Antibodies and their uses

Abstract

The present invention relates to a single domain antibody or antigen-binding fragment thereof targeting humanized MSLN or PD-L1, a multispecific antibody, specifically a multispecific antibody targeting CD3, a tumor-associated antigen, and an immune checkpoint, which has good safety and anti-tumor efficacy, and a use of the single domain antibody or multispecific antibody in the treatment of diseases.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] [Technical Field] The present invention relates to single domain antibodies that target MSLN or PD-L1. The present invention also relates to multispecific antibodies, particularly multispecific antibodies that target CD3, tumor-associated antigens, and immune checkpoints. The present invention also relates to the use of said single domain antibodies or multispecific antibodies in the treatment of diseases.

[0003] [Background technology] Mesothelin (MSLN) is a cell surface glycoprotein encoded by the MSLN gene. Compared to normal tissues, MSLN is highly expressed in various 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 aberrant 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 coinhibitory programmed death receptor 1 (PD-1). Studies have shown that PD-L1 is highly expressed in various tumor types, including melanoma, ovarian cancer, lung cancer, and renal cancer. PD-L1 plays an important role in preventing autoimmunity and maintaining peripheral tolerance in normal tissues. Tumor cells utilize the PD-1 / PD-L1 signaling pathway to evade anti-tumor immune responses, ultimately leading to proliferation and metastasis. Therefore, blocking the PD-1 / PD-L1 signaling pathway can activate endogenous antitumor immune responses and exert therapeutic effects against tumors.

[0004] Currently, most MSLN or PD-L1 antibodies in the field are conventional IgG heavy and light chain antibodies. Nanobodies, also known as single-domain antibodies (sdAbs), are composed of only heavy chains and are characterized by small molecular weight, good stability, and high permeability. However, there are currently no satisfactory nanobodies against MSLN or PD-L1 in the field, and there is a need to develop new, effective nanobodies specific to MSLN or PD-L1. Furthermore, the treatment of solid tumors with CD3 bispecific antibodies (T cell engagers, TCEs) based on T cell activation has attracted widespread attention. TCEs contain two domains: one domain binds to CD3 on T cells, and the other domain targets and binds to cancer cells. These molecules are designed to enable T cells to recognize and fully activate cancer cells, releasing cytokines such as TNFα, perforin, granzyme B, and IFN-γ, which then kill the cancer cells. MSLN is a type of TAA for solid tumors, and related multispecific antibody drugs are still being developed, among which HPN536 is currently undergoing clinical trials. However, in clinical use, the treatment of solid tumors with TCE has poor efficacy, the risk of CRS, and problems such as T cell inactivation due to excessive T cell activation. Therefore, there is a need in this field to develop T cell activation drugs with better performance.

[0005] DISCLOSURE OF THE INVENTION [Problem to be solved by the invention] In response to the above-mentioned problems, the present invention provides nanobodies targeting MSLN or PD-L1 with excellent properties, and also provides multispecific antibodies targeting CD3, tumor-associated antigens (TAA), and immune checkpoints, among which the CD3-targeting moiety moderately activates T cells, thereby improving safety in use, and furthermore, when combined with the tumor-associated antigens (TAA) and immune checkpoint-targeting moieties, significantly improving antitumor effects. Accordingly, the following aspects are provided:

[0006] [Means for solving the problem] (CD3 antibody) In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD3, which has moderate CD3 affinity, the ability to moderately activate T cells, and reduced cytokine release, thereby improving safety of use. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises an HCDR1 represented by SEQ ID NO: 55, an HCDR2 represented by SEQ ID NO: 56, and an HCDR3 represented by any one of SEQ ID NOs: 57 to 59 and 65, and / or The VL comprises an LCDR1 shown in SEQ ID NO:60, an LCDR2 shown in SEQ ID NO:61, and an LCDR3 shown in SEQ ID NO:62.

[0007] In some embodiments, the VH comprises the sequence set forth in any one of SEQ ID NOs: 40-42 and 64.

[0008] In some embodiments, the VL comprises the sequence set forth in SEQ ID NO:43.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises a VH set forth in SEQ ID NO:40 and a VL set forth in SEQ ID NO:43.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises a VH set forth in SEQ ID NO:41 and a VL set forth in SEQ ID NO:43.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises a VH set forth in SEQ ID NO:42 and a VL set forth in SEQ ID NO:43.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises a VH set forth in SEQ ID NO:64 and a VL set forth in SEQ ID NO:43.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human and / or cynomolgus CD3.

[0014] (MSLN single domain antibody) In a second aspect, the present invention relates to a humanized single-domain antibody or antigen-binding fragment thereof capable of specifically binding to MSLN. A single-domain antibody typically consists of four framework regions (FRs) and three complementarity-determining regions (CDRs), designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An antigen-binding fragment thereof comprises at least a portion of the single-domain antibody, sufficient to confer the fragment with the ability to specifically bind to an antigen (e.g., MSLN). Single-domain antibodies may be truncated at the N- or C-terminus to comprise only a portion of FR1 and / or FR4, or one or two of these framework regions may be deleted, as long as antigen binding and specificity are substantially retained. Single-domain antibodies may also be humanized to form humanized single-domain antibodies in which one or more framework regions of the VHH are replaced by substantially human framework regions. In some embodiments, the humanized single domain antibody or antigen-binding fragment thereof comprises camelid CDR regions and a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline antibody gene), and the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations of human residues to camelid residues.

[0015] In some embodiments, the humanized single domain antibody or antigen-binding fragment thereof of the invention comprises a VHH sequence set forth in any one of SEQ ID NOs: 1 to 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 to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions, preferably wherein the substitutions are conservative.

[0016] In some embodiments, the humanized single domain antibody or antigen-binding fragment thereof of the present invention comprises a CDR1 set forth in SEQ ID NO: 52, a CDR2 set forth in SEQ ID NO: 53, and a CDR3 set forth in SEQ ID NO: 54, as defined by the IMGT numbering system.

[0017] In some embodiments, the single domain antibody or antigen-binding fragment thereof specifically binds to human and / or cynomolgus MSLN.

[0018] (PD-L1 single domain antibody) In a third aspect, the present invention relates to a single domain antibody, or antigen-binding fragment thereof, capable of specifically binding to PD-L1.

[0019] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the IMGT numbering system: (1A) CDR1 comprising the sequence set forth in SEQ ID NO: 11 or 63 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 12 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof; or (1B) CDR1 comprising the sequence set forth in SEQ ID NO: 68 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 69 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 70 or a variant thereof, wherein the variants have one or more amino acid substitutions, deletions, or additions compared to the sequence from which they are derived, and preferably the substitutions are conservative substitutions. In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the IMGT numbering system: (1A) CDR1 comprising the sequence set forth in SEQ ID NO: 11 or 63, CDR2 comprising the sequence set forth in SEQ ID NO: 12, and CDR3 comprising the sequence set forth in SEQ ID NO: 13; or (1B) CDR1 comprising the sequence set forth in SEQ ID NO: 68, CDR2 comprising the sequence set forth in SEQ ID NO: 69, and CDR3 comprising the sequence set forth in SEQ ID NO: 70.

[0020] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Kabat numbering system: (2A) CDR1 comprising the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 15 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof; or (2B) CDR1 comprising the sequence set forth in SEQ ID NO: 71 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 72 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 73 or a variant thereof, wherein the variants have one or more amino acid substitutions, deletions, or additions compared to the sequence from which they are derived, and preferably the substitutions are conservative substitutions. In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Kabat numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 14, CDR2 comprising the sequence set forth in SEQ ID NO: 15, and CDR3 comprising the sequence set forth in SEQ ID NO: 16; or (2B) CDR1 comprising the sequence set forth in SEQ ID NO: 71, CDR2 comprising the sequence set forth in SEQ ID NO: 72, and CDR3 comprising the sequence set forth in SEQ ID NO: 73.

[0021] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the AbM numbering system: (3A) CDR1 comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 18 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof; or (3B) CDR1 comprising the sequence set forth in SEQ ID NO: 74 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 75 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 73 or a variant thereof, wherein the variants have one or more amino acid substitutions, deletions, or additions compared to the sequence from which they are derived, and preferably the substitutions are conservative substitutions. In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the AbM numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 17, CDR2 comprising the sequence set forth in SEQ ID NO: 18, and CDR3 comprising the sequence set forth in SEQ ID NO: 16; or (3B) CDR1 comprising the sequence set forth in SEQ ID NO: 74, CDR2 comprising the sequence set forth in SEQ ID NO: 75, and CDR3 comprising the sequence set forth in SEQ ID NO: 73.

[0022] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Chothia numbering system: (4A) CDR1 comprising the sequence set forth in SEQ ID NO: 19 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 20 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof; or (4B) CDR1 comprising the sequence set forth in SEQ ID NO: 76 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 77 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 73 or a variant thereof, wherein the variants have one or more amino acid substitutions, deletions, or additions compared to the sequence from which they are derived, and preferably the substitutions are conservative substitutions. In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Chothia numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 19, CDR2 comprising the sequence set forth in SEQ ID NO: 20, and CDR3 comprising the sequence set forth in SEQ ID NO: 16; or (4B) CDR1 comprising the sequence set forth in SEQ ID NO: 76, CDR2 comprising the sequence set forth in SEQ ID NO: 77, and CDR3 comprising the sequence set forth in SEQ ID NO: 73.

[0023] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Contact numbering system: (5A) CDR1 comprising the sequence set forth in SEQ ID NO: 21 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 22 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof, or (5B) CDR1 comprising the sequence set forth in SEQ ID NO: 78 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 79 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 80 or a variant thereof, wherein the variants have one or more amino acid substitutions, deletions, or additions compared to the sequence from which they are derived, and preferably the substitutions are conservative substitutions. In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Contact numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 21, CDR2 comprising the sequence set forth in SEQ ID NO: 22, and CDR3 comprising the sequence set forth in SEQ ID NO: 23; or (5B) CDR1 comprising the sequence set forth in SEQ ID NO: 78, CDR2 comprising the sequence set forth in SEQ ID NO: 79, and CDR3 comprising the sequence set forth in SEQ ID NO: 80.

[0024] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3 contained in a VHH set forth in any one of SEQ ID NOs: 10, 24-39, 67, and 81-88. In some embodiments, the CDRs are determined by the IMGT, Kabat, AbM, Chothia, or Contact numbering system.

[0025] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises a VHH sequence as set forth in SEQ ID NO: 10, 67 or a variant thereof, which 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 to the sequence from which it is derived, or which has one or more amino acid substitutions, deletions or additions compared thereto, preferably wherein the substitutions are conservative substitutions.

[0026] In some embodiments, the single domain antibody or antigen-binding fragment thereof is humanized, wherein one or more framework regions of its VHH are replaced by substantially human framework regions. In some embodiments, the humanized single domain antibody or antigen-binding fragment thereof comprises heavy chain framework regions derived from a human immunoglobulin (e.g., heavy chain framework regions comprised in the amino acid sequence encoded by a human heavy chain germline antibody gene), which heavy chain framework regions optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations of human residues to camelid residues.

[0027] In some embodiments, the humanized single domain antibody or antigen-binding fragment thereof comprises a VHH sequence set forth in any one of SEQ ID NOs: 24 to 39, 81 to 88, or a variant thereof, wherein the variant 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 to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions compared thereto, preferably wherein the substitutions are conservative substitutions.

[0028] In some embodiments, the single domain antibody or antigen-binding fragment thereof specifically binds to human PD-L1, mouse PD-L1, and / or cynomolgus PD-L1, for example, the single domain antibody is defined in any of the above embodiments (1A) to (5A). In some embodiments, the single domain antibody or antigen-binding fragment thereof specifically binds to human PD-L1 and / or cynomolgus PD-L1, for example, the single domain antibody is defined in any of the above embodiments (1B) to (5B).

[0029] (Polypeptide constructs) In a fourth aspect, the present invention relates to a polypeptide construct comprising a single domain antibody, or antigen-binding fragment thereof, according to any of the above aspects, and an immunoglobulin Fc domain.

[0030] In some embodiments, the polypeptide construct comprises a single domain antibody that specifically binds to MSLN according to the second aspect and an immunoglobulin Fc domain.

[0031] In some embodiments, the polypeptide construct comprises a single domain antibody that specifically binds to PD-L1 according to the third aspect and an immunoglobulin Fc domain.

[0032] As used herein, the Fc domain, also referred to as Fc region, refers to a portion of a 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 polypeptides comprising the Fc. The Fc domain may be of any antibody heavy chain constant region isotype. In some embodiments, the Fc domain is IgG1, IgG2, IgG3, or IgG4.

[0033] In some embodiments, the Fc domain contained in the polypeptide constructs of the present invention is a native Fc region comprising an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature. For example, the Fc domain may 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. The native Fc region may have an effector function. Exemplary "effector functions" include Fc receptor binding, Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), B cell activation, etc. The function may be altered by replacing at least one amino acid residue in the native Fc region with a different residue or by chemical modification, e.g., by altering the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby altering (e.g., reducing or enhancing) the effector function.

[0034] In some embodiments, the Fc domain contained in the polypeptide construct of the present invention may also be a mutant Fc region that may include one or more (e.g., 1 to 10, e.g., 1 to 5) amino acid mutations or chemical modifications to alter one or more of the following properties of the antibody of the present invention compared to a native Fc region: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0035] In some embodiments, the immunoglobulin Fc domain is optionally linked to the N-terminus and / or C-terminus (eg, the C-terminus) of the single domain antibody or antigen-binding fragment thereof by a peptide linker.

[0036] In some embodiments, the immunoglobulin Fc domain is optionally linked to the C-terminus of the single domain antibody or antigen-binding fragment thereof by a peptide linker.

[0037] In some embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain).

[0038] In some embodiments, the immunoglobulin Fc domain comprises the sequence set forth 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 thereto (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions thereto).

[0039] In some embodiments, the peptide linker is a peptide linker that includes one or more glycines and / or one or more serines. In some embodiments, the peptide linker is (G4S) n and n is 1, 2, 3, or 4. In some embodiments, the peptide linker comprises the sequence set forth in SEQ ID NO:50.

[0040] (multispecific antibody) In a fifth aspect, the present invention relates to a multispecific antibody comprising a humanised single domain antibody or antigen-binding fragment thereof targeting MSLN as described in the second aspect and / or a single domain antibody or antigen-binding fragment thereof targeting PD-L1 as described in the third aspect.

[0041] In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0042] In some embodiments, the multispecific antibody comprises the humanized single domain antibody or antigen-binding fragment thereof that targets MSLN according to the second aspect as an antigen-binding domain that targets MSLN, and at least one additional antigen-binding domain that binds to a target different from that of the antigen-binding domain.

[0043] In some embodiments, the multispecific antibody comprises a PD-L1-targeting single domain antibody or antigen-binding fragment thereof described in the third aspect as an antigen-binding domain that targets PD-L1, and at least one additional antigen-binding domain that binds to a target different from that of the antigen-binding domain.

[0044] In some embodiments, the multispecific antibody comprises, as an antigen-binding domain that targets MSLN, the humanized single domain antibody or antigen-binding fragment thereof that targets MSLN described in the second aspect, and as an antigen-binding domain that targets PD-L1, the single domain antibody or antigen-binding fragment thereof that targets PD-L1 described in the third aspect, and further comprises at least one additional antigen-binding domain that binds to a target different from the above antigen-binding domains.

[0045] 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: (i) the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to CD3 according to the first aspect; (ii) the second antigen-binding domain comprises a VHH, wherein the VHH comprises CDR1, CDR2, and CDR3 contained in a VHH shown in any one of SEQ ID NOs: 1 to 9, preferably the VHH comprises CDR1 shown in SEQ ID NO: 52, CDR2 shown in SEQ ID NO: 53, and CDR3 shown in SEQ ID NO: 54, and preferably the second antigen-binding domain comprises a 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; and / or (iii) the third antigen-binding domain comprises a single domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 according to the third aspect.

[0046] In some embodiments, in (i), the first antigen-binding domain comprises a VH and a VL, wherein the VH comprises an HCDR1 set forth in SEQ ID NO: 55, an HCDR2 set forth in SEQ ID NO: 56, and an HCDR3 set forth in any one of SEQ ID NOs: 57 to 59 and 65, and / or the VL comprises an LCDR1 set forth in SEQ ID NO: 60, an LCDR2 set forth in SEQ ID NO: 61, and an LCDR3 set forth in SEQ ID NO: 62, and preferably the VH comprises the sequence set forth in any one of SEQ ID NOs: 40 to 42 and 64, and preferably the VL comprises the sequence set forth in SEQ ID NO: 43.

[0047] In some embodiments, the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to CD3 according to 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; and 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.

[0048] In some 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 a humanized single-domain antibody or antigen-binding fragment thereof that specifically binds to MSLN according to the second aspect; and 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.

[0049] In some 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; and the third antigen-binding domain comprises a single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 according to the third aspect.

[0050] In some embodiments, the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof that specifically binds to CD3 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 described in the second aspect, and 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.

[0051] In some embodiments, the first antigen-binding domain comprises an 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; and the third antigen-binding domain comprises a single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 as described in the third aspect.

[0052] In some 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 a 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 a single-domain antibody or antigen-binding fragment thereof that specifically binds to PD-L1 as described in the third aspect.

[0053] In some embodiments, the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof that specifically binds to CD3 of the first aspect, the second antigen-binding domain comprises the humanized single-domain antibody or antigen-binding fragment thereof that specifically binds to MSLN of 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 of the third aspect.

[0054] In a 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 said first antigen-binding domain is a Fab and said second and third antigen-binding domains are VHHs.

[0055] In some embodiments, 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 said first Fc monomer is optionally linked by a linker to said first antigen-binding domain (e.g., its heavy chain CH1 domain) and its C-terminus is optionally linked by a linker to one of said second antigen-binding domains; The N-terminus of said second Fc monomer is optionally linked by a linker to another of said second antigen-binding domains, and its C-terminus is optionally linked by a linker to said third antigen-binding domain.

[0056] In some embodiments, the tumor-associated antigen is 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, Flt3, NY- The protein is selected from 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.

[0057] In some embodiments, the tumor-associated antigen is MSLN.

[0058] In some embodiments, the immune checkpoint is chosen 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.

[0059] In some embodiments, the immune checkpoint is selected from PD1, PD-L1, TIGIT, and LAG3.

[0060] In some embodiments, the immune checkpoint is selected from PD1, PD-L1, or a combination thereof, hi some embodiments, the immune checkpoint is PD-L1.

[0061] In some embodiments, the Fc domains comprised in the multispecific antibodies comprise a modification that promotes dimerization of the first Fc monomer with the second Fc monomer, such that a polypeptide comprising the first Fc monomer and a polypeptide comprising the second Fc monomer undergo (hetero)dimerization to form a complex.

[0062] Such modifications are known to those skilled in the art and may include separately modifying each of the two Fc subunits (i.e., the first and second Fc monomers) that are expected to associate, provided that the modifications are complementary to each other and thereby promote the association of the two Fc subunits. For example, a modification that promotes association can alter the structure or charge of one or two Fc subunits, thereby sterically or electrostatically promoting their association, respectively. For example, a modification that promotes association includes an amino acid mutation (e.g., amino acid substitution) in the Fc. In some embodiments, the modification is within the CH3 domain of the Fc.

[0063] In some embodiments, the CH3 domains of two monomers of said Fc domain comprise amino acid substitutions.

[0064] In some embodiments, the modification comprises a "knob" modification in one of the two Fc domain monomers and a "hole" modification in the other of the two Fc domain monomers, forming a "knob-into-hole" modification. Typically, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance fills the cavity, promoting heterodimer formation and preventing homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (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 the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0065] In some exemplary embodiments, a protuberance is formed in the CH3 domain of the first Fc monomer by substituting one amino acid residue with a larger side chain volume, and a complementary cavity having the same or similar size as the protuberance is formed in the CH3 domain of the second Fc monomer by substituting one amino acid residue with a smaller side chain volume, or a protuberance is formed in the CH3 domain of the second Fc monomer by substituting one amino acid residue with a larger side chain volume, and a complementary cavity having the same or similar size as the protuberance is formed in the CH3 domain of the first Fc monomer by substituting one amino acid residue with a smaller side chain volume,

[0066] In some embodiments, the amino acid residue with a larger side chain volume is selected from tryptophan (W), arginine (R), phenylalanine (P), and tyrosine (Y).

[0067] In some embodiments, the amino acid residue with a smaller side chain volume is selected from valine (V), alanine (A), serine (S), and threonine (T).

[0068] In some exemplary embodiments, the first Fc monomer and the second Fc monomer of the Fc domain comprise the amino acid sequences set forth in SEQ ID NOs: 46 and 47, respectively.

[0069] In some embodiments, the linker is selected from peptide linkers containing one or more glycines and / or one or more serines, e.g., (G4S) n wherein n is 1, 2, 3 or 4, and includes, for example, a sequence shown in any one of SEQ ID NOs: 49 to 51.

[0070] In some embodiments, the multispecific antibody comprises: (i) a first peptide chain comprising a VL of the first antigen-binding domain and a light chain constant region (CL), preferably wherein the CL is a kappa light chain constant region; (ii) the VH, CH1, first Fc monomer and the second antigen-binding domain of the first antigen-binding domain, preferably the first Fc monomer is an IgG, e.g., IgG1 or IgG4, preferably the first Fc monomer comprises a hinge region, CH2 and CH3, and preferably the second antigen-binding domain comprises a linker (e.g., (G4S) n a second peptide chain linked to the C-terminus of the first Fc monomer by a flexible peptide comprising: and (iii) the second antigen-binding domain, a second Fc monomer, and a third antigen-binding domain, preferably the second Fc monomer is an IgG, e.g., IgG1 or IgG4, preferably the second Fc monomer comprises a hinge region, CH2, and CH3, and preferably the second antigen-binding domain is linked to a linker (e.g., (G4S) n and preferably, the third antigen-binding domain is linked to the N-terminus of the second Fc monomer by a linker (e.g., a flexible peptide comprising (G4S) nand a third peptide chain linked to the C-terminus of the second Fc monomer by a flexible peptide comprising:

[0071] In some embodiments, the second Fc monomer of said third peptide chain is capable of forming a dimer with the first Fc monomer of said second peptide chain.

[0072] In some embodiments, the first Fc monomer of the second peptide chain and the second Fc monomer of the third peptide chain comprise a modification that promotes 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, forming a "knob-into-hole" modification.

[0073] In some embodiments, the two Fc monomers comprise the amino acid sequence set forth in SEQ ID NO: 46, 47, respectively. In some embodiments, the first Fc monomer of the second peptide chain comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the second peptide chain comprises the heavy chain constant region sequence set forth in SEQ ID NO: 45. In some embodiments, the second Fc monomer of the third peptide chain comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0074] In some embodiments, the first antigen-binding domain comprises a VH and a VL, wherein the VH comprises an HCDR1 set forth in SEQ ID NO: 55, an HCDR2 set forth in SEQ ID NO: 56, and an HCDR3 set forth in any one of SEQ ID NOs: 57 to 59 and 65, and / or the VL comprises an LCDR1 set forth in SEQ ID NO: 60, an LCDR2 set forth in SEQ ID NO: 61, and an LCDR3 set forth in SEQ ID NO: 62.

[0075] In some embodiments, the VH comprises the sequence set forth in any one of SEQ ID NOs: 40-42 and 64.

[0076] In some embodiments, the VL comprises the sequence set forth in SEQ ID NO:43.

[0077] In some embodiments, the second antigen-binding domain comprises a VHH, wherein the VHH comprises CDR1, CDR2, and CDR3 contained in a VHH set forth in any one of SEQ ID NOs: 1 to 9, and preferably the VHH comprises CDR1 set forth in SEQ ID NO: 52, CDR2 set forth in SEQ ID NO: 53, and CDR3 set forth in SEQ ID NO: 54.

[0078] In some embodiments, the second antigen-binding domain comprises a VHH set forth in any one of SEQ ID NOs: 1 to 9 or a variant thereof, and 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 to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions compared thereto, and preferably the substitutions are conservative substitutions.

[0079] In some embodiments, the third antigen-binding domain comprises a single domain antibody or antigen-binding fragment thereof capable of specifically binding to PD-L1 according to the third aspect.

[0080] In some embodiments, the multispecific antibody comprises: (i) a first peptide chain having a structure represented by [VL]-[CL]; (ii) a second peptide chain having the structure represented by [VH]-[CH]-[L1]-[VHH1]; and (iii) a third peptide chain having a structure represented by [VHH1]-[L2]-[Fc monomer]-[L3]-[VHH2]; However, one of the following items must be selected: (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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (GS) n and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) nand preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S)n and preferably, L1 and L3 are the same, and 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: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 38; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (GS) n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, the VHH1 comprises the sequence shown in SEQ ID NO: 8, the Fc monomer comprises the sequence shown in SEQ ID NO: 47, the VHH2 comprises the sequence shown in SEQ ID NO: 31, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S)n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (GS) n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (GS) n and preferably, L1 and L3 are the same, and 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (GS) n and preferably, L1 and L3 are the same, and 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, the VHH1 comprises the sequence shown in SEQ ID NO: 9, the Fc monomer comprises the sequence shown in SEQ ID NO: 47, the VHH2 comprises the sequence shown in SEQ ID NO: 10, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G4S) n and 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, 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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (GS)n Preferably, L1 and L3 are the same, and preferably, L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49, and L3 is SEQ ID NO: 51.

[0081] (Antibody production) The antibodies, single domain antibodies, polypeptide constructs or multispecific antibodies according to any of the above aspects may be produced by various methods known in the art, for example by recombinant genetic engineering techniques. For example, DNA molecules encoding them may be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules may be inserted into an expression vector, which is 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 invention.

[0082] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof according to the first aspect, or a heavy chain variable region and / or a light chain variable region thereof; a humanized single domain antibody or antigen-binding fragment thereof according to the second aspect; a single domain antibody or antigen-binding fragment thereof according to the third aspect; A polypeptide construct according to the fourth aspect, There is provided an isolated nucleic acid molecule encoding a multispecific antibody according to the fifth, sixth or seventh aspect, or a polypeptide chain thereof.

[0083] In another aspect, the present invention provides a vector comprising the above-described nucleic acid molecule. In some embodiments, the vector is a cloning vector or an expression vector.

[0084] In some embodiments, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the invention, and the nucleotide sequence encoding each peptide chain is present in the same or different vectors.

[0085] In another aspect, the present invention provides host cells comprising the above-described nucleic acid molecules or vectors, including, but not limited to, prokaryotic cells (e.g., bacterial cells such as E. coli cells), eukaryotic cells (e.g., fungal cells such as yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells, human cells).

[0086] In another aspect, the invention provides a method for producing an antibody, single domain antibody, polypeptide construct or multispecific antibody according to any of the above aspects, comprising culturing a host cell as described above under conditions allowing expression of the protein, and harvesting the antibody, single domain antibody, polypeptide construct or multispecific antibody from the culture of the cultured host cells.

[0087] (complex) The present invention provides, as one of its derivatives, a conjugate comprising an antibody, single domain antibody, polypeptide construct or multispecific antibody and binding moiety according to any of the above aspects.

[0088] In some embodiments, the binding moiety is selected from a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0089] In some embodiments, the binding moiety is selected from therapeutic agents, which in some embodiments are preferably anti-tumor agents, such as cytotoxic drugs, cytokines, toxins, radionuclides, immunoagonists, immunosuppressants, and other active substances that inhibit tumor cell proliferation or promote tumor cell apoptosis or necrosis.

[0090] (Pharmaceutical composition) The antibodies, single domain antibodies, polypeptide constructs, multispecific antibodies or conjugates (also referred to as active ingredients) disclosed herein may be incorporated into pharmaceutical compositions suitable for administration.

[0091] In another aspect, the invention relates to a pharmaceutical composition comprising an antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, or complex according to any of the above aspects, and a pharmaceutically acceptable carrier and / or excipient.

[0092] In some embodiments, the pharmaceutical composition of the invention comprises a humanized single domain antibody or antigen-binding fragment thereof according to the second aspect or a nucleic acid molecule or vector encoding it.

[0093] In some embodiments, the pharmaceutical composition of the invention comprises a single domain antibody or antigen-binding fragment thereof according to the third aspect or a nucleic acid molecule or vector encoding it.

[0094] In some embodiments, the pharmaceutical composition of the invention comprises a polypeptide construct according to the fourth aspect or a nucleic acid molecule or vector encoding it.

[0095] In some embodiments, the pharmaceutical composition of the invention comprises a multispecific antibody according to the fifth aspect or a nucleic acid molecule or vector encoding it.

[0096] In some embodiments, the pharmaceutical composition of the invention comprises a multispecific antibody according to the sixth aspect or a nucleic acid molecule or vector encoding it.

[0097] In some embodiments, the pharmaceutical composition of the invention comprises a multispecific antibody according to the seventh aspect or a nucleic acid molecule or vector encoding it.

[0098] In some embodiments, the pharmaceutical composition may further comprise another pharmaceutically active agent, for example, an anti-tumor agent, hi some embodiments, the other pharmaceutically active agent and the antibody, single domain antibody, polypeptide construct, multispecific antibody or complex, isolated nucleic acid molecule, vector or host cell of the invention are provided separately or as components of the same composition.

[0099] (therapeutic use) In another aspect, the present invention relates to a method for the prevention and / or treatment of a disease and / or a new adjunctive therapy and / or an adjunctive therapy, comprising administering to a subject in need thereof an antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, or pharmaceutical composition according to any of the above aspects. The present invention also relates to the use of an antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, or pharmaceutical composition according to any of the above aspects for the prevention and / or treatment of a disease and / or the new adjunctive therapy and / or an adjunctive therapy, or for the manufacture of a medicament for the prevention and / or treatment of a disease and / or the new adjunctive therapy and / or an adjunctive therapy.

[0100] In some embodiments, the disease is a tumor. In some embodiments, the tumor is a solid tumor or a hematological tumor. In some embodiments, the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer, and the hematological tumor comprises acute myeloid leukemia.

[0101] In another aspect, the present invention relates to a method for the prevention and / or treatment of a disease and / or new adjunctive therapy and / or adjunctive therapy, comprising administering to a subject in need thereof a humanized single domain antibody, a polypeptide construct comprising it, a multispecific antibody or complex, a nucleic acid molecule encoding them, a vector or host cell, or a pharmaceutical composition comprising any one of the above according to the second aspect. The present invention also relates to the use of a humanized single domain antibody, a polypeptide construct comprising it, a multispecific antibody or complex, a nucleic acid molecule encoding them, a vector or host cell, or a pharmaceutical composition comprising any one of the above according to the second aspect for the prevention and / or treatment of a disease and / or new adjunctive therapy and / or adjunctive therapy, or for the manufacture of a medicament for the prevention and / or treatment of a disease and / or new adjunctive therapy and / or adjunctive therapy.

[0102] In some embodiments, the disease is a tumor.

[0103] In some embodiments, the tumor is an MSLN-positive tumor.

[0104] In some 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, colon cancer, fallopian tube cancer, malignant melanoma, soft tissue tumor, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphocytic leukemia.

[0105] In some embodiments, the tumor is selected from solid tumors, such as mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer.

[0106] In some embodiments, the tumor is selected from hematological tumors, for example, acute myeloid leukemia.

[0107] In another aspect, the present invention relates to a method for the prevention and / or treatment of a disease and / or a new adjunctive therapy and / or an adjunctive therapy comprising administering to a subject in need thereof a single domain antibody, a polypeptide construct comprising thereof, a multispecific antibody or complex, a nucleic acid molecule encoding them, a vector or host cell or a pharmaceutical composition comprising any one of the above according to the third aspect. The present invention also relates to the use of a single domain antibody, a polypeptide construct comprising thereof, a multispecific antibody or complex, a nucleic acid molecule encoding them, a vector or host cell or a pharmaceutical composition comprising any one of the above according to the third aspect for the prevention and / or treatment of a disease or the use in the manufacture of a medicament for the prevention and / or treatment of a disease and / or a new adjunctive therapy and / or an adjunctive therapy.

[0108] In some embodiments, the disease is a PD-L1 associated disease, hi some embodiments, the PD-L1 associated disease is a tumor.

[0109] In some embodiments, the tumor is a PD-L1 positive tumor.

[0110] In some embodiments, the tumor is a solid tumor or a hematological tumor, hi some embodiments, the tumor is selected from gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone tumor, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, melanoma, head and neck tumor, esophageal cancer, and skin cancer.

[0111] In another aspect, the present invention relates to a method for the prevention and / or treatment of a disease and / or new adjunctive treatment and / or adjunctive therapy comprising administering to a subject in need thereof a multispecific antibody according to the fifth, sixth or seventh aspect, or a nucleic acid molecule, vector or host cell encoding same, or a pharmaceutical composition comprising any one of the above. The present invention also relates to the use of a multispecific antibody according to the fifth, sixth or seventh aspect, or a nucleic acid molecule, vector or host cell encoding same, or a pharmaceutical composition comprising any one of the above, for the prevention and / or treatment of a disease, or in the manufacture of a medicament for the prevention and / or treatment of a disease and / or new adjunctive treatment and / or adjunctive therapy.

[0112] The multispecific antibodies according to the fifth, sixth or seventh aspect of the invention can be used to treat any disease in which the effector mechanisms of cytotoxic T cells are required for the treatment of said disease. For example, when a multispecific antibody of the invention comprises a CD3-binding arm that recruits T cells and a tumor-targeting arm specific for a tumor-associated antigen (TAA), it brings the T cells into close contact with the target tumor cells, activating them locally, and subsequently perforin and granzymes released by the T cytotoxic particles destroy the target cells. When it further comprises a targeting arm specific for an immune checkpoint molecule, it activates T cells in the tumor microenvironment, subsequently killing tumor cells and enhancing the anti-tumor effect.

[0113] In some embodiments, the disease is a tumor.

[0114] In some 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, squamous cell carcinoma of the head and neck, pancreatic cancer, bile duct cancer, colon cancer, fallopian tube cancer, malignant melanoma, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphocytic leukemia.

[0115] In some embodiments, the treatment of the disease involves the effector mechanisms of T cells (e.g., cytotoxic T cells). In some embodiments, the multispecific antibody recruits T cells around target cells, activates the T cells, and induces T cell-mediated cytotoxicity (TDCC), thereby effectively killing the target cells and treating or preventing the disease.

[0116] In some embodiments, the tumor is an MSLN-positive tumor, e.g., 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, or a hematological tumor such as acute myeloid leukemia.

[0117] In some embodiments, the tumor is a PD-L1 positive tumor, for example, a solid tumor such as gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone tumor, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, melanoma, head and neck tumor, esophageal cancer, or skin cancer.

[0118] In any of the above aspects, the single domain antibody, polypeptide construct, multispecific antibody of the present invention or pharmaceutical composition comprising them may be formulated into any dosage form known in the medical field.

[0119] In any of the above aspects, the single domain antibody, polypeptide construct, multispecific antibody of the invention or pharmaceutical composition comprising same may be administered by any suitable method known in the art.

[0120] In any of the above aspects, the single domain antibody, polypeptide construct, multispecific antibody of the present invention or pharmaceutical composition comprising same may be formulated in unit dosage form for ease of administration.

[0121] In any of the above aspects, the single domain antibody, polypeptide construct, multispecific antibody of the invention or pharmaceutical composition comprising same may 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).

[0122] In any of the above aspects, the subject may be a mammal, for example a human.

[0123] (Measurement use) In another aspect, the invention provides a kit comprising an antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, or pharmaceutical composition according to any of the above aspects.

[0124] In some embodiments, the kit comprises instructions for use, for example, using the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, or pharmaceutical composition to determine the presence or level of PD-L1, MSLN, and / or CD3 in a sample and / or to diagnose or differentially diagnose a disease associated with PD-L1, MSLN, and / or CD3.

[0125] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof that specifically binds to CD3 according to the first aspect. The antibody or antigen-binding fragment thereof optionally carries a detectable label. The kit is used to determine the presence or level of CD3 in a sample and / or to diagnose or differentially diagnose a CD3-related disease.

[0126] In some embodiments, the kit comprises a humanized single domain antibody or antigen-binding fragment thereof or polypeptide construct thereof that specifically binds to MSLN according to the second aspect. The humanized single domain antibody or antigen-binding fragment thereof or polypeptide construct thereof optionally carries a detectable label. The kit is used to determine the presence or level of MSLN in a sample and / or to diagnose or differentially diagnose a disease associated with MSLN.

[0127] In some embodiments, the kit comprises a single domain antibody or antigen-binding fragment thereof or polypeptide construct thereof that specifically binds to PD-L1 according to aspect 3. The single domain antibody or antigen-binding fragment thereof or polypeptide construct thereof optionally carries a detectable label. The kit is used to determine the presence or level of PD-L1 in a sample and / or to diagnose or differentially diagnose a PD-L1 associated disease.

[0128] In some embodiments, the kit comprises a multispecific antibody according to the fifth, sixth or seventh aspect. The multispecific antibody optionally carries a detectable label. The kit is used to determine the presence or levels of PD-L1, MSLN and / or CD3 in a sample and / or to diagnose or differentially diagnose a disease associated with PD-L1, MSLN and / or CD3.

[0129] As used herein, the detectable label may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Particularly preferably, such a label is suitable for immunoassays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).

[0130] In another aspect, the present invention provides a method for determining the presence or levels of PD-L1, MSLN, and / or CD3 in a sample, the method comprising contacting the sample with an antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, or pharmaceutical composition according to any of the above aspects under conditions allowing the formation of an antibody-antigen immune complex, and detecting the formation of the complex. The formation of the complex indicates the presence of an antigen of interest or a cell expressing an antigen of interest. The antigen of interest refers to the antigen targeted by the assay reagent used. The method may be used for diagnostic or non-diagnostic purposes.

[0131] In some embodiments, the method is performed in vitro.

[0132] In some embodiments, the method is an immunoassay (eg, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).

[0133] In some embodiments, the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, or pharmaceutical composition further comprises a detectable label.

[0134] In some embodiments, the method uses an antibody or antigen-binding fragment thereof that specifically binds to CD3 according to the first aspect and is used to measure the presence or level of CD3 in a sample and / or to diagnose or differentially diagnose a CD3-related disease.

[0135] In some embodiments, the method uses a humanized single domain antibody or antigen-binding fragment thereof or polypeptide construct thereof that specifically binds to MSLN according to the second aspect and is used to determine the presence or level of MSLN in a sample and / or to diagnose or differentially diagnose a disease associated with MSLN.

[0136] In some embodiments, the method uses a single domain antibody or antigen-binding fragment thereof, or polypeptide construct thereof that specifically binds to PD-L1 as described in the third aspect, and is used to determine the presence or level of PD-L1 in a sample and / or to diagnose or differentially diagnose a PD-L1 associated disease.

[0137] In some embodiments, the method uses a multispecific antibody according to the fifth, sixth or seventh aspect to measure the presence or levels of PD-L1, MSLN and / or CD3 in a sample and / or to diagnose or differentially diagnose a disease associated with PD-L1, MSLN and / or CD3.

[0138] In another aspect, the invention provides a method for diagnosing or differentially diagnosing a disease associated with PD-L1, MSLN and / or CD3, said method comprising using an antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, or pharmaceutical composition according to any of the previous aspects.

[0139] In some embodiments, the method involves measuring the presence or levels of PD-L1, MSLN, and / or CD3 in a sample from a subject using the above-described measurement methods, thereby diagnosing or differentially diagnosing a disease.

[0140] In some embodiments, the disease is associated with, for example, characterized by, the expression or abnormal expression of, a target antigen, which refers to the antigen targeted by the assay reagent used.

[0141] In some embodiments, the method may further comprise comparing the measured 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, this indicates that the subject has a disease associated with the target antigen.

[0142] In some embodiments, the disease is a tumor. In some 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, colon cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer, and the hematological tumor comprises acute myeloid leukemia.

[0143] In another aspect, there is provided a use of the antibody, single domain antibody, polypeptide construct, multispecific antibody, isolated nucleic acid molecule, vector, host cell, complex, pharmaceutical composition or kit according to any of the above aspects in the manufacture of an assay reagent for measuring the presence or levels of PD-L1, MSLN and / or CD3 in a sample and / or for diagnosing or differentially diagnosing a disease associated with PD-L1, MSLN and / or CD3.

[0144] (Definition of terms) In the present invention, unless otherwise specified, scientific and technical terms used herein have the common meanings understood by those skilled in the art. In addition, the following provides definitions and interpretations of relevant terms for a better understanding of the present invention.

[0145] When the terms "for example," "such as," "including," "including," or variations thereof are used herein, these terms are not to be considered limiting terms and should be interpreted to mean "limited to" or "not limited to."

[0146] Unless otherwise indicated herein or clearly contradicted by context, the terms "a," "an," "one," "the," and similar referents are to be construed to cover both the singular and the plural in the context of describing the invention, particularly in the context of dealing with the claims that follow.

[0147] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen, wherein the immunoglobulin-derived molecule binds to the target antigen via at least one antigen-binding site located in its variable region. When the term "antibody" is used, unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments capable of specifically binding to a target antigen. An "intact antibody" typically consists of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain further includes a "D" region of about 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 consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various 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 of the classical complement system (C1q). The VH and VL regions can be further divided into highly variable regions (called complementarity-determining regions (CDRs)), which are separated by conserved regions called framework regions (FRs). Each V H and V L Each heavy / light chain pair consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form an antigen-binding site.

[0148] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are involved in antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be defined by various numbering systems known in the art, such as 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) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), 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, one skilled in the art can easily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art.

[0149] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention may be determined based on various numbering systems known in the art, for example, the Kabat, Chothia, IMGT, AbM, or Contact numbering systems. In some embodiments, the CDRs contained in the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention are determined by the Kabat numbering system. In some embodiments, the CDRs contained in the anti-MSLN or PD-L1 single domain antibodies or antigen-binding fragments thereof of the present invention are determined by the Kabat, Chothia, IMGT, AbM, or Contact numbering systems.

[0150] As used herein, the term "framework region" or "FR" residues refers to amino acid residues other than the above-defined CDR residues in an antibody variable region.

[0151] The term "antibody" is not limited to any particular method of antibody production. It includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies may be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0152] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificities 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 specificities for different antigens (or epitopes), and is thereby capable of binding to at least two different binding sites and / or target molecules. Each antigen-binding domain contained in a multispecific antibody may 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, an F(ab')2 fragment, or an scFv). Optionally, each antigen-binding domain is linked by a peptide linker.

[0153] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide, including a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen as the full-length antibody and / or specifically binds to an antigen in competition with the full-length antibody, and is also referred to as an "antigen-binding portion." Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of whole antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabodies, single-domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology provided by Domantis), probodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide.

[0154] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains." Here, a "full-length heavy chain" refers to a polypeptide chain consisting of, from N- to C-terminus, 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 optionally further contains a heavy chain constant region CH4 domain if the full-length antibody is an IgE isotype. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of, from N- to C-terminus, a VH, CH1, HR, CH2, and CH3. A "full-length light chain" is a polypeptide chain consisting of, from N- to C-terminus, a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full-length antibody chains are linked by a disulfide bond between the 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, each formed by a VH and VL pair, which specifically recognize or bind to the same antigen.

[0155] The term "single-domain antibody (sdAb)" as used herein has the general meaning 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), generally derived from the variable region of a heavy chain antibody (e.g., a camel or shark antibody). Typically, nanobodies consist of four framework regions and three complementarity-determining regions, with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single-domain antibodies may be truncated at the N- or C-terminus to include only a portion of FR1 and / or FR4, or one or two of these framework regions may be deleted, as long as antigen binding and specificity are substantially retained. Single-domain antibodies are also called nanobodies, and the terms may be 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 as the single domain antibody and / or that specifically binds to an antigen in competition with the single domain antibody. Antigen-binding fragments of the single domain antibodies of the present invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the single domain antibodies of the present invention. In some embodiments, the "antigen-binding fragment" of a single domain antibody may be truncated at the N- or C-terminus to comprise only a portion of FR1 and / or FR4 compared to a full-length single domain antibody, or may lack one or two of these framework regions, as long as antigen binding and specificity are substantially retained.

[0156] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by disulfide bonds in the hinge region; and the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, and consists of one complete light chain and an Fd fragment of a heavy chain (consisting of the VH and CH1 domains).

[0157] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. The six CDRs are generally believed to confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to an antigen, although the affinity may be lower than that of the complete binding site.

[0158] As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region comprising CH2 and CH3. The Fc fragment of an antibody is not involved in antigen binding but has various functions. "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. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge regulates dimerization between two polypeptides comprising the Fc. The Fc region may be any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0159] The Fc domain may comprise a native Fc region or a variant Fc region. A native Fc region comprises an amino acid sequence corresponding to the amino acid sequence of an Fc region found in nature. For example, native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, native-sequence human IgG4 Fc regions, and 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 by at least one amino acid modification. In some embodiments, the variant Fc region may have an altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to the native Fc region.

[0160] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are linked by a linker. Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeats of the amino acid sequence GGGGS or variants thereof. For example, a linker having the amino acid sequence (GGGGS)2 may be used, as may variants thereof. Optionally, an additional disulfide bond may be present between the VH and VL of the scFv.

[0161] As used herein, the term "diabody" refers to a diabody whose VH and VL domains are expressed on a single polypeptide chain, but which uses a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites.

[0162] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or specifically binds to the antigen in competition with the full-length antibody.

[0163] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from the provided antibodies (e.g., antibodies provided by the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as are intact antibodies.

[0164] As used herein, the term "identity" refers to the matching of sequences between two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps are introduced into the first amino acid or nucleic acid sequence to optimize alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If 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 two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions × 100%). In some embodiments, the two sequences are the same length.

[0165] The determination of percent identity between two sequences may also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm by Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), with an improved version described in Karlin & Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs described in Altschul et al., 1990, J. Mol. Biol. 215:403.

[0166] The term "variant," as used herein in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide containing an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" refers to a polypeptide or peptide that has been modified (by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides may 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. Variants also possess similar, identical, or improved functions as the polypeptide or peptide from which they are derived. In some 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 to the sequence from which it is derived.

[0167] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K D In the present invention, the term "K D " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0168] The specific binding properties between two molecules may be measured using methods known in the art. One of these methods involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. Both the "association rate constant (ka or k)" and the "dissociation rate constant (k or k)" can be calculated from the concentrations and the actual association and dissociation rates (see Malmqvist M, Nature, 1993, 361:186-187). The ratio k / k is the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990;59:439-473.) D In some embodiments, the dissociation constants may be measured using surface plasmon resonance (SPR) in a Biacore, biolayer interferometry, or Kinexa.

[0169] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector is called an expression vector when it is capable of expressing a protein encoded by the inserted polynucleotide. The genetic material elements contained in the vector can be introduced into a host cell by transformation, transduction, or transfection so that they are 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 (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), phages (e.g., lambda phage or M13 phage), animal viruses, and the like. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40). A single vector may contain multiple expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes, and may also contain an origin of replication site.

[0170] As used herein, the term "host cell" refers to cells 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 oryzae, 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, HEK293 cells, or human cells.

[0171] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the desired properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis, PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, e.g., substitutions using a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties (including the ability to form covalent 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, 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Amino acid residues can also be further classified by optional physical and functional properties, e.g., alcohol group-containing residues (S and T), aliphatic residues (I, L, V, and M), cycloalkenyl group-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), turn-forming residues (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); Burks et al., Proc. Natl. Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0172] The designations of the 20 common amino acids herein follow common conventions. 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" are used interchangeably. In the present invention, amino acids are generally written using one-letter and three-letter abbreviations known in the art. For example, alanine may be written as A or Ala.

[0173] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives.

[0174] As used herein, the term "prevention" refers to a method performed to prevent or delay the onset of a disease or condition or symptom in a subject. As used herein, the term "treatment" refers to a method performed to achieve beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not further worsening), delaying or mitigating disease progression, improvement or alleviation of the disease state, and alleviation of symptoms (partial or total), whether measurable or non-measurable. "Treatment" may also refer to prolonging survival compared to expected survival (in the absence of treatment). As used herein, treatment may include new adjunctive and / or adjunctive treatments. "New adjunctive treatment" refers to a therapy administered to a patient prior to a planned treatment for the disease. "Adjunctive treatment" refers to a therapy administered to a patient after a treatment for the disease.

[0175] As used herein, the term "subject" refers to a mammal, e.g., a primate mammal such as a human. In some embodiments, the subject (e.g., a human) has or is at risk of having a tumor, an inflammatory disease, or an autoimmune disease.

[0176] 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 preventively effective amount for a disease refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease, and a therapeutically effective amount for a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient with the disease. Determining this effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use is determined based on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition (e.g., age, weight, sex), the mode of drug administration, other treatments being administered at the same time, etc.

[0177] [Effects of the Invention] The beneficial effects of the present invention are as follows:

[0178] The PD-L1-targeting nanobodies of the present invention have good binding affinity and specificity to PD-L1 and can effectively promote the secretion of IL2 and IFNγ, making them useful for the prevention and / or treatment of PD-L1-related diseases or conditions. Furthermore, the humanized nanobodies of the present invention retain the functions and properties of the parent camelid antibody and are highly humanized, making them suitable for administration to human subjects. The humanized MSLN-targeting nanobodies of the present invention are highly humanized, making them less likely to cause adverse immune reactions, while also exhibiting good binding affinity and specificity to MSLN, enabling efficient targeting of MSLN-expressing tumors. Furthermore, compared with known multispecific T cell activation constructs, the multispecific antibodies of the present invention targeting CD3, tumor-associated antigens, and immune checkpoints offer distinct advantages. The multispecific antibodies of the present invention can moderately activate T cells, improving safety, and simultaneously targeting tumor-associated antigens and immune checkpoints significantly improves anti-tumor efficacy. In particular, the present invention provides a trispecific antibody that specifically targets CD3, MSLN, and PD-L1, which not only reduces cytokine release to ensure safety, but also has good anti-tumor effect.

[0179] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings and examples. It will be understood by those skilled in the art that the following drawings and examples are merely illustrative of the present invention and do not limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description based on the drawings and preferred embodiments.

[0180] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Figures 1A to 1C show the blocking activity of the 4A7 nanobody of Example 2 against the binding of human PD-L1 to human PD-1 (A), mouse PD-L1 to mouse PD-1 (B), and monkey PD-L1 to monkey PD-1 (C), respectively.

[0181] [Figure 2] Figure 2 shows the measurement of the binding activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 to human PD-L1 cells in Example 2.

[0182] [Figure 3] Figure 3 shows the measurement of the binding activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 to monkey PD-L1 cells in Example 2.

[0183] [Figure 4] Figure 4 shows the measurement of the blocking activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 against human PD-L1 and human PD-1, respectively, in Example 2.

[0184] [Figure 5] Figure 5 shows the measurement of the blocking activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 on the PD-1 / PD-L1 signaling pathway of T cells in Example 2.

[0185] [Figure 6] Figure 6 shows the measurement results of cytokine production upon T cell activation by Benchmark-ATE, 7B11, and 7B11_huGS_HM4 in Example 2, of which Figure 6a shows the measurement results of IL2 production upon T cell activation by Benchmark-ATE, 7B11, and 7B11_huGS_HM4, and Figure 6b shows the measurement results of IFNγ production upon T cell activation by Benchmark-ATE, 7B11, and 7B11_huGS_HM4.

[0186] [Figure 7] Figure 7 shows a schematic diagram of the structure of the CD3-PDL1-MSLN trispecific antibody constructed in Example 3.

[0187] [FIG. 8A] to [FIG. 8D] FIGS. 8A to 8D show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to human MC38 / MSLN cells, respectively.

[0188] [FIG. 9A] to [FIG. 9C] FIGS. 9A to 9C show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to CHO-hPDL1 cells, respectively.

[0189] [Figure 10A] to [Figure 10C] Figures 10A to 10C show the blocking activity of the CD3-PDL1-MSLN trispecific antibody of Example 5 against the binding of human PD1 and human PD-L1, respectively.

[0190] [FIG. 11A] to [FIG. 11C] FIGS. 11A to 11C respectively show the activation of Jurkat-NFAT-luc cells in the tumor-added group by the CD3-PDL1-MSLN trispecific antibody of Example 7.

[0191] [FIG. 12A] to [FIG. 12C] FIGS. 12A to 12C respectively show the activation of Jurkat-NFAT-luc cells in the tumor-free group by the CD3-PDL1-MSLN trispecific antibody of Example 7.

[0192] [FIG. 13A] to [FIG. 13C] FIGS. 13A to 13C show the TDCC activity of the CD3-PDL1-MSLN trispecific antibody of Example 8 against MSLN-expressing human tumor cells HCC1806, respectively.

[0193] 14A-14F show the results of measuring the level of IL-2 release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively.

[0194] 15A-15F show the results of measuring the level of INF-γ release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively.

[0195] [Figure 16A], [Figure 16B] Figures 16A and 16B show the in vivo killing activity and safety of the 5Y3-16 antibody of Example 10, respectively.

[0196] [Figure 17A] and [Figure 17B] Figures 17A and 17B respectively show the structural diagrams of the CD3-PDL1-MSLN trispecific antibody constructed in Example 11.

[0197] [FIG. 18A] to [FIG. 18E] FIGS. 18A to 18E show the TDCC activity of the CD3-PDL1-MSLN trispecific antibodies of Example 11 with different structures against tumor cells, respectively.

[0198] [Mode for Carrying Out the Invention] (Sequence information) The table below describes the sequence information of this application.

[0199] [Table 1] JPEG2025542154000002.jpg255166 JPEG2025542154000003.jpg255163 JPEG2025542154000004.jpg255163 JPEG2025542154000005.jpg25169

[0200] The invention will now be described with reference to the following examples which illustrate, but do not limit, the invention.

[0201] Those skilled in the art will recognize that the examples are provided to illustrate the present invention and are not intended to limit the scope of protection claimed herein. Unless otherwise specified, all experimental methods in the examples are conventional. Unless specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless the manufacturer of the reagents or equipment used is specified, they may be conventional products available on the market.

[0202] Example 1: Humanization of MSLN antibody molecule, expression of humanized antibody and measurement of dynamic affinity Alpacas were immunized with hFc-tagged human MSLN recombinant protein (huMSLN-hFc, Kactus Biosystems (Shanghai) Co., Ltd., MSL-HM280) as the immunizing antigen, and screening was performed to finally obtain camelid nanobody 3C6, which has good cross-binding activity among humans, monkeys, and mice. Its VHH sequence is shown in SEQ ID NO: 9, and CDR1 to CDR3 defined by IMGT are shown in SEQ ID NOs: 52 to 54, respectively. Furthermore, a humanized antibody of 3C6 was constructed.

[0203] 1.1 Humanization of MSLN antibody molecules The anti-MSLN nanobody 3C6 was humanized. The 3C6 sequence was compared with the IMGT offline database to identify the germline gene sequence with the highest homology. Using this germline gene sequence as a template, CDR grafting of the target sequence was performed. After grafting was completed, Vernier region residues in the framework region of the target sequence were backmutated to ensure that the affinity of the humanized antibody was not affected. The 3D structure of the humanized sequence was drawn using the Modeller program, and the energy contribution of each amino acid residue to the stability of the protein's three-dimensional structure was calculated using Chimera software. To improve the thermal stability of the humanized sequence, amino acid residues with high energy contributions were backmutated. After completing these steps, eight sequences with different degrees of humanization were obtained.

[0204] 1.2 Expression of MSLN humanized antibodies The eight sequences with different degrees of humanization described above were each attached to the N-terminus of human IgG1 Fc (SEQ ID NO: 48) using a linker (SEQ ID NO: 50), then introduced into a PTT5 expression vector (Yubao Biotechnology, lot VT2202) and transiently expressed using the Expicho-s expression system (Gibco) to obtain VHH-Fc antibodies. The supernatants from the expressing cells were collected and purified using a Protein A chromatography column to obtain multiple humanized VHH-Fc antibodies, designated 3C6hu-1, 3C6hu-2, 3C6hu-3, 3C6hu-4, 3C6hu-5, 3C6hu-6, 3C6hu-7, and 3C6hu-8. The humanized anti-MSLN nanobody variable regions are shown in SEQ ID NOs: 1-8.

[0205] 1.3 Measurement of the dynamic affinity of MSLN humanized antibody molecules Anti-DYKDDDDK antibodies were coupled to an HC30M chip via amino coupling, followed by capture of 2 μg / mL MSLN single domain antibodies. After the baseline was stabilized, gradient dilutions of huMSLN-Fc (Kactus Biosystems (Shanghai) Co., Ltd., MSL-HM280) and cynoMSLN-Fc (Kactus Biosystems (Shanghai) Co., Ltd., MSL-CM280) (gradient dilution starting from 100 nM) were passed through the chip from low to high concentrations, respectively. The association time was 8 minutes, and the dissociation time was 20 minutes. Kinetic constants were obtained by fitting using the Carteria software. The dynamic affinity results of the MSLN single domain antibodies with MSLN protein are shown in Table 2 below. The results demonstrate that the resulting humanized MSLN single domain antibodies have good binding activity to human and monkey MSLN.

[0206] [Table 2]

[0207] Example 2: Generation of PD-L1 antibodies 2.1 PD-L1-mediated immunity in alpacas To generate a humoral immune response against PD-L1 in the alpacas, the first immunization was performed using complete Freund's adjuvant (CFA), which was homogenously mixed with 0.5 mg of PD-L1-Fc (Kactus Biosystems (Shanghai) Co., Ltd., PDL-HM210) protein, followed by subcutaneous injection. The second immunization was performed using incomplete Freund's adjuvant (IFA), which was homogenously mixed with 0.25 mg of PD-L1-Fc protein, followed by subcutaneous injection. The alpacas were immunized once every three weeks for a total of four immunizations to produce antigen-specific nanobodies.

[0208] From the second immunization onward, alpaca serum was collected every 7 days and serum titers were measured. Antigen was coated overnight at 2 μg / mL in carbonate buffered saline (CBS). After washing with phosphate buffered saline (PBST), 5% skim milk was added and blocked for 1 hour at 37°C. After washing with PBST, serum dilutions (1:2000 to 2x dilutions) were added and blocked for 1 hour at 37°C. After washing with PBST, horseradish peroxidase-conjugated goat anti-alpaca secondary antibody (alpaca S001H, 1:10000 dilution in PBS) was added and incubated for 45 minutes at 37°C. After washing with PBST, TMB color development solution (Thermo, 34029) was added for color development. The reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured to obtain serum titers.

[0209] 2.2 Construction of PD-L1 antibody phage library After the third and fourth immunizations, 50 mL of peripheral blood was collected, and PBMCs were isolated and total RNA was extracted using Sample Density Separation Solution (Tianjin Haoyang Biological Manufacture Co., Ltd., HY2015) according to the manufacturer's instructions. Total RNA was extracted from the peripheral blood after the third and fourth immunizations using RNAiso Plus Reagent (Takara Bio, 9109). cDNA was synthesized and VHH sequences were amplified by nested PCR. The target fragments were then digested with SfiI using the vector pComb3XSS (Aidi Gene, 63890). The ligation products were then electrotransformed into TG1 competent cells to construct a PD-L1 nanobody library. The volume was measured and was 2.15 x 10 9 CFM. Forty-eight clones were randomly picked from the microtiter plate to measure the number of transformants in the library, and the insertion rate was 100%. The library was packaged using helper phage M13KO7 (NEB, N0315S), and the titer of the phage library was measured to be 1.02 × 10 13 cfu / mL.

[0210] 2.3 Screening of anti-PD-L1 nanobodies 2.3.1 Phage panning Protein magnetic bead-based panning was performed using streptavidin magnetic beads (Dynabeads™ M-280 Streptavidin) with 3% skim milk for 1 hour of blocking with rotation at room temperature. Five micrograms of biotinylated PD-L1 (ACRO, PD1-H82E5) antigen was then added and incubated with rotation at room temperature for 1 hour to form a magnetic bead-protein complex. Sixty microliters of the VHH phage library was then added to the magnetic bead-protein complex and incubated with rotation at room temperature for 1 hour. The complex was then washed 10 times with 0.05% PBST to remove nonspecifically bound phages. The phage were then infected with TG1 for 1 hour at 37°C, and the plate was then coated with 2YT medium and incubated overnight. The screening process was repeated twice using the same procedure to complete enrichment of positive phages.

[0211] 2.3.2 Screening for specific clones (1) Preparation of periplasmic binding proteins Ninety-six single colonies were picked from the bacterial culture containing the phage after the two rounds of panning and cultured in 2YT medium. IPTG (Sangon Biotech (Shanghai) Co., Ltd., B541007-0001) was added to a final concentration of 1 mM, and the culture was incubated overnight at 30°C to induce expression of the periplasmic binding protein. The bacterial cell wall was disrupted using lysozyme, and the periplasmic binding protein was extracted and used to screen for specific clones.

[0212] (2) Screening of positive monoclones by enzyme-linked immunosorbent assay (ELISA) CBS coating solution was used to coat 2μg / mL of human PD-L1 protein (ACRO, PD1-H5258), monkey PD-L1 protein (Kactus Biosystems (Shanghai) Co., Ltd., PDL-CM210), and / or mouse PD-L1 protein (ACRO, PD1-M5251). 50μL of the above-prepared periplasmic binding protein was added and incubated for 1 hour. Unbound non-specific clones were washed away with 0.05% PBST. Anti-mouse HA-HRP secondary antibody (Nanjing GenScript Biotechnology Co., Ltd., A01296) was added and incubated for 1 hour. Unbound non-specific clones were washed away with 0.05% PBST. TMB color development solution was added and incubated for 1 hour. The results were then read using a microplate reader at 450nm.

[0213] (3) Screening of positive monoclones by T cell signaling pathway blockade experiments One day before, GS-C2 cells (human PD-L1-overexpressing cells, Nanjing GenScript Biotechnology Co., Ltd., M00613) were plated at 4 × 10 per well. 4 Cells were seeded into a white flat-bottom 96-well plate (corning) at 100 cells per well and cultured overnight at 37°C and 5% CO2. 5 μL of the above periplasmic binding protein was added to 45 μL of 1640 medium per well for dilution. The plate seeded with GS-C2 cells one day earlier was removed, the upper layer medium discarded, and the diluted periplasmic binding protein was added to the cell culture plate. Furthermore, Jurkat-NFAT-PD1 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were obtained and cultured at 8 × 10 cells per well. 4The cells were suspended in 5% 1640 medium, added to a white cell culture plate, mixed evenly, and incubated at 37°C and 5% CO2 for 6 hours. After incubation, 100 μL of assay reagent was added to each well according to the Bio-Lite Luciferase Assay System (Nanjing Vazyme Biotech Co., Ltd., DD1201) instructions, and the system was incubated at room temperature for 3 minutes. The fluorescent signal (BMG) was then measured after loading. Two positive monoclones were obtained and designated 4A7 and 260-7-B11, respectively.

[0214] 2.4 Expression of anti-PD-L1 nanobodies The positive clones 4A7 and 260-7-B11 obtained from the above screening were cloned into prokaryotic expression vectors, and expression of nanobodies (VHH antibodies) was induced with IPTG. After purification, the nanobodies were obtained. DNA sequencing of the nanobodies was then performed. The sequencing results were analyzed to define the CDR regions. The variable region sequences of the obtained anti-PD-L1 nanobodies are shown in SEQ ID NOs: 10 and 67, and the CDR sequences are shown in the table below.

[0215] [Table 3]

[0216] 2.5 Determining the kinetic affinity of anti-PD-L1 nanobodies 2.5.1 Measurement of the dynamic affinity of the 4A7 nanobody The anti-DYKDDDDK antibody was coupled to an HC30M chip via amino coupling, followed by capture of 2μg / mL of 4A7 nanobody. After the baseline stabilized, gradient dilutions of human PD-L1 protein (ACRO, PD1-H5258), monkey PD-L1 protein (Kactus Biosystems (Shanghai) Co., Ltd., PDL-CM210), and mouse PD-L1 protein (ACRO, PD1-M5251) (gradient dilution starting from 100nM) were passed through the chip from low to high concentrations, respectively. The association time was 8 minutes, and the dissociation time was 20 minutes. Kinetic constants were obtained by fitting using Carteria software. The kinetic affinity results of 4A7 nanobody to human, mouse, and monkey PD-L1 proteins are shown in the table below.

[0217] [Table 4]

[0218] 2.5.2 Dynamic affinity measurements of the 260-7-B11 nanobody The affinity and kinetic data of the 260-7-B11 nanobody to human PD-L1 protein (ACRO, PD1-H5258) were measured using surface plasmon resonance (SPR) technology on the Carterra LSA platform. The capture kinetics process was used for the measurement. The test antibody was immobilized on the surface of a NiHC200M chip via its His-Tag. After multiple loadings of blank buffer solution to stabilize the measurement baseline, human PD-L1 protein was loaded as the analyte. Each loading included a 2-minute baseline, 8 minutes of binding, and 20 minutes of dissociation. After subtracting the reference point and zero-concentration signals from the original measurement data (Double Reference), the binding curve was fitted using Carterra LSA Kinetics software to calculate the kinetic parameters. The kinetic affinity results of the 260-7-B11 nanobody to human PD-L1 protein are shown in the table below.

[0219] [Table 5]

[0220] The results in Tables 4 and 5 reveal that the 4A7 nanobody can bind to human, mouse, and monkey PD-L1 proteins, and the 260-7-B11 nanobody can bind to human PD-L1.

[0221] 2.6 Determining the cell binding ability of anti-PD-L1 nanobodies GS-C2 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were washed with PBS and then resuspended using 1% BSA at 1 × 10 cells per well. 5 Cells were added to a V-bottom 96-well plate at 1000 x 1000 cells per well, followed by gradient dilution of the 260-7-B11 nanobody (gradient dilution starting from 33.33 nM). The cells were then incubated on ice for 1 hour, washed with PBS, and then PE-labeled goat anti-Flag fluorescent secondary antibody was added. The cells were then incubated on ice for 30 minutes, washed with PBS, and measured using a flow cytometer. The experimental data were fitted using GraphPad Prism to calculate EC values. 50 The binding affinity results of 260-7-B11 nanobody to GS-C2 cells expressing human PD-L1 are shown in the table below.

[0222] [Table 6]

[0223] The results in Table 6 showed that the 260-7-B11 nanobody could bind to GS-C2 cells overexpressing human PD-L1.

[0224] 2.7 Determining the ability of anti-PD-L1 nanobodies to block the binding of PD-L1 to PD1 2.7.1 Determining the Ability of 4A7 Nanobody to Block PD-L1 and PD1 Binding The huPDL1 sequence (NCBI accession number: NP_054862.1), mPDL1 (NCBI accession number: NP_068693), and cynoPDL1 (NCBI accession number: F6VEW6-1) were cloned into lentiviral vectors (plvx-IRES-puro, psPAX2, pMD2G), respectively. HEK293T (Thermo K1649B) cells were co-transfected with the constructed lentiviral plasmids and packaging plasmids. The supernatants were collected after 48 h and added to CHO cells (Thremo, R80007). At 24 h, 4 μg / mL puromycin was added for screening. Single cells expressing huPDL1, mPDL1, or cynoPDL1 were sorted using a cell sorter (Sony, LESH800SBP). Finally, CHO-huPDL1, CHO-mPDL1, and CHO-cynoPDL1 stable cells were obtained.

[0225] CHO-huPDL1, CHO-mPDL1, and CHO-cynoPDL1 cells were washed with PBS, resuspended in 1% BSA, and plated at 1 × 10 cells per well. 5 Cells were added to a V-bottom 96-well plate. Gradient-diluted anti-PD-L1 nanobodies (gradient dilutions starting from 33.33 nM) and a fixed concentration of 1.25 μg / mL human PD-1-hFc (ACRO, PD1-H5257), mouse PD-1-hFc (ACRO, PD1-M5251), or monkey PD-1-hFc (ACRO, PD1-C5254) protein premix were added. The cells were incubated on ice for 1 hour and washed with PBS. PE anti-human Fc (Biolegend, 410708) was added. The cells were incubated on ice for 30 minutes and washed with PBS. The IC values ​​were then measured using a flow cytometer. Experimental data were fitted using GraphPad Prism. 50 The blocking activity of 4A7 nanobody against the binding of PD-L1 to PD-1 is shown in Figures 1A to 1C and the table below.

[0226] [Table 7]

[0227] The results showed that the 4A7 nanobody could block the binding of human PD-L1 and human PD-1, could block the binding of mouse PD-L1 and mouse PD-1, and could block the binding of monkey PD-L1 and monkey PD-1.

[0228] 2.7.2 Determining the Ability of 260-7-B11 Nanobody to Block PD-L1 and PD1 Binding The simian PD-L1 (Cyno-PD-L1) sequence (NCBI accession number: F6VEW6-1) was cloned into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). HEK293T cells were co-transfected with the constructed lentiviral and packaging plasmids. The supernatant was collected after 48 hours and added to 293F cells (Nanjing Kebai Biotechnology Co., Ltd., CBP60437). At 24 hours, 4 μg / mL puromycin was added for screening. Single cells expressing Cyno-PD-L1 were sorted using a cell sorter (Sony, LESH800SBP). Finally, 293F-cyno PD-L1 cells stably overexpressing simian PD-L1 were obtained.

[0229] GS-C2 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) and 293F-cyno PD-L1 cells were washed with PBS and resuspended in 1% BSA. 1 × 10 cells were plated per well. 5Cells were added to a V-bottom 96-well plate. Gradient-diluted anti-PD-L1 nanobody (gradient dilution starting from 33.33 nM) and a fixed concentration of 1.25 μg / mL human PD-1-mFc (ACRO, PD1-H5255) or monkey PD-1-hFc (ACRO, PD1-C5254) protein premix were then added. The cells were incubated on ice for 1 hour, washed with PBS, and then a 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 then analyzed using a flow cytometer. The experimental data were fitted using GraphPad Prism to calculate the IC values. 50 The blocking activity of 260-7-B11 nanobody against PD-L1 and PD-1 binding is shown in the table below.

[0230] [Table 8]

[0231] The results in Table 8 show 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.

[0232] 2.8 Screening and measurement of PD-1 / PD-L1 signaling pathway blocking activity in T cells One day before, 4 × 10 GS-C2 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were plated per well. 4 10 cells were seeded into a white flat-bottom 96-well plate (corning) and cultured overnight at 37°C and 5% CO2. The plate seeded with GS-C2 cells one day earlier was removed, the medium on top was discarded, and gradient-diluted anti-PD-L1 nanobody (gradient dilution starting from 266.67 nM) was added. Jurket-NFAT-PD1 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were then added at 8 x 10 cells per well. 4Cells were suspended in 5% 1640 medium, added to a white cell culture plate, mixed evenly, and incubated at 37°C and 5% CO2 for 6 hours. After incubation, 100 μL of assay reagent was added to each well according to the Bio-Lite Luciferase Assay System (Nanjing Vazyme Biotech Co., Ltd., DD1201) instructions, and the system was incubated at room temperature for 3 minutes. The loading and fluorescent signal (BMG) were measured. The results of the blocking activity of the 260-7-B11 nanobody on the PD-1 / PD-L1 signaling pathway in T cells are shown in the table below.

[0233] [Table 9]

[0234] The results in Table 9 demonstrate that the 260-7-B11 nanobody has the activity of blocking the PD-1 / PD-L1 signaling pathway in T cells.

[0235] 2.9 Humanization of anti-PD-L1 nanobodies The anti-PD-L1 nanobodies 4A7 and 260-7-B11 were humanized. The 4A7 and 260-7-B11 sequences were compared with the IMGT offline database to identify the germline gene sequences with the highest homology. Using these germline gene sequences as templates, CDR grafting of the target sequences was performed. After grafting was completed, Vernier region residues in the framework regions of the target sequences were backmutated to ensure that the affinity of the humanized antibody was not affected. The 3D structure of the humanized sequences was drawn using the Modeller program, and the energy contribution of each amino acid residue to the stability of the protein's three-dimensional structure was calculated using Chimera software. To improve the thermal stability of the humanized sequences, amino acid residues with high energy contributions were backmutated. After completing these steps, multiple sequences with varying degrees of humanization were obtained. Of these, 16 sequences with varying degrees of humanization were obtained for 4A7, and 8 sequences with varying degrees of humanization were obtained for 260-7-B11.

[0236] 2.10 Expression of humanized PDL1 antibodies Sixteen sequences of the above 4A7 with varying degrees of humanization were attached to the N-terminus of human IgG1 Fc (SEQ ID NO: 48) using a linker (SEQ ID NO: 50) and then introduced into a PTT5 expression vector (Yubao Bio, lot VT2202). Eight sequences of 260-7-B11 with varying degrees of humanization were attached to the N-terminus of human IgG1 Fc (SEQ ID NO: 48) using a linker (SEQ ID NO: 50) and then introduced into a PTT5 expression vector (Yubao Bio, lot VT2202). VHH-Fc antibodies were obtained by transient expression using the Expicho-s expression system (Gibco). The supernatants from the expressing cells were collected and purified using a Protein A chromatography column to obtain multiple humanized VHH-Fc antibodies. Here, the 16 4A7 humanized VHH-Fc antibodies are 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 to 39. The eight humanized VHH-Fc antibodies of 260-7-B11 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. Here, 7B11 represents a VHH-Fc antibody in which the sequence of 260-7-B11 was fused with human IgG1 Fc (SEQ ID NO: 48) according to the method of this example.

[0237] 2.11 Dynamic affinity measurements of humanized PD-L1 nanobodies 2.11.1 Dynamic Affinity Measurements of 4A7 Humanized Nanobodies The anti-DYKDDDDK antibody was coupled to an HC30M chip via amino coupling, followed by capture of 2μg / mL humanized PD-L1 single domain antibodies. After the baseline stabilized, gradient dilutions of human PD-L1 protein (ACRO, PD1-H5258), monkey PD-L1 protein (Kactus Biosystems (Shanghai) Co., Ltd., PDL-CM210), and mouse PD-L1 protein (ACRO, PD1-M5251) (gradient dilution starting from 100nM) were passed through the chip, from low to high concentration. The association time was 8 minutes, and the dissociation time was 20 minutes. The kinetic constants were obtained by fitting using Carteria software. The results are shown in the table below. These humanized single domain antibodies have good cross-reactivity with human, mouse, and monkey PD-L1 proteins.

[0238] [Table 10]

[0239] 2.11.2 Dynamic Affinity Measurements of the 260-7-B11 Humanized Nanobody The anti-DYKDDDDK antibody was coupled to an HC30M chip via amino coupling, followed by capture of 2μg / mL humanized PD-L1 single domain antibodies. After the baseline stabilized, gradient dilutions of human PD-L1 protein (ACRO, PD1-H5258) and monkey PD-L1 protein (Kactus Biosystems (Shanghai) Co., Ltd., PDL-CM210) (gradient dilution from 100nM) were passed through the chip from low to high concentrations, respectively. The association time was 8 minutes and the dissociation time was 20 minutes. The kinetic constants were obtained by fitting using Carteria software. The results are shown in the table below. These humanized single domain antibodies have good cross-reactivity with human and monkey PD-L1 proteins.

[0240] [Table 11]

[0241] Table 11 shows that the humanized single domain antibodies in the table can bind well to human and monkey PD-L1 proteins.

[0242] 2.12 Determining the cell-binding activity of humanized anti-PD-L1 nanobodies 2.12.1 Binding activity to human PD-L1 cells GS-C2 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were washed with PBS and then resuspended using 1% BSA at 1 × 10 cells per well. 5 Cells were added to a V-bottom 96-well plate at 1000 x g, and then gradient dilutions (starting from 16.67 nM) of 7B11_huGS_HM4, 7B11, and the control antibody Benchmark-ATE (atezolizumab) were added. 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 then analyzed using a flow cytometer. Data were fitted using GraphPad Prism, and the results are shown in Figure 2.

[0243] 2.12.2 Cross-binding activity with monkey PD-L1 cells 293F-cyno PD-L1 cells (293F overexpressing monkey PD-L1) were washed with PBS and then resuspended using 1% BSA, at 1 × 10 per well. 5 Cells were added to a V-bottom 96-well plate at 1000 x g, followed by gradient dilutions of 7B11_huGS_HM4, 7B11, and Benchmark-ATE (gradient dilutions starting from 16.67 nM). The mixture was 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 mixture was incubated on ice for 30 minutes, washed with PBS, and then measured using a flow cytometer. Data were fitted using GraphPad Prism, and the results are shown in Figure 3. The cell binding activity results for the 7B11_huGS_HM4 and 7B11 nanobodies are shown in Table 12, Figures 2 and 3.

[0244] [Table 12]

[0245] Figures 2, 3 and Table 12 show that 7B11 and 7B11_huGS_HM4 have excellent binding activity to cells overexpressing human and monkey PD-L1.

[0246] 2.13 Blocking activity of humanized anti-PD-L1 nanobodies against human PD-L1 and PD-1 GS-C2 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were washed with PBS and then resuspended using 1% BSA at 1 × 10 cells per well. 5 Cells were added to a V-bottom 96-well plate at 1000 x g, followed by gradient dilutions of 7B11_huGS_HM4, 7B11, Benchmark-ATE (gradient dilution starting from 22.22 nM), and a fixed concentration of 1.25 μg / mL human PD-1-mFc protein premix. The mixture was incubated on ice for 1 hour, washed with PBS, and then a PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added. The mixture was incubated on ice for 30 minutes, washed with PBS, and then analyzed using a flow cytometer. Data were fitted using GraphPad Prism, and the results are shown in Figure 4 and Table 13.

[0247] [Table 13]

[0248] Figure 4 and Table 13 show that 7B11 and 7B11_huGS_HM4 can effectively block the binding of PD-L1 to PD-1.

[0249] 2.14 Measurement of the blocking activity of humanized anti-PD-L1 nanobodies on the PD-1 / PD-L1 signaling pathway in T cells One day before, 4 × 10 GS-C2 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were plated per well.4 The cells were seeded into a white flat-bottom 96-well plate (corning) at 100x10 cells per well and cultured overnight at 37°C and 5% CO2. The plate seeded with GS-C2 cells one day earlier was removed, the medium on top was discarded, and gradient-diluted 7B11_huGS_HM4, 7B11, and Benchmark-ATE (gradient dilution starting from 20 nM) were added. Jurket-NFAT-PD1 cells (Nanjing GenScript Biotechnology Co., Ltd., M00613) were then added at 8x10 cells per well. 4 The cells were suspended in 5% 1640 medium, added to a white cell culture plate, mixed evenly, and cultured at 37°C and 5% CO2 for 6 hours. After the culture was completed, 100 μL of measurement reagent was added to each well according to the Bio-Lite Luciferase Assay System (Nanjing Vazyme Biotech Co., Ltd.) instructions, and the system was incubated at room temperature for 3 minutes. The fluorescent signal (BMG) was then measured after loading. The results are shown in Table 14 and Figure 5.

[0250] [Table 14]

[0251] The results in Table 14 and Figure 5 demonstrate that 7B11_huGS_HM4 and 7B11 can effectively block the PD-1 / PD-L1 signaling pathway in T cells.

[0252] 2.15 Effect of humanized anti-PD-L1 nanobodies on T cell activation in lymphocyte responses DC cells (Shanghai AoNeng Biotechnology Co., Ltd., FPB-DC002F-C) and PBMC cells (Shanghai AoNeng Biotechnology Co., Ltd., FPB004F-C-MLR) were resuscitated, with 1 × 10 DC cells per well. 4 PBMC cells were 1 x 10 per well. 5The mixture was mixed in a 200 μL volume, and added to each well of a flat-bottom 96-well plate. The 96-well plate was divided into three groups, and Benchmark-ATE, 7B11, and 7B11_huGS_HM4 were added to each group. Benchmark-ATE groups received different concentrations of Benchmark-ATE (111.1 nM, 44.44 nM, 4.444 nM, and 0 nM), 7B11 groups received different concentrations of 7B11 (66.7 nM, 6.67 nM, 0.667 nM, and 0 nM), and 7B11_huGS_HM4 groups received different concentrations of 7B11_huGS_HM4 (66.7 nM, 6.67 nM, 0.667 nM, and 0 nM). The 96-well plate was incubated at 37°C and 5% CO2 for 5 days, and the concentrations of IL2 and IFNγ were measured using HUMAN IL2 KITS (Cisbio Bioassays, 62HIL02PEG) and HUMAN IFNγ KITS (Cisbio Bioassays, 62HIFNGPEG).

[0253] The results are shown in Figures 6a and 6b. Figure 6a shows the results of measuring IL2 production upon T cell activation with Benchmark-ATE, 7B11, and 7B11_huGS_HM4, and Figure 6b shows the results of measuring IFNγ production upon T cell activation with Benchmark-ATE, 7B11, and 7B11_huGS_HM4. The results demonstrate that 7B11 and 7B11_huGS_HM4 can stimulate donors to produce cytokines, indicating that 7B11 and 7B11_huGS_HM4 can effectively activate T cells.

[0254] Example 3: Generation of CD3-PDL1-MSLN trispecific antibody 3.1 Construction of CD3-PDL1-MSLN trispecific antibody As shown in Figure 7, the structure of the CD3-PDL1-MSLN trispecific antibody of this example consists 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. heavy chain 1 consisting of a humanized CD3 heavy chain variable region and the C-terminus of IgG1 variant 1 (SEQ ID NO: 45) linked to a humanized MSLN single domain antibody by (GGGGS)3, in that order; and 3. heavy chain 2 consisting of a humanized MSLN single domain antibody linked to the N-terminus of IgG1 variant 2 (SEQ ID NO: 47) by (GGGGS)1, and then the C-terminus of IgG1 variant 2 linked to a PD-L1 single domain antibody by (GGGGS)3. In this example, 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 types of trispecific antibodies (5Y3-1 to 5Y3-15, 5Y3-16) were obtained, the structures of which are shown in the table below. The nucleic acid sequences encoding the 16 types of polypeptide chains were constructed in the PTT5 plasmid vector (Yubao Bio, lot VT2202), and sufficient plasmids were extracted from each vector.

[0255] [Table 15]

[0256] 3.2 Expression and purification of CD3-PDL1-MSLN trispecific antibodies The day before transfection (D1), culture medium was used to grow the cells to a density of 2 × 10 6 On the day of transfection (D0), cells were harvested and counted (cell viability must be 95% or higher) to achieve a cell density of 4.0 × 10 6The 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 an incubator at 37°C, 120 rpm, and 8% CO2. On day 1 (D1) after transfection, prewarmed CHO gro (Mirus 6200A) complete medium was added at 1 / 5 the expression level, and the medium 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 added at 8%, 5%, and 5%, respectively. From D7 onwards, cell viability was measured daily. When viability fell below 80%, the cells were harvested, centrifuged, and the supernatant was collected and purified on a protein A column.

[0257] Example 4: Verification of binding activity of CD3-PDL1-MSLN trispecific antibody 4.1 Binding of CD3-PDL1-MSLN trispecific antibody to human Jurkat cells (1) Jurkat cells (ATCC, Cat. No. TIB-152) were diluted in PBS to 1 × 10 7 The concentration was adjusted to cells / mL, and 50 μL of cell suspension was added to each prepared sample.

[0258] (2) For 5Y3-1 to 5Y3-15, the final concentration was adjusted to 2 μg / mL with PBS and then diluted 3-fold to obtain eight gradients. For 5Y3-16, the final concentration was adjusted to 2000 nM with PBS and then diluted 3-fold to obtain seven gradients. Each well contained 50 μL of antibody.

[0259] (3) Obtain 50 μL of cell suspension, and the total number of cells is 5 × 10 5 Cells were plated at 1000 μg / well. For 5Y3-1 to 5Y3-15, the final antibody concentration was 2 μg / mL, diluted 3-fold to obtain eight wells, and incubated at 4°C for 1 hour. For 5Y3-16, the final antibody concentration was 2000 nM, diluted 3-fold to obtain seven wells, and incubated at 4°C for 1 hour.

[0260] (4) After 5 minutes at 500 g, the supernatant was gently removed and the mixture was washed twice with 200 μL of PBS.

[0261] (5) Secondary antibody PE anti-human Fc (Biolegend 410708) was added, i.e., fluorescent secondary antibody diluted with PBS (1:100), and incubated at 4°C for 30 minutes.

[0262] (6) The fluorescent secondary antibody was washed, and after 5 minutes at 500 g, the supernatant was gently removed, and 200 μL of PBS was added for one wash, after which the antibody was loaded onto a flow cytometer and measured.

[0263] The results are shown in the table below and demonstrate that the constructed trispecific antibody is capable of binding to Jurkat cells expressing CD3.

[0264] [Table 16]

[0265] 4.2 Binding of CD3-PDL1-MSLN trispecific antibodies to human MC38 / MSLN cells Human MC38 / MSLN cells were constructed as follows. The full-length human MSLN (huMSLN) sequence (cDNA, HG13128-UT, purchased from Sino Biological) was cloned into a lentiviral vector. HEK293T cells were co-transfected with the constructed lentiviral plasmid and packaging plasmid. Cell supernatants were collected at 48 and 72 hours, respectively, and then added to MC38 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60825). 24 hours later, 4 μg / mL puromycin was added for screening. Single cells highly expressing huMSLN were sorted using a cell sorter (Sony, LE-SH800SBP). Finally, stably transfected monoclonal MC38 / MSLN cells highly expressing huMSLN were obtained.

[0266] 50 μL of MC38 / MSLN cells (2 × 10 5 100 cells) were added to a V-bottom 96-well plate, and 50 μL of gradient-diluted antibody was added to each well (antibody was diluted from 100 nM). The mixture was then incubated on ice for 1 hour. After washing off the primary antibody, a PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added and incubated on ice for 1 hour. After washing, the mixture was resuspended in 200 μL of PBS per well and analyzed using a flow cytometer. EC was measured using the software Graphpad Prism. 50 was calculated and the results are shown in Figures 8A to 8D. The results show that the constructed trispecific antibody can bind to cells expressing MSLN.

[0267] 4.3 Binding of CD3-PDL1-MSLN trispecific antibody to CHO-hPDL1 cells CHO-huPDL1 cells were washed with PBS and then resuspended in 1% BSA at 1 × 10 cells per well. 5 Cells were added to a V-bottom 96-well plate. Then, gradient-diluted anti-CD3-PDL1-MSLN trispecific antibodies were added (5Y3-1 to 5Y3-15 were diluted 3-fold from 1500 nM to obtain 8 gradients, and 5Y3-16 was diluted 3-fold from 100 nM to obtain 11 gradients). After incubation on ice for 1 hour and washing with PBS, PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibodies were added, incubated on ice for 30 minutes, washed with PBS, and then measured using a flow cytometer. The experimental data were fitted using GraphPad Prism to obtain EC values. 50 The results are shown in Figures 9A-9C. The results demonstrate that the constructed trispecific antibody can bind to cells expressing PD-L1.

[0268] Example 5: Blockade of human PD1 and human PD-L1 binding by CD3-PDL1-MSLN trispecific antibodies CHO-huPDL1 cells were washed with PBS and then resuspended in 1% BSA at 1 × 10 cells per well. 5Cells were added to a V-bottom 96-well plate. Then, gradient-diluted CD3-PDL1-MSLN trispecific antibody (5Y3-1 to 5Y3-15, 3-fold gradient dilutions from 1500 nM to obtain eight gradients, and 5Y3-16, gradient dilution from 50 nM) and a fixed concentration of 1.25 μg / mL human PD-1-mFc (ACRO, PD1-H5255) protein premix were added. The cells were incubated on ice for 1 hour and washed with PBS. After that, a PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody was added. The cells were incubated on ice for 30 minutes and washed with PBS. The EC values ​​were measured using a flow cytometer. Experimental data were fitted using GraphPad Prism to calculate EC values. 50 obtained.

[0269] The results are shown in Figures 10A to 10C, and demonstrate that the constructed trispecific antibody can effectively block the binding of human PD1 and human PD-L1.

[0270] Example 6: Measurement of the dynamic affinity of CD3-PDL1-MSLN trispecific antibodies Antibody concentrations of 4, 2, and 1 μg / mL were immobilized on an HC200M chip containing an anti-human IgG Fc secondary antibody, limiting antibody binding to approximately 1000 RU. After baseline stabilization, gradient dilutions of human CD3εγ (acrobiosystems, catalog no. CDG-H52W5), human MSLN-Fc (Kactus Biosystems (Shanghai) Co., Ltd., MSL-HM280), and human PD-L1 protein (ACRO, PD1-H5258) (eight 3-fold dilutions starting from 592.5 nM) were passed through the chip at a flow rate of 1000 μL / min. The association time was 8 minutes, and the dissociation time was 20 minutes. Kinetic constants were obtained by fitting the 1:1 binding model in Carterra's built-in Kinetics software. The results are shown in the table below. The constructed trispecific antibody has good affinity for human CD3εγ, human MSLN, and human PD-L1.

[0271] [Table 17]

[0272] Example 7: Activation of the T cell activation signaling pathway by CD3-PDL1-MSLN trispecific antibodies (1) Tumor-free group For 5Y3-1 to 5Y3-15, antibodies were diluted to 2 μg / mL in 1640 + 10% FBS, and gradient dilutions were performed. 50 μL of diluted antibody was added to each well. 6 × 10 Jurkat-NFAT-luc (Nanjing Kebai Biotechnology Co., Ltd., Cat. No. CBP74020) cells in logarithmic growth phase were cultured in 1640 + 10% FBS. 5 Adjust the cell count to 3 x 10 cells / mL and add 50 µL of cells (3 x 10) to each well. 4 After gently mixing to homogenize, the plate was incubated at 37°C and 5% CO2 for 5-6 hours, and 20 μL of chemiluminescent substrate was added to the plate, followed by loading and measurement.

[0273] For 5Y3-16, the antibody was diluted to 5 nM in 1640 + 10% FBS, and gradient dilutions were performed. 50 μL of diluted antibody was added to each well. 1.6 × 10 Jurkat-NFAT-luc (Nanjing Kebai Biotechnology Co., Ltd., Cat. No. CBP74020) cells in logarithmic growth phase were cultured in 1640 + 10% FBS. 6 Adjust the platelet count to 8 x 10 cells / mL and add 50 µL of cells to each well (8 x 10 4 After gently mixing to homogenize, the plate was incubated at 37°C and 5% CO2 for 5-6 hours, and 20 μL of chemiluminescent substrate was added to each well before loading and measurement.

[0274] (2) Tumor-added group For 5Y3-1 to 5Y3-15, antibodies were diluted to 2 μg / mL in 1640 + 10% FBS, and gradient dilutions were performed. 50 μL of diluted antibodies were added to each well. 1.2 × 10 Jurkat-NFAT-luc cells in logarithmic growth phase were cultured in 1640 + 10% FBS. 6 Adjust the platelet count to 3 x 10 cells / mL and add 25 µL of cells to each well (3 x 104 cells / well), the MC38 / MSLN cells constructed in Example 4 were digested and cultured at 1.2 × 10 6 Adjust the platelet count to 3 x 10 cells / mL and add 25 µL of cells to each well (3 x 10 4 After gently mixing to homogenize, the plate was incubated at 37°C and 5% CO2 for 5-6 hours, and 20 μL of chemiluminescent substrate was added to each well before loading and measurement.

[0275] For 5Y3-16, the antibody was diluted to 1 nM in 1640 + 10% FBS, and gradient dilutions were performed. 50 μL of diluted antibody was added to each well. 3.2 × 10 Jurkat-NFAT-luc cells in logarithmic growth phase were cultured in 1640 + 10% FBS. 6 Adjust the platelet count to 8 x 10 cells / mL and add 25 µL of cells to each well (8 x 10 4 cells / well), the MC38 / MSLN cells constructed in Example 4 were digested and cultured at 1.6 × 10 6 Adjust the platelet count to 4 x 10 cells / mL and add 25 µL of cells to each well (4 x 10 4 After gently mixing to homogenize, the plate was incubated at 37°C and 5% CO2 for 5-6 hours, and 20 μL of chemiluminescent substrate was added to each well before loading and measurement.

[0276] The measurement results for the tumor-added group are shown in Figures 11A to 11C, and the measurement results for the tumor-free group are shown in Figures 12A to 12C. The results show that the CD3-PDL1-MSLN trispecific antibodies of the present invention were unable to activate Jurkat-NFAT-luc cells in the absence of tumor cells, but both antibodies were able to moderately activate Jurkat-NFAT-luc cells after tumor cell addition.

[0277] Example 8: CD3-PDL1-MSLN trispecific antibody-mediated TDCC 8.1 Preparation of HCC1806-luc cells The Luc sequence (NCBI GenBank: MF062157.1) was cloned into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). HEK293T cells were co-transfected with the constructed lentiviral and packaging plasmids. The supernatant was collected after 48 hours and added to HCC1806 cells (ATCC, CRL-2335, human breast cancer cells). At 24 hours, 4 μg / mL puromycin was added for screening. Single cells expressing Luc were sorted using a cell sorter (Sony, LESH800SBP). Finally, HCC1806-luc stable cells were obtained.

[0278] 8.2 Preparation of PBMCs Cryopreserved PBMC cells (Shanghai Saily Biotechnology Co., Ltd.) were resuscitated, centrifuged at 500g for 3 minutes, the supernatant discarded, washed once with 1640 + 10% FBS (inactivated), centrifuged at 500g for 3 minutes, the supernatant discarded, resuspended in an appropriate volume of 1640 + 10% FBS (inactivated), counted, diluted to the corresponding density, and then added to a well plate, resulting in a total number of PBMC cells of 1.25 × 10 5 25 μL was added to each well to give cells / well.

[0279] 8.3 Seeding of HCC1806-luc cells Add 25 μL of HCC1806-luc cells to the above PBMC well plate, and 1.25 × 10 4 cells / well.

[0280] 8.4 Dilution of CD3-PDL1-MSLN trispecific antibody For 5Y3-1 to 5Y3-15, the concentration was adjusted to 2 nM with medium, gradient diluted, and 50 μL of the diluted antibody was added to cells containing PBMC and HCC1806-luc. For 5Y3-16, the concentration was adjusted to 20 nM with medium, gradient diluted, and 100 μL of the diluted antibody was added to cells containing PBMC and HCC1806-luc.

[0281] 8.5 Incubation The cells were incubated in an incubator at 37°C and 5% CO2 for 2 days.

[0282] 8.6 Reading the results The well plate was removed, 50 μL of luciferase substrate was added, loaded, the results were read, and the data was analyzed.

[0283] The experimental results are shown in Figures 13A to 13C, which demonstrate that the CD3-PDL1-MSLN trispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effects on MSLN-expressing human tumor cells, HCC1806.

[0284] Example 9: Cytokine experiments of CD3-PDL1-MSLN trispecific antibodies For 5Y3-1 to 5Y3-16, 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, its sequence is shown in SEQ ID NO: 66, the HPN536 sequence was constructed into a PTT5 expression vector (Yubao Biotechnology, lot VT2202), transiently expressed using the Expicho-s expression system (Gibco), and purified on a protein A chromatography column to obtain the HPN536 antibody) were adjusted to five gradient concentrations of 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM. The diluted trispecific antibody (50 μL / well) or control antibody was added, with the last well not receiving the trispecific antibody or control antibody. The PBMC cell density was adjusted to 2 × 10 6 The cells were cultured at 50 μL per well at 37°C and 5% CO for 48 hours. After 24 and 48 hours, 30 μL of supernatant was collected and the IL2 and INF-γ contents were measured using HTRF kits (cisbio 62HIL02PEH, 62HIFNGPEH).

[0285] The results of IL2 measurement are shown in Figures 14A to 14F, and the results of INF-γ measurement are shown in Figures 15A to 15F. The results show that when co-incubated with PBMCs, the CD3-PDL1-MSLN trispecific antibody of the present invention promoted IL2 and INF-γ secretion more strongly than the control antibody, but the effect of the trispecific antibody of the present invention in promoting IL2 and INF-γ secretion was significantly lower, indicating that the trispecific antibody of the present invention has a low risk of causing side effects that induce PBMCs to produce IL2 and INF-γ and has good safety.

[0286] Example 10: PBMC immune reconstituted mouse subcutaneous HCC1806 transplant tumor model Six- to eight-week-old female NOG-dKO mice (NOG-MHC I / II-2 KO mice) (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. Each mouse received 5 × 10 7 PBMC cells were injected intraperitoneally, and 7 days later, 3 x 10 6 HCC1806 cells were subcutaneously inoculated, and tumors grew to an average of approximately 80 mm 3 When tumors reached 1000 mg / kg, they were randomly assigned to treatment groups (8 mice per group) at 0.05 mg / kg, 0.25 mg / kg, and 1 mg / kg. Treatment was administered twice a week for a total of four doses, with eight mice in the control group receiving no treatment. Tumor volume and weight were measured, and upon completion of the experiment, the mice were euthanized by cervical dislocation and the tumors were removed, weighed, and recorded.

[0287] The results are shown in Figure 16A. The 5Y3-16 antibody exhibited good efficacy, and its in vivo efficacy showed a dose-related relationship, with tumor growth inhibition rates of 81%, 101%, and 106% at three doses of 0.05 mg / kg, 0.25 mg / kg, and 1 mg / kg, respectively. As shown in Figure 16B, the tumor-bearing mice tolerated these doses well and experienced no adverse events such as weight loss.

[0288] Example 11: TDCC mediated by CD3-PDL1-MSLN trispecific antibodies of different structures 11.1 Preparation of MC38-luc, CT26-luc, H2052-luc, and HCC1806-luc cells The Luc sequence (NCBI GenBank: MF062157.1) was cloned into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). HEK293T cells were co-transfected with the constructed lentiviral plasmid and packaging plasmid. The supernatants were collected after 48 hours and added to MC38 (ATCC, CRL-2640, murine colon carcinoma cells), CT26 (ATCC, CRL-2638, murine colon carcinoma cells), and H2052 (ATCC, CRL-5915, human mesothelioma cells). At 24 hours, 4 μg / mL puromycin was added and the cells were screened. Single cells expressing Luc were sorted using a cell sorter (Sony, LESH800SBP). MC38-luc, CT26-luc, and H2052-luc stable cells were obtained, along with the HCC1806-luc cells prepared in Example 8.

[0289] 11.2 Preparation of PBMCs Cryopreserved PBMC cells (Shanghai Saily Biotechnology Co., Ltd.) were resuscitated, centrifuged at 500g for 3 minutes, the supernatant discarded, washed once with inactivated 1640 + 10% FBS, centrifuged at 500g for 3 minutes, the supernatant discarded, resuspended in an appropriate amount of inactivated 1640 + 10% FBS, counted, diluted to the corresponding density, and then added to a well plate, resulting in a total number of PBMC cells of 1.25 × 10 5 25 μL was added per well to give cells / well.

[0290] 11.3 Seeding of MC38-luc, CT26-luc, H2052-luc, and HCC1806-luc cells Add 25 µL of MC38-luc, CT26-luc, HCC1806-luc, or H2052-luc cells to the PBMC well plate from step 11.2 above, and add 1.25 × 10 cells to each well. 4 cells were added.

[0291] 11.4 Dilutions of CD3-PDL1-MSLN trispecific antibodies with different structures The structures of the trispecific antibodies 5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244 of this example are shown in Figures 17A and 17B, of which: The structures of 5Y3-168 and 5Y3-244 refer to the structure of the trispecific antibody in Example 3, 3.1.

[0292] Both 5Y3-158 and 5Y3-194 consist 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 in which a humanized CD3 heavy chain variable region and the C-terminus of IgG1 variant 1 (SEQ ID NO: 45) are linked to a humanized MSLN single domain antibody by (GGGGS)3, in that order, and 3. a heavy chain 2 in which a PDL1 single domain antibody is linked to the N-terminus of IgG1 variant 2 (SEQ ID NO: 47) by (GGGGS)1, and then the C-terminus of IgG1 variant 2 is linked to the humanized MSLN single domain antibody by (GGGGS)3. The sequences of 5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244 are shown in Table 18.

[0293] [Table 18]

[0294] Different CD3-PDL1-MSLN trispecific antibodies (5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244) were gradient diluted in medium (RPMI-1640, Gibco 61870036), and 50 μL of the diluted antibodies was added to the PBMCs obtained in step 11.3 and cells containing MC38-luc, CT26-luc, HCC1806-luc, and H2052-luc, respectively.

[0295] 11.5 Incubation The cells were incubated in an incubator at 37°C and 5% CO2 for 2 days.

[0296] 11.6 Reading the results The well plate was removed, 50 μL of luciferase substrate was added, loaded, the results were read, and the data was analyzed.

[0297] The experimental results are shown in Figures 18A to 18E. CD3-PDL1-MSLN trispecific antibodies with different structures (5Y3-158, 5Y3-168, 5Y3-194, and 5Y3-244) were able to exert T cell-mediated tumor cell killing (TDCC) effects against MSLN-expressing human tumor cells HCC1806. Among them, 5Y3-168 had a significantly better killing effect than 5Y3-194, and 5Y3-244 had a significantly better killing effect than 5Y3-158. This indicates that when the sequences are the same, the tumor killing effect is better when there is one MSLN antibody at the N-terminus and one at the C-terminus of the Fc.

[0298] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various detailed changes and modifications may be made thereto in light of all the teachings disclosed, and all of these changes are within the scope of protection of the present invention. All parts of the present invention are determined by the following claims and their equivalents. [Brief explanation of the drawings]

[0299] [Figure 1A] Figures 1A to 1C show the blocking activity of the 4A7 nanobody of Example 2 against the binding of human PD-L1 to human PD-1 (A), mouse PD-L1 to mouse PD-1 (B), and monkey PD-L1 to monkey PD-1 (C), respectively. [Figure 1B] Figures 1A to 1C show the blocking activity of the 4A7 nanobody of Example 2 against the binding of human PD-L1 to human PD-1 (A), mouse PD-L1 to mouse PD-1 (B), and monkey PD-L1 to monkey PD-1 (C), respectively. [Figure 1C] Figures 1A to 1C show the blocking activity of the 4A7 nanobody of Example 2 against the binding of human PD-L1 to human PD-1 (A), mouse PD-L1 to mouse PD-1 (B), and monkey PD-L1 to monkey PD-1 (C), respectively. [Figure 2] Figure 2 shows the measurement of the binding activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 to human PD-L1 cells in Example 2. [Figure 3] Figure 3 shows the measurement of the binding activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 to monkey PD-L1 cells in Example 2. [Figure 4] FIG. 4 shows the measurement of the blocking activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 against human PD-L1 and human PD-1 in Example 2. [Figure 5] FIG. 5 shows the measurement of the blocking activity of Benchmark-ATE, 7B11, and 7B11_huGS_HM4 on the PD-1 / PD-L1 signaling pathway of T cells in Example 2. [Figure 6] Figure 6 shows the results of measuring cytokine production by T cell activation with Benchmark-ATE, 7B11, and 7B11_huGS_HM4 in Example 2, where Figure 6a shows the results of measuring IL2 production by T cell activation with Benchmark-ATE, 7B11, and 7B11_huGS_HM4, and Figure 6b shows the results of measuring IFNγ production by T cell activation with Benchmark-ATE, 7B11, and 7B11_huGS_HM4. [Figure 7] FIG. 7 shows a schematic diagram of the structure of the CD3-PDL1-MSLN trispecific antibody constructed in Example 3. [Figure 8A] 8A to 8D show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to human MC38 / MSLN cells, respectively. [Figure 8B]8A to 8D show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to human MC38 / MSLN cells, respectively. [Figure 8C] 8A to 8D show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to human MC38 / MSLN cells, respectively. [Figure 8D] 8A to 8D show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to human MC38 / MSLN cells, respectively. [Figure 9A] 9A to 9C show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to CHO-hPDL1 cells, respectively. [Figure 9B] 9A to 9C show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to CHO-hPDL1 cells, respectively. [Figure 9C] 9A to 9C show the binding activity of the CD3-PDL1-MSLN trispecific antibody of Example 4 to CHO-hPDL1 cells, respectively. [Figure 10A] Figures 10A to 10C show the blocking activity of the CD3-PDL1-MSLN trispecific antibody of Example 5 against the binding of human PD1 and human PD-L1, respectively. [Figure 10B] Figures 10A to 10C show the blocking activity of the CD3-PDL1-MSLN trispecific antibody of Example 5 against the binding of human PD1 and human PD-L1, respectively. [Figure 10C] Figures 10A to 10C show the blocking activity of the CD3-PDL1-MSLN trispecific antibody of Example 5 against the binding of human PD1 and human PD-L1, respectively. [Figure 11A] 11A to 11C show the activation status of Jurkat-NFAT-luc cells in the tumor-added group by the CD3-PDL1-MSLN trispecific antibody of Example 7, respectively. [Figure 11B] 11A to 11C show the activation status of Jurkat-NFAT-luc cells in the tumor-added group by the CD3-PDL1-MSLN trispecific antibody of Example 7, respectively. [Figure 11C] 11A to 11C show the activation status of Jurkat-NFAT-luc cells in the tumor-added group by the CD3-PDL1-MSLN trispecific antibody of Example 7, respectively. [Figure 12A] 12A to 12C respectively show the activation status of Jurkat-NFAT-luc cells in the tumor-free group by the CD3-PDL1-MSLN trispecific antibody of Example 7. [Figure 12B] 12A to 12C respectively show the activation status of Jurkat-NFAT-luc cells in the tumor-free group by the CD3-PDL1-MSLN trispecific antibody of Example 7. [Figure 12C] 12A to 12C respectively show the activation status of Jurkat-NFAT-luc cells in the tumor-free group by the CD3-PDL1-MSLN trispecific antibody of Example 7. [Figure 13A] 13A to 13C show the TDCC activity of the CD3-PDL1-MSLN trispecific antibody of Example 8 against MSLN-expressing human tumor cells HCC1806, respectively. [Figure 13B] 13A to 13C show the TDCC activity of the CD3-PDL1-MSLN trispecific antibody of Example 8 against MSLN-expressing human tumor cells HCC1806, respectively. [Figure 13C] 13A to 13C show the TDCC activity of the CD3-PDL1-MSLN trispecific antibody of Example 8 against MSLN-expressing human tumor cells HCC1806, respectively. [Figure 14A] 14A-14F show the results of measuring the level of IL-2 release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 14B] 14A-14F show the results of measuring the level of IL-2 release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 14C] 14A-14F show the results of measuring the level of IL-2 release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 14D] 14A-14F show the results of measuring the level of IL-2 release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 14E] 14A-14F show the results of measuring the level of IL-2 release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 14F] 14A-14F show the results of measuring the level of IL-2 release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 15A] 15A-15F show the results of measuring the level of INF-γ release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 15B] 15A-15F show the results of measuring the level of INF-γ release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 15C] 15A-15F show the results of measuring the level of INF-γ release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 15D] 15A-15F show the results of measuring the level of INF-γ release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 15E] 15A-15F show the results of measuring the level of INF-γ release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 15F] 15A-15F show the results of measuring the level of INF-γ release mediated by the CD3-PDL1-MSLN trispecific antibody of Example 9, respectively. [Figure 16A] 16A and 16B show the in vivo killing activity and safety of the 5Y3-16 antibody of Example 10, respectively. [Figure 16B] 16A and 16B show the in vivo killing activity and safety of the 5Y3-16 antibody of Example 10, respectively. [Figure 17A]17A and 17B show schematic diagrams of the structure of the CD3-PDL1-MSLN trispecific antibody constructed in Example 11, respectively. [Figure 17B] 17A and 17B show schematic diagrams of the structure of the CD3-PDL1-MSLN trispecific antibody constructed in Example 11, respectively. [Figure 18A] 18A to 18E show the TDCC activity of the CD3-PDL1-MSLN trispecific antibodies of Example 11 with different structures against tumor cells, respectively. [Figure 18B] 18A to 18E show the TDCC activity of the CD3-PDL1-MSLN trispecific antibodies of Example 11 with different structures against tumor cells, respectively. [Figure 18C] 18A to 18E show the TDCC activity of the CD3-PDL1-MSLN trispecific antibodies of Example 11 with different structures against tumor cells, respectively. [Figure 18D] 18A to 18E show the TDCC activity of the CD3-PDL1-MSLN trispecific antibodies of Example 11 with different structures against tumor cells, respectively. [Figure 18E] 18A to 18E show the TDCC activity of the CD3-PDL1-MSLN trispecific antibodies of Example 11 with different structures against tumor cells, respectively.

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 set forth in any one of SEQ ID NOs: 1 to 8 or a variant thereof; provided that 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, a humanized single domain antibody or antigen-binding fragment thereof.

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

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

4. The single domain antibody or antigen-binding fragment thereof comprises a VHH sequence as set forth in SEQ ID NO: 10, 67 or a variant thereof; provided that the variant 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 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.

5. the single domain antibody or antigen-binding fragment thereof is humanized; 5. The single domain antibody or antigen-binding fragment thereof of any one of claims 2 to 4, wherein the single domain antibody or antigen-binding fragment thereof preferably comprises a heavy chain framework region of a human immunoglobulin, optionally comprising backmutations of human residues to camelid residues.

6. the single domain antibody or antigen-binding fragment thereof comprises a VHH sequence set forth in any one of SEQ ID NOs: 24 to 39, 81 to 88 or a variant thereof; provided that the variant 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 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 humanized single domain antibody or antigen-binding fragment thereof according to claim 1, and / or a 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 linked 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 by a peptide linker; Preferably, the immunoglobulin Fc domain is an IgG Fc domain (e.g., an IgG1 Fc domain); Preferably, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 48, Preferably, the peptide linker is a peptide linker containing one or more glycines and / or one or more serines, for example, (G ４ S) ｎ and n is 1, 2, 3 or 4, e.g., 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, said multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

10. a first antigen-binding domain targeting CD3, a second antigen-binding domain targeting MSLN, and a third antigen-binding domain targeting PD-L1, (i) the first antigen-binding domain comprises a VH and a VL, wherein the VH comprises an HCDR1 set forth in SEQ ID NO: 55, an HCDR2 set forth in SEQ ID NO: 56, and an HCDR3 set forth in any one of SEQ ID NOs: 57 to 59, 65, and / or the VL comprises an LCDR1 set forth in SEQ ID NO: 60, an LCDR2 set forth in SEQ ID NO: 61, and an LCDR3 set forth in SEQ ID NO: 62, preferably wherein the VH comprises a sequence set forth in any one of SEQ ID NOs: 40 to 42, 64, and preferably wherein the VL comprises a sequence set forth in SEQ ID NO: 43; (ii) the second antigen-binding domain comprises a VHH, wherein the VHH comprises CDR1, CDR2 and CDR3 contained in the VHH set forth in any one of SEQ ID NOs: 1 to 9, preferably the VHH comprises CDR1 set forth in SEQ ID NO: 52, CDR2 set forth in SEQ ID NO: 53 and CDR3 set forth in SEQ ID NO: 54, and 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) A multispecific antibody, wherein the third antigen-binding domain comprises a 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 a Fab, and the second antigen-binding domain and the third antigen-binding domain are VHHs; Preferably, said multispecific antibody further comprises an Fc domain, said Fc domain comprising a first Fc monomer and a second Fc monomer, with the proviso that: the N-terminus of said first Fc monomer is optionally linked by a linker to said first antigen binding domain (e.g., its heavy chain CH1 domain) and its C-terminus is optionally linked by a linker to one of said second antigen binding domains; a multispecific antibody, wherein the N-terminus of said second Fc monomer is optionally linked by a linker to another of said second antigen-binding domains, and its C-terminus is optionally linked by a linker to said third antigen-binding domain.

12. The multispecific antibody of claim 11 , wherein the tumor-associated antigen comprises MSLN.

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

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

15. the linker is selected from peptide linkers containing one or more glycines and / or one or more serines; Preferably, the peptide linker is (G ４ S) ｎ wherein n is 1, 2, 3 or 4, for example comprising a sequence as set forth in any one of SEQ ID NOs: 49 to 51.

16. The multispecific antibody comprises: (i) a first peptide chain comprising a VL of the first antigen-binding domain and a light chain constant region (CL), preferably wherein the CL is a kappa light chain constant region; (ii) the first antigen-binding domain comprises a VH, a CH1, a first Fc monomer, and the second antigen-binding domain, preferably the first Fc monomer is an IgG, preferably the first Fc monomer comprises a hinge region, a CH2, and a CH3, and preferably the second antigen-binding domain comprises a linker (e.g., (G ４ S) ｎ a second peptide chain linked to the C-terminus of the first Fc monomer by a flexible peptide comprising: and (iii) 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, and preferably the second antigen-binding domain is linked to a linker (e.g., (G ４ S) ｎ and preferably, the third antigen-binding domain is linked to the N-terminus of the second Fc monomer by a linker (e.g., a flexible peptide comprising (G ４ S) ｎ a third peptide chain linked to the C-terminus of the second Fc monomer by a flexible peptide comprising: The multispecific antibody of any one of claims 11 to 15, wherein 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 first Fc monomer of said second peptide chain and the second Fc monomer of said third peptide chain comprise a modification that promotes dimerization; Preferably, the modification comprises an amino acid substitution in the CH3 domain of the Fc domain; Preferably, the modifications comprise a "knob" modification on one of the two Fc monomers and a "hole" modification on the other of the two Fc monomers, forming a "knob-into-hole" modification; Preferably, the two Fc monomers comprise the amino acid sequences set forth in SEQ ID NOs: 46 and 47, respectively; Preferably, the first Fc monomer of said second peptide chain comprises the amino acid sequence set forth in SEQ ID NO: 46, and preferably, said second peptide chain comprises the heavy chain constant region sequence set forth in SEQ ID NO: 45; 17. The multispecific antibody of claim 16, wherein the second Fc monomer of the third peptide chain preferably comprises the amino acid sequence set forth in SEQ ID NO:

47.

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

43.

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

9.

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

21. The multispecific antibody comprises: (i) a first peptide chain having a structure represented by [VL]-[CL]; (ii) a second peptide chain having the structure represented by [VH]-[CH]-[L1]-[VHH1]; and (iii) a third peptide chain having the structure represented by [VHH1]-[L2]-[Fc monomer]-[L3]-[VHH2]; However, one of the following items must be selected: (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, 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; and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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: 47, and the VHH2 comprises the sequence shown in SEQ ID NO: 38, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, the VHH1 comprises the sequence shown in SEQ ID NO: 8, the Fc monomer comprises the sequence shown in SEQ ID NO: 47, the VHH2 comprises the sequence shown in SEQ ID NO: 31, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, the VHH1 comprises the sequence shown in SEQ ID NO: 9, the Fc monomer comprises the sequence shown in SEQ ID NO: 47, the VHH2 comprises the sequence shown in SEQ ID NO: 10, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and 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, 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, and the L1, L2, and L3 are peptide linkers, preferably each independently a peptide linker comprising one or more glycines and / or one or more serines (e.g., (G ４ S) ｎ and wherein L1 is SEQ ID NO: 51, L2 is SEQ ID NO: 49 and L3 is SEQ ID NO:

51.

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

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

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

25. 25. A method for producing a single domain antibody or antigen-binding fragment thereof, polypeptide construct or multispecific antibody, comprising culturing a host cell according to claim 24 under conditions allowing expression of the protein, and harvesting the single domain antibody or antigen-binding fragment thereof, polypeptide construct or multispecific antibody from the culture of the cultured host cells.

26. A humanized single domain antibody or antigen-binding fragment thereof according to claim 1, a single domain antibody or antigen-binding fragment thereof according to any one of claims 2 to 7, a polypeptide construct according to claim 8, or a multispecific antibody according to any one of claims 9 to 21, and a binding moiety linked thereto, Preferably, the binding moiety 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 drug, a cytokine, a toxin, a radionuclide, an immune agonist, an immunosuppressant, and other active substances that inhibit tumor cell proliferation or promote tumor cell apoptosis or necrosis).

27. 27. A pharmaceutical composition 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, the isolated nucleic acid molecule according to claim 22, the vector according to claim 23, the host cell according to claim 24, or the complex according to claim 26, and a pharmaceutically acceptable carrier and / or excipient.

28. 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 to 7, the polypeptide construct according to claim 8, the multispecific antibody according to any one of claims 9 to 21, the isolated nucleic acid molecule according to claim 22, the vector according to claim 23, the host cell according to claim 24, the complex according to claim 26 or the pharmaceutical composition according to claim 27 in the manufacture of a medicament for the prevention and / or treatment and / or new adjunctive treatment of a disease.

29. 28. A method for the prevention and / or treatment and / or new adjunctive therapy and / or adjunctive therapy of 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 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, the multispecific antibody according to any one of claims 9 to 21, the isolated nucleic acid molecule according to claim 22, the vector according to claim 23, the host cell according to claim 24, the complex according to claim 26, or the pharmaceutical composition according to claim 27.

30. 30. The use of claim 28 or the method of claim 29, wherein the disease is a tumor, preferably the tumor is a solid tumor or a hematological tumor, more preferably the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer, and the hematological tumor comprises acute myeloid leukemia.

31. 28. A method for measuring the presence or level of PD-L1, MSLN and / or CD3 in a sample, the method 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 to 7, the polypeptide construct of claim 8, the multispecific antibody of any one of claims 9 to 21, the isolated nucleic acid molecule of claim 22, the vector of claim 23, the host cell of claim 24, the complex of claim 26, or the pharmaceutical composition of claim 27 under conditions allowing the formation of an antibody-antigen immune complex, and detecting the formation of said complex.

32. 28. A method comprising using a humanized single domain antibody or antigen-binding fragment thereof according to claim 1, a single domain antibody or antigen-binding fragment thereof according to any one of claims 2 to 7, a polypeptide construct according to claim 8, a multispecific antibody according to any one of claims 9 to 21, an isolated nucleic acid molecule according to claim 22, a vector according to claim 23, a host cell according to claim 24, a complex according to claim 26, or a pharmaceutical composition according to claim 27, Preferably, the method comprises measuring the presence or levels of PD-L1, MSLN and / or CD3 in a sample from a subject by the method of claim 31, thereby diagnosing or differentially diagnosing 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, colon cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer, and the blood tumor includes acute myeloid leukemia. A method for diagnosing or differentially diagnosing a disease associated with PD-L1, MSLN and / or CD3.

33. 28. 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 to 7, the polypeptide construct according to claim 8, the multispecific antibody according to any one of claims 9 to 21, the isolated nucleic acid molecule according to claim 22, the vector according to claim 23, the host cell according to claim 24, the complex according to claim 26, or the pharmaceutical composition according to claim 27.

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