CO2 for 5-6 hours, and 20 μl / well of chemiluminescent substrate was added and the cells were detected by an instrument.

[0370] For 5Y3-16, the antibody was diluted to 5 nM with 1640 + 10% FBS, and 50 μl of diluted antibody was added to each well. Jurkat-NFAT-luc cells (Nanjing Kebai, Cat. No. CBP74020) in the logarithmic phase were taken and adjusted to 1.6 × 10 6 cells / ml, 50 μl of cells (8×10 4 After gently mixing, the cells were incubated at 37°C and 5% CO2 for 5-6 hours, and 20 μl / well of chemiluminescent substrate was added and the cells were detected by an instrument.

[0371] (2) Join the tumor group

[0372] For 5Y3-1 to 5Y3-15, dilute the antibody to 2 μg / ml with 1640+10% FBS, and add 50 μl of diluted antibody to each well; take Jurkat-NFAT-luc cells in the logarithmic growth phase and adjust the concentration to 1.2×10 6 cells / ml, 25 μl of cells (3×10 4 cells / well); digest the MC38 / MSLN cells constructed in Example 4, and adjust the digested MC38 / MSLN cells to 1.2×10 6 cells / ml, 25 μl of cells (3×10 4 After gently mixing, the cells were placed at 37°C and 5% CO2 for 5-6 hours, and 20 μl / well of chemiluminescent substrate was added and the cells were detected by a flow cytometer.

[0373] For 5Y3-16, dilute the antibody to 1 nM with 1640 + 10% FBS, and add 50 μl of diluted antibody to each well; take Jurkat-NFAT-luc cells in the logarithmic growth phase and adjust the volume to 3.2 × 10 6 cells / ml, 25 μl of cells (8×10 4cells / well); digest the MC38 / MSLN cells constructed in Example 4, and adjust the digested MC38 / MSLN cells to 1.6×10 6 cells / ml, 25 μl of cells (4×10 4 After gently mixing, the cells were placed at 37°C and 5% CO2 for 5-6 hours, and 20 μl / well of chemiluminescent substrate was added and the cells were detected by a flow cytometer.

[0374] The results of the tumor-addition group are shown in Figures 11A-11C, and the results of the non-tumor-addition group are shown in Figures 12A-12C. The results show that the CD3-PDL1-MSLN trispecific antibody of the present invention cannot activate Jurkat-NFAT-luc cells in the absence of tumor cells, but can moderately activate Jurkat-NFAT-luc cells after addition of tumor cells.

[0375] Example 8: CD3-PDL1-MSLN trispecific antibody-mediated TDCC

[0376] 8.1 HCC1806-luc cell preparation

[0377] The Luc sequence (NCBI: GenBank: MF062157.1) was cloned into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). The constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells. After 48 hours, the cell supernatant was collected and added to HCC1806 cells (ATCC, CRL-2335, human breast cancer cells). After 24 hours, 4 μg / ml puromycin was added for selection. Single cells expressing Luc were sorted using a cell sorter (Sony, LESH800SBP) to obtain stable HCC1806-luc cells.

[0378] 8.2 PBMC Preparation

[0379] Resuscitated frozen PBMC cells (Shanghai Saili) were centrifuged at 500g for 3 min, and the supernatant was removed. The cells were then washed once with 1640+10% FBS (inactivated) at 500g for 3 min, and the supernatant was removed. The cells were resuspended with an appropriate amount of 1640+10% FBS (inactivated), counted, and diluted to the corresponding density. The cells were added to the well plate, and 25 μl was added to each well to make the total number of PBMC cells 1.25×10 5 cells / well.

[0380] 8.3 Plating of HCC1806-luc cells

[0381] Take 25 μl of HCC1806-luc cells and add them to the above PBMC well plate, 1.25×10 4 cells / well;

[0382] 8.4 CD3-PDL1-MSLN trispecific antibody dilution

[0383] For 5Y3-1 to 5Y3-15, adjust the concentration to 2 nM with culture medium and perform serial dilutions. Add 50 μl of the diluted antibodies to the culture medium containing PBMC and HCC1806-luc cells.

[0384] For 5Y3-16, adjust the concentration to 20 nM with culture medium and perform serial dilutions. Add 100 μl of the diluted antibody to the culture medium containing PBMC and HCC1806-luc cells.

[0385] 8.5 Incubation

[0386] Incubate in a 37°C, 5% CO2 incubator for 2 days;

[0387] 8.6 Reading

[0388] Remove the plate, add 50 μl of luciferase substrate, read and analyze the data on the computer;

[0389] The experimental results are shown in Figures 13A-13C. The CD3-PDL1-MSLN trispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effect on human tumor cells HCC1806 expressing MSLN.

[0390] Example 9: CD3-PDL1-MSLN trispecific antibody cytokine assay

[0391] For 5Y3-1 to 5Y3-16, the concentrations of the CD3-PDL1-MSLN trispecific antibody and the positive control antibody HPN536 (HPN536 is a trispecific antibody targeting CD3 / MSLN / HAS developed by Harpoon Therapeutics, whose sequence is shown in SEQ ID NO: 66. The HPN536 sequence was constructed into the PTT5 expression vector (Ubao Bio, lot: VT2202) and transiently expressed using the Expicho-s expression system (Gibco), followed by purification via a protein A column to obtain the HPN536 antibody) were adjusted to 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM in a five-step gradient. Diluted trispecific antibodies (50 μl / well) or control antibodies were added, and no trispecific antibodies or control antibodies were added to the last well. The PBMC cell density was adjusted to 2 × 10 6cells / ml, 50 μl / well; culture at 37°C, 5% CO2 for 48 h. Take 30 μl of supernatant at 24 h and 48 h, respectively, and detect the IL2 and INF-γ contents in the supernatant using HTRF kits (cisbio 62HIL02PEH, 62HIFNGPEH).

[0392] The IL2 detection results are shown in Figures 14A-14F, and the INF-γ detection results are shown in Figures 15A-15F. The results show that the CD3-PDL1-MSLN trispecific antibody of the present invention, when co-incubated with PBMCs, strongly promoted the secretion of IL2 and INF-γ compared to the antibody in the control group, while the trispecific antibody of the present invention promoted the secretion of IL2 and INF-γ much less strongly, indicating that the trispecific antibody of the present invention has a low risk of side effects in inducing PBMC to produce IL2 and INF-γ, and has a good safety profile.

[0393] Example 10: PBMC immune reconstitution of subcutaneous HCC1806 transplanted tumor model in mice

[0394] Female NOG-dKO (NOG-MHC I / II-2 KO mice) aged 6 to 8 weeks were selected (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) and injected intraperitoneally with 5×10^ 7 PBMC cells were subcutaneously inoculated into each mouse 7 days later at a dose of 3×10^ 6 HCC1806 cells, wait until the tumor grows to an average of about 80 mm 3 At the time of the experiment, mice were randomly divided into 8 groups, each receiving 0.05 mg / kg, 0.25 mg / kg, and 1 mg / kg doses, respectively. Dosing was repeated twice a week for a total of 4 doses. A control group of 8 mice remained untreated. Tumor volume and body weight were monitored. At the end of the experiment, mice were sacrificed by cervical dislocation, and tumors were removed, weighed, and recorded.

[0395] The results, shown in Figure 16A, demonstrate that the 5Y3-16 antibody exhibited excellent efficacy and a dose-dependent in vivo response, with tumor inhibition rates of 81%, 101%, and 106% at doses of 0.05 mg / kg, 0.25 mg / kg, and 1 mg / kg, respectively. As shown in Figure 16B, tumor-bearing mice tolerated these doses well, with no adverse reactions such as weight loss.

[0396] Example 11: TDCC mediated by trispecific antibodies with different CD3-PDL1-MSLN structures

[0397] 11.1 Preparation of MC38-luc, CT26-luc, H2052-luc, and HCC1806-luc cells

[0398] The Luc sequence (NCBI: GenBank: MF062157.1) was cloned into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). The constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells. The cell supernatant was collected 48 hours later and added to MC38 cells (ATCC, CRL-2640, mouse colon cancer cells), CT26 (ATCC, CRL-2638, mouse colon cancer cells), and H2052 (ATCC, CRL-5915, human mesothelioma cells). 4 μg / ml puromycin was added for selection 24 hours later. Single cells expressing Luc were sorted using a cell sorter (Sony, LESH800SBP) to obtain stable MC38-luc, CT26-luc, and H2052-luc cells, and the HCC1806-luc cells prepared in Example 8 were also obtained.

[0399] 11.2 PBMC Preparation

[0400] Resuscitated frozen PBMC cells (Shanghai Saili) were centrifuged at 500g for 3 min, and the supernatant was removed. The cells were then washed once with inactivated 1640+10% FBS; centrifuged at 500g for 3 min, and the supernatant was removed. The cells were resuspended with an appropriate amount of inactivated 1640+10% FBS, counted, and diluted to the corresponding density. The cells were then added to the well plate, with 25 μl added to each well to make the total number of PBMC cells 1.25×10 5 cells / well.

[0401] 11.3 Plating MC38-luc, CT26-luc, H2052-luc, and HCC1806-luc Cells

[0402] Take 25 μl of MC38-luc, CT26-luc, HCC1806-luc, and H2052-luc cells respectively and add them to the PBMC well plate prepared in step 11.2 above. Add 1.25×10 4 cells / well;

[0403] 11.4 Dilution of CD3-PDL1-MSLN trispecific antibodies with different structures

[0404] The structures of the trispecific antibodies 5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244 of this embodiment are shown in Figures 17A-17B, wherein:

[0405] The structures of 5Y3-168 and 5Y3-244 are the same as the structure of the trispecific antibody in 3.1 in Example 3.

[0406] 5Y3-158 and 5Y3-194 are each composed of three chains: 1. A light chain consisting of the CD3 antibody light chain variable region and the light chain CL1 constant region (SEQ ID NO: 44); 2. A heavy chain 1 consisting of the humanized CD3 heavy chain variable region, followed by the C-terminus of IgG1 mutant 1 (SEQ ID NO: 45) connected to the humanized MSLN single domain antibody via (GGGGS)3; and 3. A heavy chain 2 consisting of the PDL1 single domain antibody connected to the N-terminus of IgG1 mutant 2 (SEQ ID NO: 47) via (GGGGS)1, followed by the C-terminus of IgG1 mutant 2 connected to the humanized MSLN single domain antibody via (GGGGS)3. The sequences of 5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244 are shown in Table 18.

[0407] Table 18. Trispecific antibody sequence combinations

[0408] Prepare trispecific antibodies with different CD3-PDL1-MSLN structures (5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244) and serially dilute them in culture medium (RPMI-1640, Gibco 61870036). Add 50 μl of the diluted antibodies to the PBMCs and MC38-luc, CT26-luc, HCC1806-luc, and H2052-luc cells obtained in step 11.3.

[0409] 11.5 Incubation

[0410] Incubate in a 37°C, 5% CO2 incubator for 2 days;

[0411] 11.6 Reading

[0412] Remove the plate, add 50 μl of luciferase substrate, read and analyze the data on the computer;

[0413] The experimental results are shown in Figures 18A-18E. The trispecific antibodies with different structures of CD3-PDL1-MSLN (5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244) can exert T cell-mediated tumor cell killing (TDCC) on human tumor cells HCC1806 expressing MSLN, among which the killing effect of 5Y3-168 is significantly better than that of 5Y3-194, and the killing effect of 5Y3-244 is significantly better than that of 5Y3-158; this indicates that when the sequence is the same, the tumor killing effect is better when the MSLN antibody is located at the N-terminus and C-terminus of the Fc.

[0414] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A humanized single-domain antibody or antigen-binding fragment thereof capable of specifically binding to MSLN, wherein the humanized single-domain antibody or antigen-binding fragment thereof comprises: a VHH sequence as shown in any one of SEQ ID NOs: 1-8 or a variant thereof, in, The variant has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence from which it is derived, Alternatively, the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

2. A single-domain antibody or antigen-binding fragment thereof that can specifically bind to PD-L1, comprising: CDR1, CDR2, and CDR3 contained in the VHH set forth in any one of SEQ ID NOs: 10, 24-39, 67, and 81-88; Preferably, the CDRs are identified by the IMGT, Kabat, AbM, Chothia, or Contact numbering systems.

3. The single-domain antibody or antigen-binding fragment thereof according to claim 2, wherein the single-domain antibody or antigen-binding fragment thereof comprises: (1) The following CDRs are defined by the IMGT numbering system: (1A) a CDR1 comprising the sequence shown in SEQ ID NO: 11 or 63, a CDR2 comprising the sequence shown in SEQ ID NO: 12, and a CDR3 comprising the sequence shown in SEQ ID NO: 13; or, (1B) a CDR1 comprising the sequence set forth in SEQ ID NO: 68, a CDR2 comprising the sequence set forth in SEQ ID NO: 69, and a CDR3 comprising the sequence set forth in SEQ ID NO: 70; (2) The following CDRs defined by the Kabat numbering system: (2A) a CDR1 comprising the sequence set forth in SEQ ID NO: 14, a CDR2 comprising the sequence set forth in SEQ ID NO: 15, and a CDR3 comprising the sequence set forth in SEQ ID NO: 16; or, (2B) a CDR1 comprising the sequence set forth in SEQ ID NO: 71, a CDR2 comprising the sequence set forth in SEQ ID NO: 72, and a CDR3 comprising the sequence set forth in SEQ ID NO: 73; (3) The following CDRs defined by the AbM numbering system: (3A) a CDR1 comprising the sequence set forth in SEQ ID NO: 17, a CDR2 comprising the sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the sequence set forth in SEQ ID NO: 16; or, (3B) a CDR1 comprising the sequence set forth in SEQ ID NO: 74, a CDR2 comprising the sequence set forth in SEQ ID NO: 75, and a CDR3 comprising the sequence set forth in SEQ ID NO: 73; (4) The following CDRs defined by the Chothia numbering system: (4A) a CDR1 comprising the sequence shown in SEQ ID NO: 19, a CDR2 comprising the sequence shown in SEQ ID NO: 20, and a CDR3 comprising the sequence shown in SEQ ID NO: 16; or, (4B) a CDR1 comprising the sequence set forth in SEQ ID NO: 76, a CDR2 comprising the sequence set forth in SEQ ID NO: 77, and a CDR3 comprising the sequence set forth in SEQ ID NO: 73; or, (5) The following CDRs defined by the Contact Numbering System: (5A) a CDR1 comprising the sequence shown in SEQ ID NO: 21, a CDR2 comprising the sequence shown in SEQ ID NO: 22, and a CDR3 comprising the sequence shown in SEQ ID NO: 23; or, (5B) CDR1 comprising the sequence shown in SEQ ID NO: 78, CDR2 comprising the sequence shown in SEQ ID NO: 79, and CDR3 comprising the sequence shown in SEQ ID NO:

80.

4. The single-domain antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein The single-domain antibody or antigen-binding fragment thereof comprises the VHH sequence shown in SEQ ID NO: 10, 67 or a variant thereof, wherein the variant has a sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived, Or, wherein the variant has one or more amino acid substitutions, deletions or additions compared thereto; preferably, the substitutions are conservative substitutions.

5. The single domain antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, which is humanized; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain framework region of a human immunoglobulin, The framework regions of the chains optionally contain back mutations from human to camelid residues.

6. The single-domain antibody or antigen-binding fragment thereof according to claim 5, wherein The single-domain antibody or antigen-binding fragment thereof comprises a VHH sequence shown in any one of SEQ ID NOs: 24-39, 81-88 or a variant thereof, wherein the variant has a sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived, Alternatively, the variant has one or more amino acid substitutions, deletions or additions compared thereto; preferably, the substitutions are conservative substitutions.

7. The single-domain antibody or antigen-binding fragment thereof according to any one of claims 2 to 6, wherein: The PD-L1 is selected from human PD-L1, mouse PD-L1, camel PD-L1 and / or monkey PD-L1.

8. A polypeptide construct comprising the humanized single-domain antibody or antigen-binding fragment thereof according to claim 1, and / or the single-domain antibody or antigen-binding fragment thereof according to any one of claims 2 to 7, and an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is optionally connected to the N-terminus and / or C-terminus (e.g., C-terminus) of the humanized single-domain antibody or antigen-binding fragment thereof or the single-domain antibody or antigen-binding fragment thereof via a peptide linker; Preferably, the immunoglobulin Fc domain is the Fc domain of IgG (eg, the Fc domain of IgG1); Preferably, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 48; Preferably, the peptide linker is a peptide linker comprising one or more glycine and / or one or more serine, for example, (G4S)n, where n is 1, 2, 3 or 4, for example, the sequence shown in SEQ ID NO:

50.

9. A multispecific antibody comprising: the humanized single-domain antibody or antigen-binding fragment thereof capable of specifically binding to MSLN according to claim 1 and / or the single-domain antibody or antigen-binding fragment thereof capable of specifically binding to PD-L1 according to any one of claims 2 to 7; Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

10. A multispecific antibody comprising: a first antigen-binding domain targeting CD3, a second antigen-binding domain targeting MSLN, and a third antigen-binding domain targeting PD-L1, wherein: (i) The first antigen-binding domain comprises VH and VL, wherein the VH comprises SEQ ID NO: 55 The present invention further comprises the HCDR1 shown in SEQ ID NO: 54, the HCDR2 shown in SEQ ID NO: 56, and the HCDR3 shown in any one of SEQ ID NOs: 57-59, 65; and / or the VL comprises the LCDR1 shown in SEQ ID NO: 60, the LCDR2 shown in SEQ ID NO: 61, and the LCDR3 shown in SEQ ID NO: 62; preferably, the VH comprises the sequence shown in any one of SEQ ID NOs: 40-42, 64; preferably, the VL comprises the sequence shown in SEQ ID NO: 43; (ii) the second antigen-binding domain comprises a VHH, wherein the VHH comprises the CDR1, CDR2 and CDR3 contained in the VHH set forth in any one of SEQ ID NOs: 1-9; preferably, the VHH comprises the CDR1 set forth in SEQ ID NO: 52, the CDR2 set forth in SEQ ID NO: 53, and the CDR3 set forth in SEQ ID NO: 54; preferably, the second antigen-binding domain comprises the humanized single-domain antibody or antigen-binding fragment thereof according to claim 1, or comprises the VHH sequence set forth in SEQ ID NO: 9; and / or, (iii) the third antigen-binding domain comprises the single-domain antibody or antigen-binding fragment thereof according to any one of claims 2 to 7.

11. A multispecific antibody comprising: a first antigen-binding domain targeting CD3, a second antigen-binding domain targeting a tumor-associated antigen (TAA), and a third antigen-binding domain targeting an immune checkpoint, wherein: The first antigen-binding domain is Fab, and the second and third antigen-binding domains are VHH; Preferably, the multispecific antibody further comprises an Fc domain, wherein the Fc domain comprises a first Fc monomer and a second Fc monomer; wherein: The N-terminus of the first Fc monomer is optionally connected to the first antigen-binding domain (e.g., the heavy chain CH1 domain thereof) via a linker, and the C-terminus thereof is optionally connected to one of the second antigen-binding domains via a linker; The N-terminus of the second Fc monomer is optionally connected to another second antigen-binding domain via a linker, and the C-terminus thereof is optionally connected to the third antigen-binding domain via a linker.

12. The multispecific antibody according to claim 11, wherein The tumor-associated antigen comprises MSLN.

13. The multispecific antibody according to claim 11 or 12, wherein The immune checkpoint is selected from PD1, PD-L1 or a combination thereof; Preferably, the immune checkpoint is PD-L1.

14. The multispecific antibody according to any one of claims 11 to 13, wherein The Fc domain comprises modifications to promote dimerization of the first Fc monomer and the second Fc monomer; Preferably, the modification comprises an amino acid substitution in the CH3 domain of the Fc domain; Preferably, the modification comprises a "knob" modification in one of the two monomers and a "hole" modification in the other of the two monomers to form a "knob-into-hole" modification; Preferably, the first Fc monomer and the second Fc monomer of the Fc domain comprise the amino acid sequences shown in SEQ ID NOs: 46 and 47, respectively.

15. The multispecific antibody according to any one of claims 11 to 14, wherein The linker is selected from a peptide linker comprising one or more glycine and / or one or more serine; Preferably, the peptide linker comprises (G4S)n, where n is 1, 2, 3 or 4, for example, comprises the sequence shown in any one of SEQ ID NOs: 49-51.

16. The multispecific antibody according to any one of claims 11 to 15, wherein The multispecific antibody comprises: (i) a first peptide chain comprising the VL and light chain constant region (CL) of the first antigen-binding domain; preferably, the CL is a kappa light chain constant region; (ii) a second peptide chain comprising the VH, CH1 and first Fc monomer of the first antigen-binding domain and the second antigen-binding domain; preferably, the first Fc monomer is IgG; preferably, the first Fc monomer comprises a hinge region, CH2 and CH3; preferably, the second antigen-binding domain is connected to the C-terminus of the first Fc monomer via a linker (e.g., a flexible peptide comprising (G4S)n); and (iii) a third peptide chain comprising the second antigen-binding domain, a second Fc monomer, and a third antigen-binding domain; preferably, the second Fc monomer is IgG; preferably, the second Fc monomer comprises a hinge region, CH2, and CH3; preferably, the second antigen-binding domain is linked to the N-terminus of the second Fc monomer via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the third antigen-binding domain is linked to the C-terminus of the second Fc monomer via a linker (e.g., a flexible peptide comprising (G4S)n); Preferably, the second Fc monomer of the third peptide chain is capable of forming a dimer with the first Fc monomer of the second peptide chain.

17. The multispecific antibody according to claim 16, wherein The first Fc monomer of the second peptide chain and the second Fc monomer of the third peptide chain comprise modifications to promote dimerization; Preferably, the modification comprises an amino acid substitution in the CH3 domain of the Fc domain; Preferably, the modification comprises a "knob" modification in one of the two Fc monomers and a "hole" modification in the other of the two Fc monomers to form a "knob-into-hole" modification; Preferably, the two Fc monomers comprise the amino acid sequences shown in SEQ ID NOs: 46 and 47, respectively; Preferably, the first Fc monomer of the second peptide chain comprises the amino acid sequence shown in SEQ ID NO: 46; preferably, the second peptide chain comprises the heavy chain constant region sequence shown in SEQ ID NO: 45; Preferably, the second Fc monomer of the third peptide chain comprises the amino acid sequence shown in SEQ ID NO:

47.

18. The multispecific antibody according to any one of claims 11 to 17, wherein The first antigen-binding domain targeting CD3 comprises a VH and a VL, wherein the VH comprises the HCDR1 of SEQ ID NO: 55, the HCDR2 of SEQ ID NO: 56, and the HCDR3 of any one of SEQ ID NOs: 57-59, 65; and / or the VL comprises the LCDR1 of SEQ ID NO: 60, the LCDR2 of SEQ ID NO: 61, and the LCDR3 of SEQ ID NO: 62; Preferably, the VH comprises the sequence shown in any one of SEQ ID NOs: 40-42, 64; Preferably, the VL comprises the sequence shown in SEQ ID NO:

43.

19. The multispecific antibody according to any one of claims 11 to 18, wherein The second antigen-binding domain targeting MSLN comprises a VHH, wherein the VHH comprises the CDR1, CDR2, and CDR3 contained in the VHH shown in any one of SEQ ID NOs: 1-9; preferably, the VHH comprises the CDR1 shown in SEQ ID NO: 52, the CDR2 shown in SEQ ID NO: 53, and the CDR3 shown in SEQ ID NO: 54; Preferably, the second antigen-binding domain comprises the humanized single-domain antibody or antigen-binding fragment thereof according to claim 1 or comprises the VHH sequence shown in SEQ ID NO:

9.

20. The multispecific antibody according to any one of claims 11 to 19, wherein The third antigen-binding domain targeting PD-L1 comprises the single-domain antibody or antigen-binding fragment thereof capable of specifically binding to PD-L1 according to any one of claims 2 to 7.

21. The multispecific antibody according to any one of claims 11 to 20, wherein The multispecific antibody comprises: (i) a first peptide chain having the structure [VL]-[CL], (ii) a second peptide chain having the structure [VH]-[CH]-[L1]-[VHH1], and (iii) a third peptide chain having the structure [VHH1]-[L2]-[Fc monomer]-[L3]-[VHH2]; One of the following: (1) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 81; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (2) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 29; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (3) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 29; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (4) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 29; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (5) The VL comprises the sequence shown in SEQ ID NO: 43, and the CL comprises the sequence shown in SEQ ID NO:

44. the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 30; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (6) the VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 30; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (7) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 30; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (8) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 38; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (9) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: NO:47, the VHH2 comprises the sequence shown in SEQ ID NO:38; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO:51, L2 is SEQ ID NO:49, and L3 is SEQ ID NO:51; (10) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 38; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (11) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 31; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (12) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 31; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (13) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 31; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide comprising one or more glycine and / or one or more serine. A linker (e.g., a peptide linker represented by (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (14) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 40, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 39; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (15) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 41, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 39; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (16) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 42, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 8; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 39; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; (17) The VL comprises the sequence shown in SEQ ID NO: 43, the CL comprises the sequence shown in SEQ ID NO: 44; the VH comprises the sequence shown in SEQ ID NO: 64, the CH comprises the sequence shown in SEQ ID NO: 45, and the VHH1 comprises the sequence shown in SEQ ID NO: 9; the Fc monomer comprises the sequence shown in SEQ ID NO: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 10; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (e.g., a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51; or, (18) The VL comprises the sequence shown in SEQ ID NO:43, the CL comprises the sequence shown in SEQ ID NO:44; the VH comprises the sequence shown in SEQ ID NO:40, the CH comprises the sequence shown in SEQ ID NO:45, and the VHH1 comprises the sequence shown in SEQ ID NO:9; the Fc monomer comprises the sequence shown in SEQ ID NO:47, and the VHH2 comprises the sequence shown in SEQ ID NO:10; the L1, L2 and L3 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and / or one or more serine (for example, a peptide linker shown in (G4S)n), preferably, L1 is SEQ ID NO:51, L2 is SEQ ID NO:49, and L3 is SEQ ID NO:

51.

22. An isolated nucleic acid molecule encoding: (i) the humanized single-domain antibody or antigen-binding fragment thereof of claim 1, (ii) the single-domain antibody or antigen-binding fragment thereof of any one of claims 2-7, (iii) the polypeptide construct of claim 8, or (iv) the multispecific antibody or polypeptide chain thereof of any one of claims 9-21.

23. A vector comprising the isolated nucleic acid molecule of claim 22; Preferably, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody, and the nucleotide sequence encoding each peptide chain is present on the same or different vectors.

24. A host cell comprising the isolated nucleic acid molecule of claim 22 or the vector of claim 23.

25. A method for preparing a single-domain antibody or an antigen-binding fragment thereof, a polypeptide construct, or a multispecific antibody, comprising: Cultivating the host cell of claim 24 under conditions that allow protein expression, and collecting the single-domain antibody or antigen-binding fragment thereof, the polypeptide construct, or the multispecific antibody from the culture of the cultured host cell.

26. A conjugate comprising: the humanized single-domain antibody or antigen-binding fragment thereof according to claim 1, the single-domain antibody or antigen-binding fragment thereof according to any one of claims 2 to 7, the polypeptide construct according to claim 8, or the multispecific antibody according to any one of claims 9 to 21, and a coupling moiety linked thereto; Preferably, the coupling portion is selected from a detectable label (such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme) or a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin, a radionuclide, an immune agonist, an immunosuppressant, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis).

27. A pharmaceutical composition comprising the humanized single-domain antibody or antigen-binding fragment thereof of claim 1, the single-domain antibody or antigen-binding fragment thereof of any one of claims 2-7, the polypeptide construct of claim 8, or the multispecific antibody of any one of claims 9-21, the isolated nucleic acid molecule of claim 22, the vector of claim 23, the host cell of claim 24, or the conjugate of claim 26, and a pharmaceutically acceptable carrier and / or excipient.

28. Use of the humanized single-domain antibody or antigen-binding fragment thereof according to claim 1, the single-domain antibody or antigen-binding fragment thereof according to any one of claims 2-7, the polypeptide construct according to claim 8, the multispecific antibody according to any one of claims 9-21, the isolated nucleic acid molecule according to claim 22, the vector according to claim 23, the host cell according to claim 24, the conjugate according to claim 26, or the pharmaceutical composition according to claim 27 in the preparation of a medicament for the prevention and / or treatment and / or neoadjuvant treatment and / or adjuvant treatment of a disease.

29. A method for preventing and / or treating and / or neoadjuvant treating and / or adjuvant treating a disease in a subject, comprising administering to a subject in need thereof an effective amount of the humanized single-domain antibody or antigen-binding fragment thereof of claim 1, the single-domain antibody or antigen-binding fragment thereof of any one of claims 2-7, the polypeptide construct of claim 8, the multispecific antibody of any one of claims 9-21, the isolated nucleic acid molecule of claim 22, the vector of claim 23, the host cell of claim 24, the conjugate of claim 26, or the pharmaceutical composition of claim 27.

30. The use according to claim 28 or the method according to claim 29, wherein The disease is a tumor; preferably, the tumor is a solid tumor or a blood tumor; more preferably, the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer; the blood tumor includes acute myeloid leukemia.

31. A method for detecting the presence or level of PD-L1, MSLN and / or CD3 in a sample, comprising contacting the sample with the humanized single-domain antibody or antigen-binding fragment thereof of claim 1, the single-domain antibody or antigen-binding fragment thereof of any one of claims 2-7, the polypeptide construct of claim 8, the multispecific antibody of any one of claims 9-21, the isolated nucleic acid molecule of claim 22, the vector of claim 23, the host cell of claim 24, the conjugate of claim 26, or the pharmaceutical composition of claim 27 under conditions that allow the formation of an antibody-antigen immune complex, and detecting the formation of the complex.

32. A method for diagnosing or differentially diagnosing a disease associated with PD-L1, MSLN, and / or CD3, the method comprising using the humanized single-domain antibody or antigen-binding fragment thereof according to claim 1, the single-domain antibody or antigen-binding fragment thereof according to any one of claims 2-7, the polypeptide construct according to claim 8, the multispecific antibody according to any one of claims 9-21, the isolated nucleic acid molecule according to claim 22, the vector according to claim 23, the host cell according to claim 24, the conjugate according to claim 26, or the pharmaceutical composition according to claim 27; Preferably, the method detects the presence or level of PD-L1, MSLN and / or CD3 in a sample from a subject by the method of claim 31 to diagnose or differentially diagnose a disease; Preferably, the disease is a tumor; preferably, the tumor is a solid tumor or a blood tumor; more preferably, the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer; the blood tumor includes acute myeloid leukemia.

33. A kit comprising the humanized single-domain antibody or antigen-binding fragment thereof according to claim 1, the single-domain antibody or antigen-binding fragment thereof according to any one of claims 2-7, the polypeptide construct according to claim 8, the multispecific antibody according to any one of claims 9-21, the isolated nucleic acid molecule according to claim 22, the vector according to claim 23, the host cell according to claim 24, the conjugate according to claim 26, or the pharmaceutical composition according to claim 27.

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