Single-domain antibody specifically binding to CDCP1 and use thereof

Abstract

A single-domain antibody specifically binding to CDCP1 or an antigen-binding fragment thereof, a polypeptide construct and a conjugate containing the single-domain antibody or the antigen-binding fragment thereof, a nucleic acid molecule encoding the single-domain antibody or the antigen-binding fragment thereof and a host cell containing same, and a related use.

Description

Single domain antibody specifically binding to CDCP1 and its application Technical Field

[0001] The present invention relates to a single-domain antibody or an antigen-binding fragment thereof that specifically binds to CDCP1, a polypeptide construct and a conjugate containing the single-domain antibody or the antigen-binding fragment thereof, a nucleic acid molecule encoding the single-domain antibody or the antigen-binding fragment thereof, a host cell containing the same, and related uses. Background Art

[0002] CUB-domain containing protein 1 (CDCP1) is a type I transmembrane protein consisting of 836 amino acids. CDCP1 encodes a 135 kDa protein with three CUB domains in its extracellular region. Serine proteases cleave the CUB1 and CUB2 domains at amino acid positions 368 / 369 or 369 / 370 to produce a 70 kDa fragment (cleaved CDCP1). Overexpression and proteolytic activation of CDCP1 contribute to loss of cell adhesion, increased migration, and poor prognosis / survival in cancer patients. The N-terminal end of the extracellular domain contains 14 N-glycosylation sites. N-glycosylation has been observed to be a prerequisite for CDCP1 protein stability and plasma membrane localization, and the degree of glycosylation is correlated with the metastatic potential of cancer cells. The cytoplasmic domain of CDCP1 contains five conserved tyrosine residues, which serve as phosphorylation sites for Src family kinases (SFKs, such as Src, Fyn, and Yes).

[0003] CDCP1 is highly expressed in tumor cells such as colorectal cancer, renal cancer, lung cancer, pancreatic cancer, breast cancer, and ovarian cancer. It increases the migration and invasiveness of cancer cells as well as cell survival by interacting with important signal transduction pathways in tumor invasiveness. Overexpression of CDCP1 is associated with poor tumor prognosis.

[0004] Patent US20080008719A1 describes an anti-hCDCP1 monoclonal antibody (clone name: 25A11) and methods for screening, diagnosing, and treating prostate cancer using the antibody. It discloses that an ADC (antibody-drug-conjugate) antibody conjugated to 25A11 exhibits cytotoxic activity against PC-3 cancer cell lines in vitro, and that intravenous administration of the ADC-conjugate antibody exhibits significant tumor growth inhibitory activity. Patent US20200181281A1 describes multiple anti-CDCP1 antibodies, disclosing that an anti-hCDCP1 antibody ADC-conjugated with MMAE (monomethyl auristatin E) exhibits cytotoxic activity against human colon cancer cell lines or breast cancer cell lines in vitro, and exhibits anti-tumor activity in mouse xenograft models transplanted with human colon cancer cell lines or breast cancer cell lines. Patent CN115176013A describes multiple anti-CDCP1 antibodies that bind to human CDCP1 and have low binding to human CD34-positive cells. It discloses that anti-hCDCP1 antibodies ADC-conjugated with pyrrolobenzodiazepine (PBD) exhibit cytotoxic activity against different tumor cell lines in vitro and anti-tumor activity in mouse transplanted tumor models transplanted with human colon cancer cell lines or prostate cancer cell lines.

[0005] Conventional antibodies have a molecular weight of approximately 150 kDa. Single-domain antibodies and nanobodies derived from alpacas, when fused to human Fc, have a molecular weight of only about 75 kDa, half that of conventional antibodies. This allows them to penetrate deeper into tumors, maximizing the chances of antibody drugs binding to tumor cells and resulting in better diagnostic and therapeutic outcomes.

[0006] Therefore, providing a single-domain antibody against CDCP1 will have better prospects for diagnosing and treating tumors.

[0007] Summary of the Invention

[0008] After extensive research, the inventors of this application have screened and obtained a series of anti-CDCP1 single-domain antibodies and polypeptide constructs. These single-domain antibodies or polypeptide constructs have high binding activity to CDCP1. In particular, the single-domain antibodies or polypeptide constructs of the present invention can increase the endocytic activity of tumor cells and have strong anti-tumor cell killing activity. Furthermore, humanized single-domain antibodies or polypeptide constructs prepared from these single-domain antibodies still possess these outstanding activities. Furthermore, these single-domain antibodies or polypeptide constructs also have the advantages of low molecular weight and easy production.

[0009] Based on this, the present application also provides a conjugate containing the single-domain antibody or its antigen-binding fragment, a nucleic acid molecule encoding the single-domain antibody or its antigen-binding fragment, a host cell containing the same, and related uses.

[0010] Single domain antibodies and antigen-binding fragments thereof

[0011] Therefore, in one aspect, the present application provides a single domain antibody or an antigen-binding fragment thereof that can specifically bind to CUB domain-containing protein 1 (CDCP1), wherein the single domain antibody or the antigen-binding fragment thereof comprises:

[0012] (a) CDR1 having: a sequence as shown in any one of SEQ ID NOs: 1, 4, 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 1, 4, 7;

[0013] (b) CDR2 having: a sequence as shown in any one of SEQ ID NOs: 2, 5, 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 2, 5, 8; and

[0014] (c) CDR3, which has: a sequence as shown in any one of SEQ ID NOs: 3, 6, and 9, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence shown in any one of SEQ ID NOs: 3, 6, and 9.

[0015] In certain embodiments, the substitutions are conservative substitutions.

[0016] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO: 1; a CDR2 as shown in SEQ ID NO: 2; and a CDR3 as shown in SEQ ID NO: 3.

[0017] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO:4; a CDR2 as shown in SEQ ID NO:5; and a CDR3 as shown in SEQ ID NO:6.

[0018] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: a CDR1 as set forth in SEQ ID NO:7; a CDR2 as set forth in SEQ ID NO:8; and a CDR3 as set forth in SEQ ID NO:9.

[0019] On the other hand, the present application provides a single-domain antibody or an antigen-binding fragment thereof that specifically binds to CUB domain-containing protein 1 (CDCP1), wherein the single-domain antibody or the antigen-binding fragment thereof comprises: three CDRs contained in the heavy chain variable region (VHH) as shown in any one of SEQ ID NOs: 10-27.

[0020] In certain embodiments, the three CDRs contained in the VHH are defined by the Kabat, IMGT, or Chothia numbering systems.

[0021] In certain embodiments, the single-domain antibody or antigen-binding fragment thereof as described above comprises an amino acid sequence selected from the group consisting of:

[0022] (i) a sequence as shown in any one of SEQ ID NOs: 10-27;

[0023] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 10-27; or

[0024] (iii) a sequence having 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%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 10-27.

[0025] In certain embodiments, the substitutions are conservative substitutions.

[0026] Single domain antibodies and antigen-binding fragments thereof

[0027] In another aspect, the present application provides a polypeptide construct that specifically binds to CDCP1, comprising the single-domain antibody or antigen-binding fragment thereof as described above, and an immunoglobulin Fc domain.

[0028] In certain embodiments, the immunoglobulin Fc domain is optionally linked to the N-terminus and / or C-terminus (eg, C-terminus) of the single-domain antibody or antigen-binding fragment thereof via a peptide linker.

[0029] In certain embodiments, the immunoglobulin Fc domain is an IgG Fc domain, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0030] In certain embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 28 or 29, or a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared thereto (e.g., a sequence having up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, a sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof).

[0031] In certain embodiments, the polypeptide construct has a sequence as shown in any one of SEQ ID NOs: 30-47.

[0032] Isolated nucleic acid molecules

[0033] In another aspect, the present application also provides an isolated nucleic acid molecule encoding the single domain antibody or antigen-binding fragment thereof as described above, or the polypeptide construct as described above.

[0034] carrier

[0035] In another aspect, the present application also provides a vector comprising the nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0036] host cells

[0037] On the other hand, the application also provides a host cell comprising a nucleic acid molecule or a vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0038] Preparation method

[0039] The single-domain antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, a DNA molecule encoding the single-domain antibody of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, and then transformed / transfected into a host cell. The transformed / transfected host cell is then cultured under specific conditions to express the single-domain antibody of the present invention.

[0040] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact nanobody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). Those skilled in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0041] On the other hand, the present application also provides a method for preparing the single-domain antibody or its antigen-binding fragment as described above or the polypeptide construct as described above, which comprises culturing the host cell as described above under conditions that allow protein expression, and recovering the single-domain antibody or its antigen-binding fragment or recovering the polypeptide construct as described above from the cultured host cell culture.

[0042] Bispecific or multispecific antibodies

[0043] In another aspect, the present application also provides a bispecific or multispecific antibody comprising the single domain antibody or antigen-binding fragment thereof as described above or the polypeptide construct as described above.

[0044] In certain embodiments, the bispecific or multispecific antibody specifically binds CDCP1 and additionally specifically binds one or more other targets.

[0045] In certain embodiments, the bispecific or multispecific antibody further comprises at least one second antibody with a second binding specificity for a second target.

[0046] Conjugate

[0047] In one aspect, the present application also provides a conjugate comprising the single domain antibody or antigen-binding fragment thereof as described above or the polypeptide construct as described above, and a therapeutic agent linked to the single domain antibody or antigen-binding fragment thereof.

[0048] In certain embodiments, the therapeutic agent is selected from a cytotoxic agent.

[0049] In certain embodiments, the therapeutic agent is selected from the group consisting of an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, and any combination thereof.

[0050] In certain embodiments, the conjugate is an antibody-drug conjugate (ADC).

[0051] Pharmaceutical composition

[0052] In one aspect, the present application also provides a pharmaceutical composition comprising a single-domain antibody or an antigen-binding fragment thereof as described above, or a polypeptide construct as described above, or a bispecific or multispecific antibody as described above, or a conjugate as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0053] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0054] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0055] In certain embodiments, the single domain antibody or antigen-binding fragment thereof, bispecific or multispecific antibody or conjugate and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.

[0056] Reagent test kit

[0057] In another aspect, the present application also provides a kit comprising the single domain antibody or antigen-binding fragment thereof as described above or the polypeptide construct as described above.

[0058] In certain embodiments, the single domain antibody or antigen-binding fragment thereof or the polypeptide construct carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0059] In certain embodiments, the kit further comprises a second antibody that specifically recognizes the single domain antibody or antigen-binding fragment thereof as described above or the polypeptide construct as described above.

[0060] In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.

[0061] Chimeric antigen receptor

[0062] In another aspect, the present application also provides a chimeric antigen receptor, which comprises the antigen-binding domain of the single-domain antibody or antigen-binding fragment thereof as described above, or the polypeptide construct as described above.

[0063] In certain embodiments, the antigen binding domain is expressed by immune effector cells (e.g., T cells).

[0064] Isolated nucleic acid molecules

[0065] In another aspect, the present application also provides an isolated nucleic acid molecule encoding the chimeric antigen receptor as described above.

[0066] carrier

[0067] In another aspect, the present application also provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is used to prepare chimeric antigen receptor T cells.

[0068] host cells

[0069] On the other hand, the application also provides a host cell comprising an isolated nucleic acid molecule as described above or a vector as described above. In certain embodiments, the host cell is an immune effector cell (e.g., T cell or NK cell). In certain embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0070] use

[0071] On the other hand, the present application also provides the use of the single-domain antibody or antigen-binding fragment thereof as described above, or the polypeptide construct as described above, or the bispecific or multispecific antibody as described above, or the conjugate as described above, or the pharmaceutical composition as described above, or the kit as described above, or the chimeric antigen receptor as described above in the preparation of a medicament for preventing and / or treating tumors in a subject.

[0072] In certain embodiments, the tumor expresses CDCP1.

[0073] In certain embodiments, the medicament is used to inhibit the growth of tumor cells expressing CDCP1 and / or kill the tumor cells.

[0074] In certain embodiments, the medicament further comprises an additional pharmaceutically active agent.

[0075] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0076] In certain embodiments, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

[0077] In certain embodiments, the subject is a mammal, such as a human.

[0078] In another aspect, the present application also provides use of the aforementioned single-domain antibody or antigen-binding fragment thereof or the aforementioned polypeptide construct in preparing a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting CDCP1.

[0079] In certain embodiments, the antibody or antigen-binding fragment thereof is detectably labeled.

[0080] In certain embodiments, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

[0081] method

[0082] In another aspect, the present application also provides a method for detecting the presence or amount of CDCP1 (eg, human CDCP1) in a sample, comprising the following steps:

[0083] (1) contacting the sample with the single domain antibody or antigen-binding fragment thereof as described above or the polypeptide construct as described above;

[0084] (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof or the polypeptide construct and CDCP1 or detecting the amount of the complex.

[0085] In certain embodiments, the antibody or antigen-binding fragment thereof or the polypeptide construct is detectably labeled.

[0086] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0087] On the other hand, the present application also provides a method for reducing the expression level of CDCP1 on the cell surface, comprising contacting the cell with the single-domain antibody or antigen-binding fragment thereof as described above, or the polypeptide construct as described above, or the bispecific or multispecific antibody as described above, or the conjugate as described above, or the pharmaceutical composition as described above, or the kit as described above, so that the expression level of CDCP1 on the cell surface is reduced; wherein, the cell expresses CDCP1 on its surface.

[0088] In certain embodiments, the cell is a tumor cell expressing CDCP1.

[0089] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0090] On the other hand, the present application also provides a method for preventing and / or treating tumors, which comprises administering to a subject in need thereof an effective amount of a single-domain antibody or an antigen-binding fragment thereof as described above, or a polypeptide construct as described above, or a bispecific or multispecific antibody as described above, or a conjugate as described above, or a pharmaceutical composition as described above, or a kit as described above, or a chimeric antigen receptor as described above.

[0091] In certain embodiments, the tumor expresses CDCP1.

[0092] In certain embodiments, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

[0093] In certain embodiments, the subject is a mammal, such as a human.

[0094] In another aspect, the present application also provides a method for detecting whether a tumor can be treated by anti-tumor therapy targeting CDCP1, the method comprising administering an effective amount of the single domain antibody or antigen-binding fragment thereof or the polypeptide construct as described above to a subject in need thereof.

[0095] In certain embodiments, the single domain antibody or antigen-binding fragment thereof is detectably labeled.

[0096] In certain embodiments, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

[0097] Definition of terms

[0098] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0099] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0100] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0101] As used herein, the term "single domain antibody" has a meaning generally understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A nanobody can be truncated at the N-terminus or C-terminus so that it comprises only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Single domain antibodies are also referred to as nanobodies, and the two are used interchangeably.

[0102] As used herein, the term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind to the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of the single-domain antibody may be truncated at the N-terminus or C-terminus compared to the full-length single-domain antibody so that it comprises only part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

[0103] Antigen-binding fragments of single-domain antibodies can be obtained from a given single-domain antibody (such as the Nanobodies provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of single-domain antibodies can be screened for specificity in the same manner as for intact Nanobodies.

[0104] Herein, unless the context clearly indicates otherwise, when referring to the term "single domain antibody", it includes not only intact single domain antibodies, but also antigen-binding fragments of single domain antibodies.

[0105] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to 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), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In this context, the CDRs of a Nanobody are preferably determined by the Kabat, Chothia and / or IMGT numbering systems.

[0106] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0107] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0108] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0109] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0110] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D(See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0111] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0112] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0113] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0114] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0115] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0116] Advantageous Effects of the Invention

[0117] The anti-CDCP1 single-domain antibodies or polypeptide constructs provided herein have high binding activity to CDCP1. Specifically, the single-domain antibodies or polypeptide constructs of the present invention can increase the endocytic activity of tumor cells and exhibit strong anti-tumor cell cytotoxicity. Furthermore, humanized single-domain antibodies or polypeptide constructs prepared from these single-domain antibodies or polypeptide constructs also exhibit these outstanding activities. Furthermore, these single-domain antibodies or polypeptide constructs have the advantages of low molecular weight and ease of production.

[0118] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] FIG1 shows the binding ability of 18 nanobody constructs of the present application to human CDCP1 antigen detected by ELISA.

[0120] FIG2 shows the binding ability of 8 nanobody constructs of the present application to human CDCP1 antigen detected by ELISA.

[0121] Figure 3 shows the binding activity of the Nanobody constructs of the present application to HCT116 cells (Figure 3A) and SW480 cells (Figure 3B) in FACS experiments.

[0122] Figure 4 shows the binding activity of the Nanobody constructs of the present application to PL45 cells in a FACS experiment.

[0123] FIG5 shows the results of endocytosis experiments in HCT116 cells ( FIG5A ) and SW480 cells ( FIG5B ).

[0124] FIG6 shows the killing activity of the nanobody construct of the present application on HCT116 cells.

[0125] FIG7A and FIG7B show the binding activity of the humanized Nanobody construct of the present application to the human CDCP1 antigen detected by ELISA.

[0126] Figure 8 shows the binding activity of the humanized Nanobody constructs of the present application to HCT116 cells in a FACS experiment.

[0127] FIG9 shows the results of an endocytosis experiment in HCT116 cells.

[0128] FIG10 shows the killing activity of the humanized nanobody constructs of the present application on HCT116 cells.

[0129] FIG11 shows the binding activity of the humanized Nanobody construct of the present application to human CDCP1 antigen detected by ELISA.

[0130] Figure 12 shows the binding activity of the humanized Nanobody constructs of the present application to HCT116 cells in a FACS experiment.

[0131] FIG13 shows the results of an endocytosis experiment in HCT116 cells.

[0132] Figure 14 shows the killing activity of the humanized nanobody constructs of the present application on HCT116 cells.

[0133] FIG15 shows the binding ability of the humanized nanobody construct of the present application to CDCP1 antigens of different species detected by ELISA.

[0134] Figure 16 shows the binding activity of the humanized Nanobody constructs of the present application to HCT116 cells in a FACS experiment.

[0135] Figure 17 shows the binding activity of the humanized Nanobody constructs of the present application to SW480 cells in a FACS experiment.

[0136] Figure 18 shows the binding activity of the humanized Nanobody constructs of the present application to PL45 cells in a FACS experiment.

[0137] Figure 19 shows the binding activity of the humanized Nanobody constructs of the present application to HT-29 cells in a FACS experiment.

[0138] Figure 20 shows the binding activity of the humanized Nanobody constructs of the present application to LNCaP cells in a FACS experiment.

[0139] Figure 21 shows the killing activity of the humanized Nanobody constructs of the present application on HCT116 cells.

[0140] Sequence information

[0141] A description of the sequences involved in this application is provided in the table below.

[0142] Table 1: Sequence information DETAILED DESCRIPTION

[0143] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0144] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0145] Example 1. Production of CDCP1-specific antibodies

[0146] 1.1. Production of CDCP1-specific antibody 25A11 (MHAB10-PC1)

[0147] The variable regions of the 25A11 (MHAB10-PC1) antibody are derived from patent US20080008719A1. The VH and VL sequences are integrated into the CH and CL sequences of human IgG1-kappa, respectively. The heavy chain sequence of the integrated antibody is shown in SEQ ID NO:48, and the light chain sequence is shown in SEQ ID NO:49. After transient expression in CHO cells, the antibody was purified using Protein A and dissolved in PBS. The antibody solution had a purity (SEC, 280 nm) of 98.887% and an endotoxin level (EU / mg) of <1. After aliquoting, store frozen at -80°C. This antibody served as a positive control antibody in subsequent screening experiments.

[0148] 1.2. Production of CDCP1-specific antibody h14A043 (MHAB10-PC2)

[0149] The variable region of the h14A043 (MHAB10-PC2) antibody is derived from patent US20230050380A1. The VH and VL sequences are integrated into the CH and CL sequences of Human IgG1-Kappa, respectively. The sequence of the heavy chain of the integrated antibody is shown in SEQ ID NO: 50, and the sequence of the light chain of the integrated antibody is shown in SEQ ID NO: 51. After transient expression in CHO, the antibody was purified by Protein A and dissolved in PBS. The purity of the antibody solution (SEC, 280nm) was 79.69%. This antibody was used as a positive control antibody for the subsequent anti-human CDCP1 humanized nanoantibody MHAB10-16-14 activity detection experiment.

[0150] Example 2. Acquisition of anti-human CDCP1 nanobody sequences

[0151] 2.1. Alpaca immunization and serum titer testing

[0152] A blank alpaca was immunized with CDCP1 antigen (Genebank ID: Q9H5V8-1). Before the initial immunization, 10 mL of blood was collected as negative serum. For the initial immunization, a mixture of 0.5 mg of CDCP1 antigen and complete Freund's adjuvant in a 1:1 ratio was injected subcutaneously at multiple sites. Secondary, third, and fourth immunizations were performed at weeks three, five, and seven, respectively, using a mixture of 0.25 mg of CDCP1 antigen and incomplete Freund's adjuvant in a 1:1 ratio. Before the third and fourth immunizations, 10 mL of peripheral blood was collected, serum was isolated, and immune responses were monitored using ELISA. Five days after the fourth immunization, 50 mL of peripheral blood was collected for subsequent nanobody library construction (the decision to perform this blood collection was determined based on the serum immune response).

[0153] Table 2. Immunization information

[0154] 2.2. Screening and identification of specific nanoantibodies

[0155] Peripheral blood was collected from immunized alpacas, RNA was extracted, and cDNA samples were prepared. VHH antibody-encoding genes were cloned by PCR to construct a single-domain antibody phage display library. Randomly selected clones were then analyzed for library capacity and diversity.

[0156] A single-domain antibody phage display library was panned and enriched using the CDCP1 antigen (KACTUS, Cat. No. CDC-HM101). Single clones were selected for Phage-ELISA testing after screening to identify candidate clones with different amino acid sequences in the CDR region. Ultimately, 37 unique sequences were identified. See Table 3 for details.

[0157] 2.3. Unique Sequence Solubility Verification and Antibody Expression

[0158] The plasmids of the 37 positive clones were extracted and transformed into E. coli Rosetta. Expression was induced overnight with 0.4 mM IPTG at 20°C and 200 rpm. The next day, the precipitate was centrifuged and resuspended in PBS. Ultrasonic disruption was performed and the supernatants of the 37 clones were obtained by centrifugation. The binding activity of the supernatants of the 37 clones to human CDCP1 antigen was detected by ELISA (OD 450 as shown in Table 3 below).

[0159] Table 3. OD of 37 clones 450 result

[0160] Sequences with strong binding activity to human CDCP1 were selected and the VHH sequence was integrated into the Fc (LALA) sequence of human IgG1 (SEQ ID NO: 29). Shanghai Baiying Biotechnology Co., Ltd. was commissioned to perform transient CHO expression services to produce MHAB10 candidate antibodies (expression numbers and unique sequences from Nanchang Dajia Technology Co., Ltd. are shown in Table 4 below. Subsequent descriptions of MHAB10 candidate antibodies will use expression numbers MHAB10-1 to 26). After expression, the antibodies were purified using Protein A and dissolved in PBS. The antibody solutions achieved a purity (SEC, 280 nm) of >95% and an endotoxin level (EU / mg) of <1. Aliquots were stored at -80°C.

[0161] Table 4. Expression numbers corresponding to unique sequences

[0162] Example 3. Anti-human CDCP1 nanobody activity detection

[0163] ELISA binding assay

[0164] The binding ability of the candidate MHAB10 antibody (anti-human CDCP1 nanobody Fc fusion protein) to the human CDCP1 antigen was tested using an ELISA method. CDCP1, His Tag (KACTUS, Cat. No. CDC-HM101) was diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plate was coated overnight at 2°C-8°C. Rinse 3 times with PBST, add 1% BSA / PBST to block at room temperature for 1 hour, then rinse 5 times with PBST, add the test antibody (test antibody number is MHAB10, positive control antibody is 25A11, i.e. MHAB10-PC1), the initial concentration is 10μg / mL, and 10-fold serial dilution is performed), incubate for 1 hour, rinse 7 times with PBST, wash away the unbound antibody, add 1:50,000 dilution of Goat Anti-Human IgG Fc (HRP) (Abcam, Cat.No.ab97225), incubate at room temperature for 30 minutes. After washing away the excess secondary antibody, add 1-Step TMUltra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) (100 μL / well) was added and developed for 10 min at room temperature in the dark. The reaction was then terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472). Absorbance was read at 450 nm using a microplate reader, and four-parameter curves were plotted using GraphPad. All experimental results are expressed as mean ± SEM (standard error of the mean). Graphs and data analysis were performed using Prism 6 (GraphPad) software.

[0165] ELISA binding results (Figures 1 and 2) show that the top 10 MHAB10 candidate antibodies with the strongest binding activity to the human CDCP1 antigen are MHAB10-2, 3, 8, 9, 10, 11, 13, 16, 17, and 26. These 10 antibodies all exhibited stronger binding activity to the human CDCP1 antigen than the positive control antibody 25A11 (MHAB10-PC1). These 10 antibodies will be subsequently tested for binding activity by FACS.

[0166] FACS binding assay

[0167] In the FACS experiment, human colorectal cancer HCT116 cells and SW480 cells were cultured using HCT116 complete cell culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No.30-2007 + 10% FBS, Sigma-Aldrich, Cat.no.F8687 + 1% P / S, Gibco, Cat.no.15140-122) and SW480 complete cell culture medium (ATCC-formulated Leibovitz's L-15 Medium, Catalog No.30-2008 + 10% FBS, Sigma-Aldrich, Cat.no.F8687 + 1% P / S, Gibco, Cat.no.15140-122), respectively. Furthermore, to prevent trypsin from cleaving fl-CDCP1, HCT116 and SW480 cells were digested with Versene (Gibco, Cat. no. 15040-066). Binding of the candidate antibodies was then tested using HCT116 and SW480 cells, both of which endogenously express human CDCP1. The antibody was serially diluted (30,000 ng / mL, 7,500 ng / mL, 1,875 ng / mL, 468.8 ng / mL, 117.2 ng / mL, 29.3 ng / mL, 7.3 ng / mL, and 1.8 ng / mL). Binding was detected using 100 μL of FITC anti-human IgG Fc Antibody secondary antibody (Biolegend, Cat. no. 410720). FITC fluorescence intensity (MFI) was measured by flow cytometry. All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0168] Binding curves are shown in Figure 3, with Figure 3A showing the results for HCT116 cells and Figure 3B showing the results for SW480 cells. FACS binding results indicate that the candidate MHAB10 antibodies have similar binding activity to both HCT116 and SW480 cells, with MHAB10-11 and MHAB10-16 showing the strongest binding activity. These two antibodies will be subsequently tested for endocytosis and endocytosis-mediated cell cytotoxicity.

[0169] 3.3. FACS Binding Assay to Detect Binding Activity of Selected Antibodies to Human Cleaved CDCP1

[0170] For FACS experiments, human pancreatic ductal adenocarcinoma PL45 cells were cultured in complete PL45 cell culture medium (ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). Binding of the candidate antibodies was then detected using PL45 cells endogenously expressing human cleaved CDCP1. The test antibodies were serially diluted (30,000 ng / mL, 7,500 ng / mL, 1,875 ng / mL, 468.8 ng / mL, 117.2 ng / mL, 29.3 ng / mL, 7.3 ng / mL, and 1.8 ng / mL) and 100 μL of FITC anti-human IgG Fc antibody (Biolegend, Cat. no. 410720) were used to detect the binding of the test antibodies, and FITC fluorescence intensity (MFI) was measured by flow cytometry. All experimental results are expressed as mean ± SEM (standard error of the mean). Graphs were generated and data analyzed using Prism 6 (GraphPad) software.

[0171] The binding curves are shown in Figure 4. The FACS binding results show that the binding activity of the MHAB10 candidate antibodies to PL45 cells is consistent with the binding activity trend of human colorectal cancer HCT116 cells and SW480 cells that endogenously express human CDCP1. Among them, MHAB10-11 and MHAB10-16 have the strongest binding activity.

[0172] Endocytosis assay

[0173] Endocytosis activity was detected using human colorectal cancer HCT116 cells and SW480 cells that endogenously express human CDCP1. 1×10 6Prepare a 1.5 mL Eppendorf tube containing 10 μg / mL of the test antibody solution and 2 μg / mL of the anti-CDCP1 suspension. Centrifuge at 1200 rpm at 4°C for 3 minutes and discard the supernatant. Add 1.5 mL of the test antibody solution to the cells and incubate on ice for 30 minutes. Wash four times with 200 μL / well of pre-chilled FACS buffer. Resuspend the cells in 1.5 mL of complete culture medium, mix thoroughly, and add the cells to a 96-well plate at 100 μL / well. Incubate at 4°C and 37°C for 0, 1, 2, and 4 hours (in duplicate). After incubation, centrifuge at 1200 rpm and 4°C for 3 min, add 100 μL of FITC anti-human IgG Fc Antibody diluent (Biolegend, Cat. no. 410720), and incubate at 4°C for 30 min. After washing away unbound secondary antibody, the FITC fluorescence intensity (mean fluorescence intensity, MFI) was measured using flow cytometry, and the endocytosis rate was calculated using the following formula:

[0174] Internalization(%)=100%-MFI T / MFI T0 *100%

[0175] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0176] The endocytosis percentage curves are shown in Figure 5, with Figure 5A showing the results for HCT116 cells and Figure 5B showing the results for SW480 cells. The endocytosis results indicate that the MHAB10 candidate antibodies exhibited robust endocytosis activity in both HCT116 and SW480 cells at 37°C, with consistent trends. Specifically, MHAB10-16 exhibited significantly greater endocytosis activity than both MHAB10-11 and the positive control antibody (25A11) (MHAB10-PC1).

[0177] 3.5. Fab-ZAP assay for antibody endocytosis-mediated cell killing

[0178] Human colorectal cancer HCT116 cells were digested and resuspended in complete culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). The cell density was adjusted to 1E4 cells / mL and 50 μL was added to each well of a 96-well cell culture plate. The cell culture plate was incubated at 37°C for 16 hours. The next day, ZAP diluent containing 9 nM ZAP-Fab was prepared in culture medium. The antibody was then serially diluted with ZAP diluent to a working solution ranging from 1.6 nM to 0.5 pM (6 concentration steps, 1:5 dilution). After incubation at 37°C for 15 minutes, the working solution was added to the cell culture plate at 50 μL per well, mixed, and the cell culture plate was incubated at 37°C for another 120 hours. 7.5 μL Triton-X 100 was added to the cell wells not treated with antibody-Fab-ZAP in advance and incubated for 30 minutes. This well served as a positive control. The cell wells not treated with antibody-Fab-ZAP or Triton-X100 (NT) served as negative controls. 20 μL MTS (Promega, Cat: G3598B) was then added to each well of the cell culture plate and incubated in a 37°C cell culture incubator for 2 hours. The plate was shaken for 10 seconds and the data was read using a microplate reader. The detection wavelength was A490. The killing rate (%) was calculated using the formula: 100% - (OD sample -OD Triton-X100) / (OD NT -OD Triton-X100)*100% to calculate the cell killing efficiency.

[0179] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0180] The results of endocytosis-mediated cell killing are shown in Table 5 and Figure 6. MHAB10-16-Fab-ZAP has good killing activity against HCT116 cells. Based on the FACS binding and endocytosis results, MHAB10-16 will be humanized and further screened.

[0181] Table 5. Endocytosis-mediated cell killing results

[0182] Example 4. Humanization and activity detection of anti-human CDCP1 nanobody

[0183] Preparation of MHAB10-16 Nanobodies and Humanized Nanobodies

[0184] After sequencing and analysis, the CDR sequence (defined according to the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003)), VHH sequence and complete sequence of the MHAB10-16 nanobody were obtained. The specific sequence information is shown in Table 1 and Table 6 below.

[0185] Table 6. MHAB10-16 Nanobody Sequence Information

[0186] Furthermore, by performing database sequence analysis on the sequence of the original camel-derived antibody MHAB10-16, the humanized sequence Germeline was clearly referenced and the humanized sequence was designed. After the first round of humanization, the degree of humanization was at least 82%. It was then constructed into a nanobody-Fc fusion protein (10 molecules, MHAB10-16-1 to MHAB10-16-10, were constructed) and transiently expressed in CHO cells. A second round of humanization was performed based on the degree of humanization, affinity, and expression level. After the second round of humanization, it was constructed into a nanobody-Fc fusion protein (7 molecules, MHAB10-16-11 to MHAB10-16-17, were constructed), transiently expressed in CHO cells, and its binding activity was verified.

[0187] A total of 17 humanized nanobodies were prepared and named MHAB10-16-1 to MHAB10-16-17. The specific sequence information is shown in Table 1.

[0188] ELISA binding assay

[0189] The binding ability of the humanized MHAB10-16 sequence to the human CDCP1 antigen was tested using an ELISA method. CDCP1, His Tag (KACTUS, Cat. No. CDC-HM101) was diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plate was coated overnight at 2°C-8°C. Rinse 3 times with PBST, add 1% BSA / PBST to block at room temperature for 1 hour, then wash 5 times with PBST, add MHAB10-16 humanized antibody (antibody number is MHAB10-16-1 to 8, positive control antibody is 25A11, i.e. MHAB10-PC1), initial concentration is 10 μg / mL, perform 10-fold serial dilution), incubate for 1 hour, rinse 7 times with PBST, wash away unbound antibody, add 1:50,000 dilution of Goat Anti-Human IgG Fc (HRP) (Abcam, Cat.No.ab97225), incubate at room temperature for 30 minutes. After washing away excess secondary antibody, add 1-Step TM Ultra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) (100 μL / well) was added and developed at room temperature in the dark for 15 min. The reaction was then terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472). The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0190] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0191] The ELISA binding results ( FIG. 7A , FIG. 7B and Table 7 ) show that the binding activity of the humanized MHAB10-16 antibody to the human CDCP1 antigen is substantially equivalent to that of the parental MHAB10-16, and is higher than that of the positive control antibody 25A11, i.e., MHAB10-PC1.

[0192] ELISA binding EC of MHAB10-16 and MHAB10-16-1 to MHAB10-16-8 50 The results are shown in Table 7 below.

[0193] Table 7. ELISA binding to EC 50 result

[0194] Table 7. ELISA binding EC 50 result

[0195] Note: MHAB10-16-9* and MHAB10-16-10* are intermediate sequences between the first and second rounds of humanization to explore changes in expression levels (the sequences are two variants of MHAB10-16-1, respectively mutated; see Table 1 for sequence information). ELISA binding activity was not tested.

[0196] FACS binding assay

[0197] In the FACS experiment, the binding of MHAB10-16 humanized antibodies was detected using human colorectal cancer HCT116 cells endogenously expressing human CDCP1. The test antibody was serially diluted (7500ng / mL, 1875ng / mL, 375ng / mL, 75ng / mL, 15ng / mL, 3ng / mL and 0.6ng / mL) and 100μL Alexa Fluor The binding of the test antibody was detected using 488AffiniPure Goat Anti-Human IgG (H+L) secondary antibody (YEASEN, Cat. no. 33126ES60), and the fluorescence intensity (MFI) of the bound secondary antibody was measured by flow cytometry.

[0198] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0199] The binding curve is shown in Figure 8. 50 The results are shown in Table 8. The FACS binding results show that the binding activity of MHAB10-16 humanized antibody to HCT116 cells is comparable to that of parental MHAB10-16. 50 and humanization levels, and three antibodies with the best development potential (MHAB10-16-3, 4, and 7) were selected for subsequent endocytosis and endocytosis-mediated cell killing activity testing.

[0200] ELISA binding EC of MHAB10-16 and MHAB10-16-1 to MHAB10-16-8 50 The results are shown in Table 8 below.

[0201] Table 8. FACS binding EC 50 result

[0202] Table 8. FACS binding EC 50 result

[0203] 4.4. Endocytosis experiments

[0204] Endocytosis activity was detected using human colorectal cancer HCT116 cells that endogenously express human CDCP1. 1×10 6 / mL cell suspension and 10μg / mL anti-CDCP1 test antibody solution, add the cell suspension to a 1.5mL EP tube, centrifuge at 1200rpm, 4℃ for 3min, and discard the supernatant. Pipette 1.5mL of the test antibody solution and add it to the cells, incubate on ice for 30min, and wash four times with pre-cooled 200μL / well FACS buffer. Resuspend the cells with 1.5mL complete culture medium, mix well and add the cells to a 96-well plate, 100μL / well. Incubate at 4℃ and 37℃ for 0h, 1h, 2h and 4h (double wells). After incubation, centrifuge at 1200rpm, 4℃ for 3min, add 100μL Alexa Fluor 488AffiniPure Goat Anti-Human IgG (H+L) secondary antibody (YEASEN, Cat. no. 33126ES60) was diluted and incubated at 4°C for 30 min. After washing away the unbound secondary antibody, the FITC fluorescence intensity (mean fluorescence intensity, MFI) was measured using flow cytometry and the endocytosis rate was calculated using the following formula:

[0205] Internalization(%)=100%-MFI T / MFI T0 *100%

[0206] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0207] The endocytosis percentage curve is shown in Figure 9. The endocytosis results show that at 37°C, the humanized MHAB10-16 antibody has good endocytosis activity in HCT116 cells. The endocytosis activities of MHAB10-16-3, 4, and 7 are basically equivalent to that of the parental MHAB10-16, and are all higher than the positive control antibody 25A11, i.e., MHAB10-PC1.

[0208] 4.5. Fab-ZAP assay for antibody endocytosis-mediated cell killing

[0209] Human colorectal cancer HCT116 cells were digested and resuspended in complete culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). The cell density was adjusted to 1E4 cells / mL and 50 μL was added to each well of a 96-well cell culture plate. The cell culture plate was incubated at 37°C for 16 hours. The next day, ZAP diluent containing 9 nM ZAP-Fab was prepared in culture medium. The antibody was then serially diluted with ZAP diluent to a working solution ranging from 1.6 nM to 0.5 pM (6 concentration steps, 1:5 dilution). After incubation at 37°C for 15 minutes, the working solution was added to the cell culture plate at 50 μL per well, mixed, and the cell culture plate was incubated at 37°C for another 120 hours. 7.5 μL Triton-X 100 was added to the cell wells not treated with antibody-Fab-ZAP in advance and incubated for 30 minutes. This well served as a positive control. The cell wells not treated with antibody-Fab-ZAP or Triton-X100 (NT) served as negative controls. 20 μL MTS (Promega, Cat: G3598B) was then added to each well of the cell culture plate and incubated in a 37°C cell culture incubator for 2 hours. The plate was shaken for 10 seconds and the data was read using a microplate reader. The detection wavelength was A490. The killing rate (%) was calculated using the formula: 100% - (OD sample -OD Triton- X100 ) / (OD NT -OD Triton-X100 )*100% to calculate the cell killing efficiency.

[0210] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0211] The results of endocytosis-mediated cell killing are shown in Table 9 and Figure 10. Compared with the parental MHAB10-16, the MHAB10-16 humanized antibody had similar killing activity against HCT116.

[0212] Table 9. Endocytosis-mediated cell killing results

[0213] Example 5. Second round of humanization and activity detection of anti-human CDCP1 nanobody

[0214] ELISA binding assay

[0215] The binding ability of the humanized MHAB10-16 sequence to the human CDCP1 antigen was tested using an ELISA method. CDCP1, His Tag (KACTUS, Cat. No. CDC-HM101) was diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plate was coated overnight at 2°C-8°C. Rinse 3 times with PBST, add 1% BSA / PBST to block at room temperature for 1 hour, then rinse 5 times with PBST, add MHAB10-16 humanized antibody (antibody numbers are MHAB10-16-11 to 17, the positive control antibody is 25A11, i.e. MHAB10-PC1), the initial concentration is 10μg / mL, and a 10-fold serial dilution is performed). After incubation for 1 hour, rinse 7 times with PBST to wash away unbound antibodies, add 1:50,000 diluted Goat Anti-Human IgG Fc (HRP) (Abcam, Cat.No.ab97225), and incubate at room temperature for 30 minutes. After washing away excess secondary antibody, add 1-Step TM Ultra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) (100 μL / well) was added and developed at room temperature in the dark for 15 min. The reaction was then terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472). The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0216] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0217] The ELISA binding results ( FIG. 11 and Table 10 ) show that the binding activity of the humanized MHAB10-16 antibody to the human CDCP1 antigen is substantially equivalent to that of the parental MHAB10-16, and is higher than that of the positive control antibody 25A11, i.e., MHAB10-PC1.

[0218] ELISA binding EC of MHAB10-16 and MHAB10-16-11 to MHAB10-16-17 50 The results are shown in Table 10 below.

[0219] Table 10. ELISA binding to EC 50 result

[0220] Table 10. ELISA binding EC 50 result

[0221] FACS binding assay

[0222] In the FACS experiment, the binding of MHAB10-16 humanized antibodies was detected using human colorectal cancer HCT116 cells endogenously expressing human CDCP1. The antibody to be tested was serially diluted (30000ng / mL, 7500ng / mL, 1875ng / mL, 375ng / mL, 75ng / mL, 15ng / mL, 3ng / mL, 0.6ng / mL, 0.12ng / mL and 0.024ng / mL) and 100μL Alexa Fluor The binding of the test antibody was detected using 488 AffiniPure Goat Anti-Human IgG (H+L) secondary antibody (YEASEN, Cat. no. 33126ES60), and the fluorescence intensity (MFI) of the bound secondary antibody was measured by flow cytometry.

[0223] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0224] The binding curve is shown in Figure 12. 50 The results are shown in Table 11. The FACS binding results show that the binding activity of MHAB10-16 humanized antibody to HCT116 cells was not significantly weakened compared with parental MHAB10-16. 50 and humanization levels, and the four antibodies with the best development potential (MHAB10-16-13, 14, 15, and 16) were selected for subsequent endocytosis and endocytosis-mediated cell killing activity testing.

[0225] FACS binding of MHAB10-16 and MHAB10-16-11 to MHAB10-16-17 to EC 50 The results are shown in Table 11 below.

[0226] Table 11. FACS binding EC 50 result

[0227] Table 11. FACS combined with EC 50 result

[0228] 5.3. Endocytosis assay

[0229] Endocytosis activity was detected using human colorectal cancer HCT116 cells that endogenously express human CDCP1. 1×10 6 / mL cell suspension and 10μg / mL anti-CDCP1 test antibody solution, add the cell suspension to a 1.5mL EP tube, centrifuge at 1200rpm, 4℃ for 3min, and discard the supernatant. Pipette 1.5mL of the test antibody solution and add it to the cells, incubate on ice for 30min, and wash four times with pre-cooled 200μL / well FACS buffer. Resuspend the cells with 1.5mL complete culture medium, mix well and add the cells to a 96-well plate, 100μL / well. Incubate at 4℃ and 37℃ for 0h, 1h, 2h and 4h (double wells). After incubation, centrifuge at 1200rpm, 4℃ for 3min, add 100μL Alexa Fluor 488 AffiniPure Goat Anti-Human IgG (H+L) secondary antibody (YEASEN, Cat. no. 33126ES60) was diluted and incubated at 4°C for 30 min. After washing away the unbound secondary antibody, the FITC fluorescence intensity (mean fluorescence intensity, MFI) was measured using flow cytometry and the endocytosis rate was calculated using the following formula:

[0230] Internalization(%)=100%-MFI T / MFI T0 *100%

[0231] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0232] The endocytosis percentage curve is shown in Figure 13. The endocytosis results show that at 37°C, the humanized MHAB10-16 antibody has good endocytosis activity in HCT116 cells. The endocytosis activities of MHAB10-16-13, 14, 15, and 16 are similar, and the endocytosis activities of MHAB10-16-13, 14, 15, and 16 are basically equivalent to those of the parental MHAB10-16. They are all significantly higher than the positive control antibody 25A11, i.e., MHAB10-PC1.

[0233] 5.4. Fab-ZAP assay for antibody endocytosis-mediated cell killing

[0234] Human colorectal cancer HCT116 cells were digested and resuspended in complete culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). The cell density was adjusted to 1E4 cells / mL and 50 μL was added to each well of a 96-well cell culture plate. The cell culture plate was incubated at 37°C for 16 hours. The next day, ZAP diluent containing 9 nM ZAP-Fab was prepared in culture medium. The antibody was then serially diluted with ZAP diluent to a working solution ranging from 1.6 nM to 0.5 pM (6 concentration steps, 1:5 dilution). After incubation at 37°C for 15 minutes, the working solution was added to the cell culture plate at 50 μL per well, mixed, and the cell culture plate was incubated at 37°C for another 120 hours. 7.5 μL Triton-X 100 was added to the cell wells not treated with antibody-Fab-ZAP in advance and incubated for 30 minutes. This well served as a positive control. The cell wells not treated with antibody-Fab-ZAP or Triton-X100 (NT) served as negative controls. 20 μL MTS (Promega, Cat: G3598B) was then added to each well of the cell culture plate and incubated in a 37°C cell culture incubator for 2 hours. The plate was shaken for 10 seconds and the data was read using a microplate reader. The detection wavelength was A490. The killing rate (%) was calculated using the formula: 100% - (OD sample -OD Triton- X100 ) / (OD NT -OD Triton-X100 )*100% to calculate the cell killing efficiency.

[0235] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0236] The endocytosis-mediated cell killing curve is shown in Figure 14 . The endocytosis activities of MHAB10-16-13, 14, 15, and 16 were basically equivalent to that of parental MHAB10-16.

[0237] Example 6. Activity detection of anti-human CDCP1 humanized nanobody MHAB10-16-14

[0238] ELISA binding assay

[0239] The binding ability of the anti-human CDCP1 humanized nanobody MHAB10-16-14 and negative control isotype to CDCP1 antigens from different species was tested using ELISA. Human CDCP1, His Tag (KACTUS, Cat. No. CDC-HM101) and rhesus macaque CDCP1, His Tag (KACTUS, Cat. No. CDC-CM101) were diluted to 0.5 μg / mL in PBS and added to an ELISA plate (Corning, Cat. No. 9018) at 100 μL / well. The plates were coated overnight at 2°C-8°C. Rinse 3 times with PBST, add 1% BSA / PBST to block at room temperature for 1 hour, then wash 5 times with PBST, add humanized antibody MHAB10-16-14 (negative control antibody is Isotype antibody, initial concentration is 10μg / mL, 10-fold serial dilution), incubate for 1 hour, rinse 7 times with PBST, wash away unbound antibody, add 1:50,000 dilution of Goat Anti-Human IgG Fc (HRP) (Abcam, Cat.No.ab97225), incubate at room temperature for 30 minutes. After washing away excess secondary antibody, add 1-Step TM Ultra TMB-ELISA Substrate Solution (Absin, Cat. No. 9178) (100 μL / well) was added and developed at room temperature in the dark for 15 min. The reaction was then terminated by adding 100 μL of TMB Stop Solution (Absin, Cat. No. abs9472). The absorbance was read at 450 nm using a microplate reader, and a four-parameter curve was plotted using GraphPad.

[0240] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0241] The ELISA binding results (Table 12 and Figure 15) show that MHAB10-16-14 can specifically bind to human and rhesus monkey CDCP1 antigens with comparable binding activity.

[0242] Table 12. ELISA binding to EC 50 result

[0243] 6.2. FACS binding assay

[0244] In the FACS assay, binding of the humanized antibody MHAB10-16-14 was detected using human colorectal cancer cells HCT116, SW480, PL45, HT-29, and LNCaP, which endogenously express human CDCP1 (the positive control antibody was h14A043, i.e., MHAB10-PC2). The test antibody was serially diluted (30,000 ng / mL, 6,000 ng / mL, 1,200 ng / mL, 240 ng / mL, 48 ng / mL, 9.6 ng / mL, 1.92 ng / mL, and 0.384 ng / mL). Binding of the test antibody was detected using 100 μL of FITC anti-human IgG Fc Antibody secondary antibody (Biolegend, Cat. no. 366916). The fluorescence intensity (MFI) of the bound secondary antibody was measured by flow cytometry.

[0245] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0246] Binding curves are shown in Figures 16-20 and Table 13. The FACS binding results show that MHAB10-16-14 can specifically bind to different human tumor cells (taking human colorectal cancer HCT116, human colorectal cancer SW480, human pancreatic ductal adenocarcinoma PL45, human colorectal cancer HT-29, and human prostate cancer LNCaP cells as examples), and the binding activity of MHAB10-16-14 is stronger than that of the positive control antibody h14A043.

[0247] Table 13. FACS binding EC 50 result

[0248] 6-3Fab-ZAP assay to detect cell killing mediated by MHAB10-16-14 antibody endocytosis

[0249] Human colorectal cancer HCT116 cells were digested and resuspended in complete culture medium (ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 + 10% FBS, Sigma-Aldrich, Cat. no. F8687 + 1% P / S, Gibco, Cat. no. 15140-122). The cell density was adjusted to 1E4 cells / mL and added to a 96-well cell culture plate at 50 μL per well. The cell culture plate was placed in a 37°C cell culture incubator and cultured for 16 hours. The next day, ZAP diluent containing 9 nM ZAP-Fab was prepared in culture medium. The antibody was then serially diluted with ZAP diluent to a working solution ranging from 2 nM to 0.16 pM (the positive control antibody was h14A043, also known as MHAB10-PC2). The concentration setting was as follows: 8 concentrations, with a 1:5 dilution at the first three points, followed by two 1:2 dilutions, and then a final 1:5 dilution). After incubation at 37°C for 15 minutes, the working solution was added to the cell culture plate, 50 μL per well, mixed, and the cell culture plate was placed in a 37°C cell culture incubator for an additional 120 hours. 7.5 μL of Triton-X 100 was added to a well of cells not treated with antibody-Fab-ZAP in advance and incubated for 30 minutes. This well served as a positive control. Wells of cells not treated with antibody-Fab-ZAP or Triton-X 100 (NT) served as negative controls. Then, 20 μL of MTS (Promega, Cat: G3598B) was added to each well of the cell culture plate and incubated in a 37°C cell culture incubator for 2 h. The plate was shaken for 10 s and the data was read using a microplate reader. The detection wavelength was A490. The killing rate was calculated using the formula: 100% - (OD sample -OD Triton-X100 ) / (OD NT -OD Triton-X100 )*100% to calculate the cell killing efficiency.

[0250] All experimental results are expressed as mean ± SEM (standard error of the mean), and Prism 6 (GraphPad) software was used for plotting and data analysis.

[0251] The endocytosis-mediated cell killing curve is shown in FIG21 and Table 14 . The killing activity of MHAB10-16-14 against HCT116 is significantly stronger than that of the positive control antibody h14A043.

[0252] Table 14. Endocytosis-mediated cell killing results

[0253] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A single domain antibody or an antigen-binding fragment thereof capable of specifically binding to CUB domain-containing protein 1 (CDCP1), the single domain antibody or the antigen-binding fragment thereof comprising: (a) CDR1, which has: a sequence as shown in any one of SEQ ID NOs: 1, 4, 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 1, 4, 7; (b) CDR2, which has: a sequence as shown in any one of SEQ ID NOs: 2, 5, 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 2, 5, 8; and (c) CDR3, which has: a sequence as shown in any one of SEQ ID NOs: 3, 6, 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 3, 6, 9; Preferably, the substitution is a conservative substitution; Preferably, the single domain antibody or antigen-binding fragment thereof comprises: (1) CDR1 as shown in SEQ ID NO: 1; CDR2 as shown in SEQ ID NO: 2; and CDR3 as shown in SEQ ID NO: 3; (2) CDR1 as shown in SEQ ID NO:4; CDR2 as shown in SEQ ID NO:5; and CDR3 as shown in SEQ ID NO:6; or, (3) CDR1 as shown in SEQ ID NO:7; CDR2 as shown in SEQ ID NO:8; and CDR3 as shown in SEQ ID NO:

9.

2. A single domain antibody or antigen binding fragment thereof that specifically binds to CUB domain-containing protein 1 (CDCP1), wherein the single domain antibody or antigen binding fragment thereof comprises: three CDRs contained in a heavy chain variable region (VHH) as shown in any one of SEQ ID NOs: 10-27; Preferably, the three CDRs contained in the VHH are defined by the Kabat, IMGT or Chothia numbering systems.

3. The single domain antibody or antigen binding fragment thereof according to claim 1 or 2, comprising an amino acid sequence selected from the group consisting of: (i) a sequence as shown in any one of SEQ ID NOs: 10-27; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 10-27; or (iii) a sequence having 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%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 10-27; Preferably, the substitutions are conservative substitutions.

4. A polypeptide construct that specifically binds to CDCP1, comprising the single-domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is optionally connected to the N-terminus and / or C-terminus (e.g., C-terminus) of the single domain antibody or antigen-binding fragment thereof via a peptide linker; Preferably, the immunoglobulin Fc domain is an IgG Fc domain, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; Preferably, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 28 or 29, or a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared thereto (e.g., a sequence having at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions or additions or any combination thereof; for example, a sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); Preferably, the polypeptide construct has a sequence as shown in any one of SEQ ID NOs: 30-47.

5. An isolated nucleic acid molecule encoding the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3, or the polypeptide construct according to claim 4.

6. A vector comprising the nucleic acid molecule according to claim 5; preferably, the vector is a cloning vector or an expression vector.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A method for preparing the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4, comprising culturing the host cell according to claim 7 under conditions that allow protein expression, and recovering the single domain antibody or antigen binding fragment thereof or the polypeptide construct from the cultured host cell culture.

9. A bispecific or multispecific antibody comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4; Preferably, the bispecific or multispecific antibody specifically binds to CDCP1, and additionally specifically binds to one or more other targets; Preferably, the bispecific or multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target.

10. A conjugate comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4, and a therapeutic agent linked to the single domain antibody or antigen binding fragment thereof; Preferably, the therapeutic agent is selected from a cytotoxic agent; Preferably, the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and any combination thereof; Preferably, the conjugate is an antibody-drug conjugate (ADC).

11. A pharmaceutical composition comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4, or the bispecific or multispecific antibody according to claim 9 or the conjugate according to claim 10, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor or an oncolytic virus; Preferably, the single domain antibody or antigen binding fragment thereof, bispecific or multispecific antibody or conjugate and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.

12. A kit comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4; Preferably, the single domain antibody or antigen binding fragment thereof or the polypeptide construct carries a detectable label, such as an enzyme (e.g. horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g. chemiluminescent substance) or biotin; Preferably, the kit further comprises a second antibody that specifically recognizes the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4; Preferably, the second antibody further comprises a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.

13. A chimeric antigen receptor comprising the antigen binding domain of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4; Preferably, the antigen binding domain is expressed by immune effector cells (eg, T cells).

14. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 13.

15. A vector comprising the isolated nucleic acid molecule of claim 14; preferably, it is used for preparing chimeric antigen receptor T cells.

16. A host cell comprising the isolated nucleic acid molecule of claim 14 or the vector of claim 15; Preferably, the host cell is an immune effector cell (e.g., a T cell or a NK cell); Preferably, the host cell is a chimeric antigen receptor T cell (CAR-T).

17. Use of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3, or the polypeptide construct according to claim 4, or the bispecific or multispecific antibody according to claim 9, or the conjugate according to claim 10, or the pharmaceutical composition according to claim 11, or the kit according to claim 12, or the chimeric antigen receptor according to claim 13 in the preparation of a medicament for preventing and / or treating a tumor in a subject; preferably, the tumor expresses CDCP1; Preferably, the drug is used to inhibit the growth of tumor cells expressing CDCP1 and / or kill the tumor cells; Preferably, the medicament further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a molecular targeted drug, an immune checkpoint inhibitor or an oncolytic virus; Preferably, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axial tumors, brainstem gliomas; Preferably, the subject is a mammal, such as a human.

18. Use of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4 in the preparation of a kit for detecting whether a tumor can be treated by an anti-tumor therapy targeting CDCP1; Preferably, the single domain antibody or antigen-binding fragment thereof carries a detectable label; Preferably, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

19. A method for detecting the presence or amount of CDCP1 (eg, human CDCP1) in a sample, comprising the following steps: (1) contacting the sample with the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4; (2) detecting the formation of a complex between the single domain antibody or antigen binding fragment thereof or the polypeptide construct and CDCP1 or detecting the amount of the complex; Preferably, the single domain antibody or antigen-binding fragment thereof or the polypeptide construct carries a detectable label.

20. A method for reducing the expression level of CDCP1 on the cell surface, comprising contacting the cell with the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3, or the polypeptide construct according to claim 4, or the bispecific or multispecific antibody according to claim 9, or the conjugate according to claim 10, or the pharmaceutical composition according to claim 11, or the kit according to claim 12, so that the expression level of CDCP1 on the cell surface is reduced; wherein, The cells express CDCP1 on their surface; Preferably, the cell is a tumor cell expressing CDCP1.

21. A method for preventing and / or treating tumors, the method comprising administering to a subject in need thereof an effective amount of the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3, or the polypeptide construct according to claim 4, or the bispecific or multispecific antibody according to claim 9, or the conjugate according to claim 10, or the pharmaceutical composition according to claim 11, or the kit according to claim 12, or the chimeric antigen receptor according to claim 13; Preferably, the tumor expresses CDCP1; Preferably, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axial tumors, brainstem gliomas; Preferably, the subject is a mammal, such as a human.

22. A method for detecting whether a tumor can be treated by an anti-tumor therapy targeting CDCP1, the method comprising administering an effective amount of the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the polypeptide construct according to claim 4 to a subject in need thereof; Preferably, the single domain antibody or antigen-binding fragment thereof carries a detectable label; Preferably, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, breast cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides ... fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem gliomas.

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