PTK7 antigen-binding protein and use thereof

Abstract

Provided is an isolated antigen-binding protein, the antigen-binding protein being capable of targeting PTK7, and the antigen-binding protein comprising at least one CDR in an antibody heavy chain variable region. Also provided are a pharmaceutical composition comprising the antigen-binding protein, a chimeric antigen receptor, an immunoconjugate, a nucleic acid encoding the antigen-binding protein, a vector comprising the nucleic acid molecule, and a cell comprising the vector. Also provided is a use of the antigen-binding protein in the prevention and / or treatment of disease.

Description

PTK7 antigen binding protein and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting PTK7. Background Art

[0002] Protein tyrosine kinase 7 (PTK7), also known as colon cancer kinase 4 (CCK4), is a highly conserved member of the receptor protein tyrosine kinase family. It consists of seven extracellular immunoglobulin-like domains, a transmembrane region, a juxtamembrane region, and a catalytically inactive cytoplasmic tyrosine kinase domain. PTK7 regulates numerous physiological processes within cells and is involved in several important biological processes, including cell proliferation, differentiation, apoptosis, cell signaling, cell adhesion, and cell migration. Genetic and biochemical studies have demonstrated a critical function for PTK7 in noncanonical Wnt signaling, with PTK7-deficient embryos exhibiting severe developmental defects in planar cell polarity. There is also evidence that PTK7 has additional, potentially context-dependent, functions in the vascular endothelial growth factor (VEGF), semaphorin / plexin, and canonical Wnt signaling pathways. PTK7 has been shown to have an oncogenic role in colon, lung, and esophageal cancers, and it can promote cell survival and chemotherapy resistance in acute myeloid leukemia. By querying the Cancer Genome Atlas (TCGA) database, it was confirmed that PTK7 was overexpressed in primary breast tumors, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, head and neck cancer, esophageal cancer and other cancers. The average expression level of mRNA in tumors was significantly higher than that in the corresponding normal tissues. In non-small cell lung cancer, high PTK7 expression was associated with shorter survival.

[0003] Therefore, as a potentially attractive target for tumor treatment, PTK7 urgently needs to develop more therapeutic drugs that can effectively combine with it to meet the treatment needs of a wide range of diseases. Summary of the Invention

[0004] The present application provides an antigen-binding protein targeting PTK7. In the present application, the antigen-binding protein has one or more of the following properties: (1) the ability to specifically bind to the PTK7 antigen and have good binding activity; (2) the ability to specifically bind to PTK7 expressed on the surface of target cells; (3) a certain degree of endocytosis. The present application also provides an ADC comprising the antigen-binding protein, which has one or more of the following properties: (1) the ability to specifically bind to the PTK7 antigen and have good binding activity; (2) the ability to specifically bind to PTK7 expressed on the surface of target cells; (3) the ability to kill tumor cells.

[0005] In one aspect, the present application provides an isolated antigen-binding protein capable of binding to PTK7, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, wherein the VH comprises HCDR3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3.

[0006] In certain embodiments, the VH comprises HCDR2, and the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2.

[0007] In certain embodiments, the VH comprises HCDR1, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:1.

[0008] In certain embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3.

[0009] In certain embodiments, the VH comprises H-FR1, and the amino acid sequence of the H-FR1 is shown in SEQ ID NO:4 or SEQ ID NO:17.

[0010] In certain embodiments, the VH comprises H-FR2, and the amino acid sequence of H-FR2 is shown in SEQ ID NO:5 or SEQ ID NO:18.

[0011] In certain embodiments, the VH comprises H-FR3, and the amino acid sequence of the H-FR3 is shown in SEQ ID NO:6, SEQ ID NO:19 or SEQ ID NO:27.

[0012] In certain embodiments, the VH comprises H-FR4, and the amino acid sequence of the H-FR4 is shown in SEQ ID NO:7 or SEQ ID NO:20.

[0013] In certain embodiments, the VH comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 are selected from any one of the following combinations:

[0014] (1) The amino acid sequence of H-FR1 is shown in SEQ ID NO: 4, the amino acid sequence of H-FR2 is shown in SEQ ID NO: 5, the amino acid sequence of H-FR3 is shown in SEQ ID NO: 6, and the amino acid sequence of H-FR4 is shown in SEQ ID NO: 7;

[0015] (2) the amino acid sequence of H-FR1 is shown in SEQ ID NO: 17, the amino acid sequence of H-FR2 is shown in SEQ ID NO: 18, the amino acid sequence of H-FR3 is shown in SEQ ID NO: 19, and the amino acid sequence of H-FR4 is shown in SEQ ID NO: 20; and

[0016] (3) The amino acid sequence of the H-FR1 is shown in SEQ ID NO: 17, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 18, the amino acid sequence of the H-FR3 is shown in SEQ ID NO: 27, and the amino acid sequence of the H-FR4 is shown in SEQ ID NO: 20.

[0017] In certain embodiments, the amino acid sequence of the VH is shown in SEQ ID NO:8, SEQ ID NO:21 or SEQ ID NO:28.

[0018] In certain embodiments, the antigen binding protein comprises an antibody light chain variable region VL, wherein the VL comprises LCDR3, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:11.

[0019] In certain embodiments, the VL comprises LCDR2, the amino acid sequence of which is shown in SEQ ID NO: 10 (GAS).

[0020] In certain embodiments, the VL comprises LCDR1, and the amino acid sequence of the LCDR1 is shown in SEQ ID NO:9.

[0021] In certain embodiments, the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:9, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:10 (GAS), and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:11.

[0022] In certain embodiments, the VL comprises L-FR1, and the amino acid sequence of the L-FR1 is shown in SEQ ID NO: 12 or SEQ ID NO: 22.

[0023] In certain embodiments, the VL comprises L-FR2, and the amino acid sequence of the L-FR2 is shown in SEQ ID NO: 13 or SEQ ID NO: 23.

[0024] In certain embodiments, the VL comprises L-FR3, and the amino acid sequence of the L-FR3 is shown in SEQ ID NO: 14 or SEQ ID NO: 24.

[0025] In certain embodiments, the VL comprises L-FR4, and the amino acid sequence of the L-FR4 is shown in SEQ ID NO: 15 or SEQ ID NO: 25.

[0026] In certain embodiments, the VL comprises L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1, L-FR2, L-FR3, and L-FR4 are selected from any one of the following combinations:

[0027] (1) the amino acid sequence of L-FR1 is shown in SEQ ID NO: 12, the amino acid sequence of L-FR2 is shown in SEQ ID NO: 13, the amino acid sequence of L-FR3 is shown in SEQ ID NO: 14, and the amino acid sequence of L-FR4 is shown in SEQ ID NO: 15; and

[0028] (2) The amino acid sequence of the L-FR1 is shown in SEQ ID NO: 22, the amino acid sequence of the L-FR2 is shown in SEQ ID NO: 23, the amino acid sequence of the L-FR3 is shown in SEQ ID NO: 24, and the amino acid sequence of the L-FR4 is shown in SEQ ID NO: 25.

[0029] In certain embodiments, the amino acid sequence of the VL is shown in SEQ ID NO: 16 or SEQ ID NO: 26.

[0030] In certain embodiments, the antigen binding protein comprises an antibody heavy chain variable region VH and an antibody light chain variable region VL, the VH comprises HCDR1-3, the VL comprises LCDR1-3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10 (GAS) and SEQ ID NO: 11, respectively.

[0031] In certain embodiments, the antigen binding protein comprises VH and VL, the amino acid sequence of VH is shown in SEQ ID NO:8, SEQ ID NO:21 or SEQ ID NO:28, and the amino acid sequence of VL is shown in SEQ ID NO:16 or SEQ ID NO:26.

[0032] In certain embodiments, the antigen binding protein comprises a VH and a VL, wherein the VH and VL are selected from any one of the following combinations:

[0033] The amino acid sequence of the VH is shown in SEQ ID NO: 8, and the amino acid sequence of the VL is shown in SEQ ID NO: 16;

[0034] The amino acid sequence of the VH is shown in SEQ ID NO: 21, and the amino acid sequence of the VL is shown in SEQ ID NO: 26; and

[0035] The amino acid sequence of the VH is shown in SEQ ID NO: 28, and the amino acid sequence of the VL is shown in SEQ ID NO: 26.

[0036] In certain embodiments, the antigen binding protein further comprises an immunoglobulin constant region.

[0037] In certain embodiments, the immunoglobulin constant region is a human immunoglobulin constant region.

[0038] In certain embodiments, the immunoglobulin constant region is a human IgG heavy chain constant region and / or a human light chain constant region.

[0039] In certain embodiments, the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0040] In certain embodiments, the human light chain constant region is a human kappa (Kappa) or lambda (Lambda) light chain constant region.

[0041] In certain embodiments, the antigen binding protein is an antibody or an antigen binding fragment thereof.

[0042] In certain embodiments, the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH and / or dAb fragment of the antibody.

[0043] In certain embodiments, the antigen binding protein is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0044] In certain embodiments, the antigen binding protein is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0045] In certain embodiments, the antigen binding protein is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

[0046] In another aspect, the present application provides an immunoconjugate comprising the antigen-binding protein.

[0047] In certain embodiments, the immunoconjugate comprises the antigen binding protein, a linker, and a payload.

[0048] In certain embodiments, the payload is a cytotoxic drug, a polymer, a protein, a radioisotope, a nucleic acid compound, and / or a glucocorticoid.

[0049] In certain embodiments, the payload is a cytotoxic drug.

[0050] In certain embodiments, the payload is a chemotherapeutic drug, a tubulin inhibitor, a DNA damaging agent, and / or a topoisomerase I inhibitor.

[0051] In certain embodiments, the payload is MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), DM1, DM4, Tubulysins, PBD, IBD, Dxd, SN-38, Bcl-xL inhibitors, thailanstatin A, α-amanitin, β-amanitin, carmamycin A and / or carmamycin B.

[0052] In certain embodiments, the payload is MMAE.

[0053] In certain embodiments, the payload is Dxd.

[0054] In certain embodiments, the immunoconjugate is an antibody drug conjugate (ADC).

[0055] In certain embodiments, the ADC is a single-load ADC, a dual-load ADC, or a multi-load ADC.

[0056] In certain embodiments, the linker is a non-cleavable linker, an enzyme-cleavable linker, an acid-cleavable linker, a GSH-cleavable reducing linker, an Fe(II)-cleavable linker, a photoresponsive cleavable linker, and / or a bioorthogonal cleavable linker.

[0057] In certain embodiments, the linker is MC-VC-PAB or GGFG (SEQ ID NO: 29).

[0058] In certain embodiments, the immunoconjugate comprises an antigen binding protein, MC-VC-PAB and MMAE, wherein the antigen binding protein comprises VH and VL, wherein the VH comprises HCDR1-3, and the VL comprises LCDR1-3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10 (GAS) and SEQ ID NO: 11, respectively.

[0059] In certain embodiments, the immunoconjugate comprises an antigen binding protein, GGFG and Dxd, the antigen binding protein comprises VH and VL, the VH comprises HCDR1-3, the VL comprises LCDR1-3, and the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10 (GAS) and SEQ ID NO: 11, respectively.

[0060] On the other hand, the present application provides a chimeric antigen receptor (CAR) comprising the antigen binding protein.

[0061] In another aspect, the present application provides a modified immune cell comprising the chimeric antigen receptor.

[0062] In another aspect, the present application provides an isolated nucleic acid molecule encoding the antigen binding protein and / or the chimeric antigen receptor.

[0063] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0064] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0065] On the other hand, the present application provides a pharmaceutical composition comprising the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0066] On the other hand, the present application provides a pharmaceutical combination comprising the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition and a therapeutic agent.

[0067] On the other hand, the present application provides a detection kit comprising the antigen binding protein, and the detection kit is used to detect the presence and / or content of PTK7 in a sample or a subject.

[0068] In another aspect, the present application provides a method for detecting the presence and / or content of PTK7, which comprises using the antigen binding protein.

[0069] On the other hand, the present application provides the use of the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug combination in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0070] In certain embodiments, the disease and / or condition is a tumor.

[0071] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0072] In certain embodiments, the tumor is a PTK7-positive tumor.

[0073] In certain embodiments, the tumor is a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer, prostate cancer, and / or esophageal cancer.

[0074] On the other hand, the present application provides a method for preventing and / or treating a disease and / or condition, comprising administering the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug combination to a subject in need thereof.

[0075] In certain embodiments, the disease and / or condition is a tumor.

[0076] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0077] In certain embodiments, the tumor is a PTK7-positive tumor.

[0078] In certain embodiments, the tumor is a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer, prostate cancer, and / or esophageal cancer.

[0079] On the other hand, the present application provides the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug combination, which are used to prevent and / or treat diseases and / or disorders.

[0080] In certain embodiments, the disease and / or condition is a tumor.

[0081] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0082] In certain embodiments, the tumor is a PTK7-positive tumor.

[0083] In certain embodiments, the tumor is a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer, prostate cancer, and / or esophageal cancer.

[0084] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0086] FIG1 shows the results of the endocytic ability test of the anti-PTK7 antibody described in the present application.

[0087] FIG2 shows the binding results of the ADC and control antibodies described in this application to PANC-1 target cells.

[0088] FIG3 shows the killing results of the ADC described in this application on BxPC-3 target cells.

[0089] FIG4 shows the killing results of the ADC described in this application on HGC-27 target cells.

[0090] FIG5 shows the anti-tumor efficacy test results of the ADC described in this application in the PANC-1 CDX pancreatic cancer mouse efficacy model.

[0091] FIG6 shows the anti-tumor efficacy test results of the ADC described in this application in the PDX pancreatic cancer mouse model.

[0092] FIG7 shows the anti-tumor efficacy test results of the ADC described in this application in the PDX lung cancer mouse model.

[0093] FIG8 shows the anti-tumor efficacy test results of the ADC described in this application in the PDX gastric cancer mouse model.

[0094] Figure 9 shows the anti-tumor efficacy test results of the ADC described in this application in the PDX colorectal cancer mouse model.

[0095] FIG10 shows the killing results of the ADC described in this application on NCI-H446 target cells.

[0096] FIG11 shows the anti-tumor efficacy test results of the ADC described in this application in the NCI-H446 lung cancer mouse model. DETAILED DESCRIPTION

[0097] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0098] Definition of terms

[0099] In this application, the terms "PTK7," "protein tyrosine kinase 7," and "colon cancer kinase 4 (CCK4)" are generally used interchangeably and generally refer to a highly conserved member of the receptor protein tyrosine kinase family, consisting of seven extracellular immunoglobulin-like domains, a transmembrane region, a juxtamembrane region, and a catalytically inert cytoplasmic tyrosine kinase domain. In this application, the PTK7 may be intact PTK7 and functionally active fragments, homologs, analogs, variants, or derivatives thereof. For example, the PTK7 may be a truncated PTK7 that retains the activity of the intact PTK7. In this application, the PTK7 may be of any species. For example, the PTK7 may be human PTK7. In this application, the PTK7 may be wild-type or artificially modified. For example, the PTK7 may be a modified PTK7.

[0100] In this application, the term "antigen binding protein" generally refers to a protein with the ability to bind to an antigen. In this application, the antigen binding protein may include a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes the antigen binding portion to bind to the antigen. In this application, the antigen binding protein may be wild type or artificially modified. In this application, the antigen binding protein may be artificially synthesized. In this application, the antigen binding protein may include, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with a transplanted CDR or CDR derivative. In this application, the antigen binding protein may be an antibody or an antigen binding fragment thereof, as well as variants, homologs, derivatives or analogs thereof. In this application, the antigen binding protein may include, but is not limited to, antibodies, antigen binding fragments, single domain antibodies, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they show the desired antigen binding activity.

[0101] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof that can undergo a specific binding reaction with a corresponding antigen. For example, the antibody may comprise a protein of at least two heavy chains and two light chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. In this application, the heavy chain may comprise a heavy chain variable region (VH) and a heavy chain constant region (CH). In this application, the heavy chain constant region may comprise three domains, CH1, CH2, and CH3. In this application, the light chain may comprise a light chain variable region (VL) and a light chain constant region (CL). In this application, the light chain constant region may comprise one domain. In this application, the VH and VL may comprise hypervariable regions and more conserved regions, the hypervariable regions being referred to as complementarity determining regions (CDRs) or hypervariable regions (HVRs), which alternate with more conserved regions, referred to as framework regions (FRs). In the present application, the VH and VL can each include three CDRs and four FRs, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. For example, the variable domains of the heavy chain and the light chain can each include four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4). In the present application, the CDRs can be determined by a variety of coding systems. For example, the CDRs can be identified by CCG, Kabat, Chothia, IMGT, AbM, Kabat / Chothia, etc. In the present application, the CDRs encompass CDR sequences obtained by dividing according to any CDR division method. For example, the CDRs encompass variants. For example, the amino acid sequence of the CDR may be substituted, deleted and / or added with one or more amino acids, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions. For example, the CDR encompasses homologs. For example, the homolog may be an amino acid sequence having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR. In the present application, the antibody may be wild-type or artificially modified. For example, the antibody may be artificially synthesized. For example, the antibody may be derived from plasma, a hybridoma or a recombinant cell line. In the present application, the antibody may be of animal or non-animal origin. For example, the antibody may be of murine origin. For example, the antibody can be human. In the present application, the antibody can be a monoclonal antibody or a polyclonal antibody. In the present application, the antibody can be a monospecific antibody, a bispecific antibody or a multispecific antibody.In the present application, the antibody may be a chimeric antibody, a humanized antibody or a fully human antibody. In the present application, the antibody may be a recombinant, hybrid, mutated or transplanted antibody.

[0102] In this application, the term "antigen-binding fragment" generally refers to a portion of an intact antibody and includes the variable region of an intact antibody. For example, the antigen-binding fragment may include Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragments. In this application, the antigen-binding fragment can be obtained using conventional techniques known to those skilled in the art, and the function of the fragment can be evaluated in the same manner as for intact antibodies. For example, the antigen-binding fragment can be obtained by in vitro display technology.

[0103] In this application, the terms "immunoglobulin constant region" and "constant region" are generally used interchangeably and generally refer to the region of the non-variable region of an antibody. In this application, the constant region may not directly participate in antigen binding, but may exhibit various effector functions. In this application, the constant region may be the entire non-variable region of an antibody, or it may be a portion of the non-variable region of an antibody. For example, the constant region may be a heavy chain constant region. For example, the constant region may be a light chain constant region. In this application, the antibody heavy chain constant region may be an IgG heavy chain constant region. For example, the IgG heavy chain constant region may be an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In this application, the antibody light chain constant region may be a kappa (kappa) or lambda (lambda) light chain constant region. In this application, the constant region may be derived from an animal. For example, the constant region may be derived from human, goat, rabbit, rat, or guinea pig. For example, the heavy chain constant region may be a human IgG heavy chain constant region. For example, the heavy chain constant region of human IgG can be a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. For example, the light chain constant region can be a human light chain constant region. For example, the human light chain constant region can be a human kappa (Kappa) or lambda (Lambda) light chain constant region.

[0104] The proteins and / or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs with the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the amino acid sequence of the protein (e.g., the antigen-binding protein described in this application). For example, the functional variant may include a protein or polypeptide that has been subjected to amino acid changes by at least 1, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant can substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution can be a conservative substitution.

[0105] In the present application, a portion of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class. For example, the variable region and constant region of an antibody may both be derived from the variable region and constant region of an antibody from a single animal species (e.g., human).

[0106] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., the antigen-binding protein described herein).

[0107] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating other active ingredients to the antigen-binding protein. In the present application, the active ingredient can be covalently linked to the antigen-binding protein through a linker. In the present application, the immunoconjugate can be a conjugation of an antigen-binding protein with a payload. For example, the payload can be a cytotoxic drug, a polymer, a protein, a radioisotope, a nucleic acid compound, or a glucocorticoid. In the present application, the immunoconjugate can be an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted nuclear medicine (RDC). For example, the immunoconjugate can be an ADC. In the present application, the conjugate can be specifically bound to the antigen on the target cell by the antigen-binding protein, and the payload is delivered to the target cell. For example, the target cell can be a tumor cell.

[0108] In this application, the terms "antibody-drug conjugate," "antibody-drug conjugate," and "ADC" are generally used interchangeably. These conjugates are typically composed of an antigen-binding protein coupled to a cytotoxic drug via a linker. In this application, the ADC can utilize the antigen-binding protein's ability to recognize an antigen to transport the drug molecule to the vicinity of target cells and release it. In this application, the antigen-binding protein can be an antibody or an antigen-binding fragment thereof. For example, the antigen-binding protein can be an IgG antibody. For example, the antibody can be an IgG1 or IgG4 antibody. For example, the antigen-binding protein can bind to PTK7. In this application, the linker can be a cleavable linker or a non-cleavable linker. For example, the linker can be a non-cleavable linker, an enzyme-cleavable linker, an acid-cleavable linker, a GSH-cleavable reducing linker, an Fe(II)-cleavable linker, a photoresponsive cleavable linker, and / or a bioorthogonal cleavable linker. For example, the linker can be MC-VC-PAB. For example, the linker can be GGFG (SEQ ID NO: 29). In the present application, the cytotoxic drug can be a chemotherapeutic drug, a tubulin inhibitor, a DNA damaging agent and / or a topoisomerase I inhibitor. For example, the cytotoxic drug can be MMAE (monomethyl auristatin E). For example, the cytotoxic drug can be Dxd. In the present application, the ADC can be a single-load ADC, a dual-load ADC or a multi-load ADC.

[0109] In this application, the terms "chimeric antigen receptor" and "CAR" are generally used interchangeably, generally referring to the ability to anchor specific molecules that recognize target cell surface antigens on immune cells through artificially modified receptors, so that immune cells can recognize tumor antigens or viral antigens and kill tumor cells or viral infections. For example, the specific molecule can be an antigen binding protein. For example, the immune cell can be a T cell, NK cell, NKT cell, dendritic cell, macrophage, TIL cell, iNKT cell, CIK cell, γδT cell or DNT cell. In this application, CAR may sequentially include an optional signal peptide, an antigen binding domain, a hinge region, a transmembrane domain region and an intracellular signaling domain. For example, the CAR may also include an optional intracellular costimulatory domain. In this application, the antigen binding domain may be artificially synthesized. In this application, the antigen binding domain may include an antigen binding protein. In this application, the antigen binding protein may be an antibody or an antigen binding fragment thereof. For example, the antigen-binding fragment can be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment.

[0110] As used herein, the term "pharmaceutically acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. For example, the pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dose and concentration employed. For example, a physiologically acceptable carrier can be water, salt, protein, polysaccharide, lipid, or inactive viral particles.

[0111] In this application, the term "prevention and / or treatment" generally refers to the prevention and / or treatment of a disease. For example, the prevention and / or treatment may include preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith. In this application, the disease may be a neoplastic disease. For example, the onset of one or more symptoms associated with a tumor may be prevented or alleviated, and the severity and duration of a tumor and symptoms associated therewith may be reduced.

[0112] As used herein, the term "tumor" generally refers to any new, pathological tissue growth that harbors tumor antigens that can be recognized by the immune system. As used herein, the tumor may include benign or malignant tumors (cancers). As used herein, the cancer may be metastatic or non-metastatic. As used herein, the tumor may include solid tumors and hematologic tumors. As used herein, the solid tumor generally refers to a tangible tumor that can be detected by clinical examination. For example, the solid tumor may include a neoplasm or solid lesion formed by abnormal cell growth. As used herein, the hematologic tumor generally refers to a class of hematopoietic diseases. As used herein, the hematologic tumor may include various leukemias, multiple myeloma, or malignant lymphomas. As used herein, the tumor may be a tumor that positively expresses PTK7. For example, the tumor may be a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, bladder cancer, prostate cancer, and / or esophageal cancer.

[0113] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0114] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0115] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0116] Detailed Description of the Invention

[0117] Antigen binding proteins

[0118] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity to the amino acid sequence of the CDR. For example, the CDRs of the isolated antigen-binding proteins described herein can be determined using IMGT.

[0119] In one aspect, the present application provides an isolated antigen-binding protein, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, wherein the VH comprises a heavy chain complementary determining region HCDR1, HCDR2 and / or HCDR3. For example, the VH may comprise a heavy chain complementary determining region HCDR1, HCDR2 and HCDR3.

[0120] In the present application, the antigen binding protein has one or more of the following properties: (1) it can specifically bind to the PTK7 antigen and has good binding activity; (2) it can specifically bind to PTK7 expressed on the surface of target cells; (3) it has a certain endocytosis effect; and (4) it can have a killing effect on tumor cells.

[0121] In the present application, the antigen binding protein can bind to PTK7 on the target cell with a higher affinity / binding activity than the control antibodies 900797 and BMS (7C8). For example, the isolated antigen binding protein can bind to the PTK7 protein with a lower KD value than the control antibodies 900797 and BMS (7C8). For example, the isolated antigen binding protein can bind to the PTK7 protein with an affinity / binding activity of 5×10 -9 M or lower KD value to bind to PTK7 protein. For example, the KD value can be less than 4.5×10 -9 M, less than 5.0×10 - 9 M is less than 3.5×10 -9 M or less than 3.0×10 -9 M.

[0122] In the present application, the EC50 value or KD value can be determined by conventional techniques in the art. In the present application, the KD value can be determined by Octet, SPR, ELISA, competitive ELISA, BIACORE, or KINEXA. In the present application, the isolated antigen-binding protein is capable of specifically binding to PTK7. In the present application, the specific binding can be determined by FACS.

[0123] In the present application, the antigen binding protein can have an endocytic effect comparable to or better than that of the control antibody 900797. In the present application, the antigen binding protein can have a certain internalization effect.

[0124] In the present application, the antigen binding protein can bind to PTK7, and the antigen binding protein may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 21 or SEQ ID NO: 28.

[0125] In the present application, the antigen-binding protein may comprise a HCDR3, which may comprise the amino acid sequence set forth in SEQ ID NO:3. In the present application, the antigen-binding protein may comprise a HCDR2, which may comprise the amino acid sequence set forth in SEQ ID NO:2. In the present application, the antigen-binding protein may comprise a HCDR1, which may comprise the amino acid sequence set forth in SEQ ID NO:1. In the present application, the HCDR1, HCDR2, and / or HCDR3 may be wild-type sequences. For example, one or more amino acid sequences in HCDR1, HCDR2, and / or HCDR3 may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the HCDR1, HCDR2, and / or HCDR3 may be variants thereof, wherein the variant comprises one or more amino acids substituted, deleted, and / or added to the amino acid sequence of the HCDR1, HCDR2, and / or HCDR3.

[0126] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

[0127] In the present application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and / or H-FR4. In the present application, the antigen-binding protein may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:17. In the present application, the antigen-binding protein may comprise H-FR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:18. In the present application, the antigen-binding protein may comprise H-FR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:19 or SEQ ID NO:27. In the present application, the antigen-binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:20. In the present application, the H-FR1, H-FR2, H-FR3 and / or H-FR4 may be wild-type sequences. For example, one or more amino acid sequences in H-FR1, H-FR2, H-FR3, and / or H-FR4 can be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the H-FR1, H-FR2, H-FR3, and / or H-FR4 can be variants thereof, wherein the variant comprises one or more amino acids substituted, deleted, and / or added to the amino acid sequence of the H-FR1, H-FR2, H-FR3, and / or H-FR4.

[0128] In the present application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:17, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:18, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:19 or SEQ ID NO:27, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:20.

[0129] In the present application, the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4, H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0130] In the present application, the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17, H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 18, H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 19, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 20.

[0131] In the present application, the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17, H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 18, H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 27, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 20.

[0132] In the present application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4. In the present application, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 may be located between the HCDR1 and the HCDR2, the H-FR3 may be located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 may be directly or indirectly connected to the C-terminus of the HCDR3.

[0133] In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and / or H-FR4, and the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3.

[0134] In the present application, the antigen-binding protein may comprise an antibody light chain variable region VL, wherein the VL comprises light chain complementary determining regions LCDR1, LCDR2 and / or LCDR3. For example, the VL may comprise light chain complementary determining regions LCDR1, LCDR2 and LCDR3.

[0135] In the present application, the antigen-binding protein may comprise at least one CDR in the antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 26.

[0136] In the present application, the antigen-binding protein may comprise LCDR3, which may comprise the amino acid sequence set forth in SEQ ID NO:11. In the present application, the antigen-binding protein may comprise LCDR2, which may comprise the amino acid sequence set forth in SEQ ID NO:10 (GAS). In the present application, the antigen-binding protein may comprise LCDR1, which may comprise the amino acid sequence set forth in SEQ ID NO:9. In the present application, the LCDR1, LCDR2, and / or LCDR3 may be wild-type sequences. For example, one or more amino acid sequences in LCDR1, LCDR2, and / or LCDR3 may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the LCDR1, LCDR2, and / or LCDR3 may be variants thereof, wherein the variant comprises one or more amino acids substituted, deleted, and / or added to the amino acid sequence of the LCDR1, LCDR2, and / or LCDR3.

[0137] In the present application, the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:10 (GAS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:11.

[0138] In the present application, the antigen-binding protein may comprise L-FR1, L-FR2, L-FR3, and / or L-FR4. In the present application, the antigen-binding protein may comprise L-FR1, and the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:22. In the present application, the antigen-binding protein may comprise L-FR2, and the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:13 or SEQ ID NO:23. In the present application, the antigen-binding protein may comprise L-FR3, and the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:14 or SEQ ID NO:24. In the present application, the antigen-binding protein may comprise L-FR4, and the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:25. In the present application, the L-FR1, L-FR2, L-FR3, and / or L-FR4 may be wild-type sequences. For example, one or more amino acid sequences in L-FR1, L-FR2, L-FR3, and / or L-FR4 can be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the L-FR1, L-FR2, L-FR3, and / or L-FR4 can be variants thereof, wherein the variant comprises one or more amino acids substituted, deleted, and / or added to the amino acid sequence of L-FR1, L-FR2, L-FR3, and / or L-FR4.

[0139] In the present application, the antigen-binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 22, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 23, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 24, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 25.

[0140] In the present application, the antigen-binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12, L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0141] In the present application, the antigen-binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 22, L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 23, L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 24, and L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0142] In the present application, the antigen-binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4. In the present application, the C-terminus of the L-FR1 may be directly or indirectly linked to the N-terminus of the LCDR1, the L-FR2 may be located between the LCDR1 and the LCDR2, the L-FR3 may be located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 may be directly or indirectly linked to the C-terminus of the LCDR3.

[0143] In the present application, the isolated antigen-binding protein may comprise LCDR1, LCDR2, LCDR3, L-FR1, L-FR2, L-FR3 and / or L-FR4, and the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, the L-FR2 is located between the LCDR1 and the LCDR2, the L-FR3 is located between the LCDR2 and the LCDR3, and the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3.

[0144] In the present application, the direct or indirect connection may be connected through intermolecular forces, or may be connected through a linker.

[0145] In the present application, the amino acid sequence of the FR may be FR of any species, for example, the FR may be FR of mouse, rabbit, goat, alpaca or human origin.

[0146] In the present application, the amino acid sequence of the FR can be adjusted as needed. For example, the FR can be a wild-type sequence. For example, during the process of antibody humanization, the amino acid sequence of the FR can be changed without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, during the process of antibody humanization, one or more amino acid sequences in the FR can be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the FR can be a variant thereof, wherein the variant includes one or more amino acids in the amino acid sequence of the FR having been substituted, deleted, and / or added. For example, 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. For example, the FR can be a homolog, which can be an amino acid sequence having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity to the amino acid sequence of the FR.

[0147] In the present application, the antigen-binding protein may comprise a VH, which may comprise the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:21, or SEQ ID NO:28. For example, the VH may be a wild-type sequence. For example, one or more amino acid sequences in the VH may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the VH may be a variant thereof, wherein the variant comprises a substitution, deletion, and / or addition of one or more amino acids in the amino acid sequence of the FR. For example, the VH may also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology to the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:21, or SEQ ID NO:28.

[0148] In the present application, the antigen-binding protein may comprise a VL, which may comprise the amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 26. For example, the VL may be a wild-type sequence. For example, one or more amino acid sequences in the VL may be mutated or optimized without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the VL may be a variant thereof, comprising a substitution, deletion, and / or addition of one or more amino acids to the amino acid sequence of the FR. For example, the VL may also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology to the amino acid sequence set forth in either SEQ ID NO: 16 or SEQ ID NO: 26.

[0149] In the present application, the antigen binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 21 or SEQ ID NO: 28, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 26.

[0150] In the present application, the antigen-binding protein may comprise VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 8, and the VL comprises the amino acid sequence shown in SEQ ID NO: 16. In the present application, the antigen-binding protein may comprise VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26. In the present application, the antigen-binding protein may comprise VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26.

[0151] In the present application, the antigen-binding protein may further comprise an immunoglobulin constant region. In the present application, the immunoglobulin constant region may be an IgG heavy chain constant region and / or an antibody light chain constant region. In the present application, the IgG heavy chain constant region may be an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. For example, the IgG heavy chain constant region may be an IgG1 heavy chain constant region. In the present application, the antibody light chain constant region may be a kappa (Kappa) or lambda (Lambda) light chain constant region.

[0152] In the present application, the immunoglobulin constant region can be an immunoglobulin constant region of any species. For example, the immunoglobulin constant region can be an immunoglobulin constant region of mouse, rabbit, goat or human origin. For example, the immunoglobulin constant region can be a human immunoglobulin constant region. In the present application, the immunoglobulin constant region can be a heavy chain constant region of human IgG and / or a light chain constant region of a human antibody. In the present application, the heavy chain constant region of human IgG can be a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. For example, the heavy chain constant region of human IgG can be a human IgG1 heavy chain constant region. In the present application, the light chain constant region of the human antibody can be a human kappa (Kappa) or lambda (Lambda) light chain constant region.

[0153] In the present application, the antigen-binding protein may be an antibody or an antigen-binding fragment thereof. In the present application, the antigen-binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment of the antibody.

[0154] In the present application, the antigen-binding protein can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen-binding protein can be mutated / optimized in the FR and constant regions without reducing the binding activity / affinity of the antigen-binding protein to PTK7. For example, the antigen-binding protein can have low immunogenicity without reducing the binding activity / affinity of the antigen-binding protein to PTK7.

[0155] In the present application, the antigen-binding protein may be a monospecific antibody, a bispecific antibody, or a multispecific antibody. For example, the antigen-binding protein may further comprise a second domain. For example, the second domain may target PTK7 or another antigen. For example, the second domain may target the same epitope or a different epitope of PTK7. In the present application, the second domain may be an antibody or antigen-binding fragment thereof with the same or different antigenic specificity. In the present application, the second domain may target a tumor-associated antigen or a tumor-specific antigen. For example, the second domain may target a hematologic tumor or a solid tumor antigen. For example, the antigen-binding protein may further comprise a third domain. For example, the third domain may target PTK7 or another antigen. For example, the third domain may target the same epitope or a different epitope of PTK7. In the present application, the third domain may be an antibody or antigen-binding fragment thereof with the same or different antigenic specificity. In the present application, the third domain may target a tumor-associated antigen or a tumor-specific antigen. For example, the third domain may target a hematologic tumor or a solid tumor antigen.

[0156] In the present application, the antigen-binding protein can be a monovalent antibody, a bivalent antibody, or a multivalent antibody. For example, the antigen-binding protein can have one antigen-binding site. For example, the antigen-binding protein can have two antigen-binding sites. For example, the antigen-binding protein can have multiple antigen-binding sites. In the present application, the antigen-binding site is the site where the antigen-binding protein binds to the antigen molecule.

[0157] In the present application, the antigen-binding protein can be expressed as a fusion protein. For example, the antigen-binding protein can be fused to a peptide, polypeptide, amino acid, or protein. In the present application, the fusion protein can be a Fab fusion protein, an Fc fusion protein, or a single-chain antibody fusion protein. For example, the antigen-binding protein can be fused to one or more functional molecules. For example, the functional molecules can be one or more antigen-binding proteins. For example, the functional molecules can be one or more Fc regions.

[0158] In the present application, the antigen-binding protein may be wild-type. For example, the antigen-binding protein may be produced by a B cell. For example, the antigen-binding protein may comprise a wild-type sequence. In the present application, the antigen-binding protein may be recombinant. For example, the antigen-binding protein may be an antibody produced using recombinant DNA technology. In the present application, the antigen-binding protein may be modified. For example, the antigen-binding protein may be mutated or optimized. For example, the antigen-binding protein may be substituted, deleted, and / or added with one or more amino acids, such as 1-30, 1-20, or 1-10, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions.

[0159] Immunoconjugates

[0160] On the other hand, the present application provides an immunoconjugate, which may include the antigen binding protein. In the present application, the immunoconjugate can specifically bind to PTK7. For example, the immunoconjugate can specifically bind to PTK7-positive target cells.

[0161] In the present application, the immunoconjugate may comprise an antigen-binding protein, a linker, and a payload as described herein. In the present application, the parts of the immunoconjugate are directly or indirectly connected. For example, the parts of the immunoconjugate are indirectly connected. For example, the parts of the immunoconjugate are connected via a linker.

[0162] In the present application, the antigen binding protein can target PTK7. For example, the antigen binding protein can be the aforementioned antigen binding protein.

[0163] In the present application, the payload may be a cytotoxic drug, a polymer, a protein, a radioisotope, a nucleic acid compound or a glucocorticoid.

[0164] In the present application, the immunoconjugate can be an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC) or a targeted nuclear drug (RDC). For example, the immunoconjugate can be an antibody drug conjugate.

[0165] In the present application, the PROTAC-antibody conjugate may comprise the antigen binding protein, a linker, and a PROTAC. In the present application, the PROTAC may comprise a target protein ligand, a linker, and an E3 ligase ligand. In the present application, the E3 ligase ligand may be CRBN, VHL, IAP, MDM2, DCF15, RNF114, DCAF16, KEAP1, or FEM1B.

[0166] In the present application, the targeted nuclear medicine may comprise the antigen binding protein, a linker and a radioactive isotope. In the present application, the radioactive isotope may be iodine-131, radium-223, thallium-201 or arsenic-211.

[0167] On the other hand, the present application also provides an antibody-drug conjugate comprising the antigen-binding protein, which has one or more of the following properties: (1) the ability to specifically bind to the PTK7 antigen with good binding activity; (2) the ability to specifically bind to PTK7 expressed on the surface of target cells; and (3) the ability to kill tumor cells. In the present application, the antibody-drug conjugate (ADC) can be a single-loaded, dual-loaded ADC, or multi-loaded ADC.

[0168] In the present application, the immunoconjugate may comprise one or more payloads. In the present application, the immunoconjugate may comprise one or more payloads. For example, the immunoconjugate may comprise one, two or more (e.g., 3, 4, 5, 6, 7 or 8) cytotoxic drugs.

[0169] In the present application, the immunoconjugate may comprise the antigen binding protein, a linker, and a cytotoxic drug. In the present application, the cytotoxic drug may be a chemotherapeutic drug, a tubulin inhibitor, a DNA damaging agent, or a topoisomerase I inhibitor. In the present application, the chemotherapeutic drug may be vinblastine, adriamycin, or a derivative thereof. In the present application, the tubulin inhibitor may be auristatin, eribulin, maytansine, tubulysin, cryptocolistin, an anti-mitotic EG5 inhibitor, or a derivative thereof. For example, the cytotoxic drug may be MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), DM1, DM4, or Tubulysins. In the present application, the DNA damaging agent may be calicheamicin, duocarmycin, anthramycin derivatives, pyrrolobenzazepines, indolechlorobenzazepines, or a derivative thereof. For example, the DNA damaging agent may be PBD or IBD. In the present application, the cytotoxic drug can be an apoptosis inducer (such as a Bcl-xL inhibitor), thailanstatin and its analogs (such as thailanstatin A), amanita toxin (such as α-amanitin glycoprotein or β-amanitin toxin), nicotinamide phosphoribosyltransferase or carmamycin (such as carmamycin A and carmamycin B). In the present application, the topoisomerase I inhibitor can be camptothecin, hydroxycamptothecin, irinotecan, topotecan or its derivatives. For example, the topoisomerase I inhibitor can be Dxd or SN-38. For example, the cytotoxic drug can be MMAE or Dxd. For example, the topoisomerase I inhibitor can be an irinotecan derivative having a structure shown in formula (Ia), formula (Ib) or formula (Ic),

[0170] or a pharmaceutically acceptable salt, isotope label, stereoisomer or prodrug thereof; wherein R1, R2, R4, R5 and R7 are independently selected from hydrogen, substituted or unsubstituted C1-C6 straight-chain alkane, substituted or unsubstituted C1-C6 branched alkane, substituted or unsubstituted three- to six-membered ring, or substituted or unsubstituted PEG chains of different lengths; or, R1 and R2 form a substituted or unsubstituted three- to six-membered ring or spiro ring with the nitrogen atom of the urea group to which they are attached; or, R4 and R5 form a substituted or unsubstituted three- to six-membered ring with the nitrogen atom of the thiourea group to which they are attached. wherein R1 and R2 are not hydrogen at the same time, R4 and R5 are not hydrogen at the same time, and when R1 and R3 are hydrogen, R2 is not -CH2CH2OH, when R2 and R3 are hydrogen, R1 is not -CH2CH2OH, and when R4 and R6 are hydrogen, R5 is not -CH2CH2OH, and when R5 and R6 are hydrogen, R4 is not -CH2CH2OH.

[0171] For example, the topoisomerase I inhibitor may have the structure shown below and its isomers:

[0172] For example, the topoisomerase I inhibitor may have the structure shown below and its isomers:

[0173] In the present application, the payload can be a cytotoxic drug. For example, the cytotoxic drug can be MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), DM1, DM4, Tubulysins, PBD, IBD, Dxd, SN-38, Bcl-xL inhibitor, thailanstatin A, α-amanitin glycoprotein, β-amanitin toxin, carmamycin A and / or carmamycin B. For example, the payload can be MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), DM1, DM4, Tubulysins, PBD, IBD, Dxd, SN-38, Bcl-xL inhibitor, thailanstatin A, α-amanitin glycoprotein, β-amanitin toxin, carmamycin A and / or carmamycin B. For example, the payload can be MMAE or Dxd.

[0174] In the present application, the antigen binding protein and the cytotoxic drug are connected by a linker. In the present application, the linker can be a non-cleavable linker or a cleavable linker. In the present application, the cleavable linker can be broken in the target cell and release the payload. In the present application, the cleavable linker can be an enzyme-cleavable linker, an acid-cleavable linker or a GSH-cleavable reducing linker. In the present application, the linker can be an Fe(II)-cleavable linker, a photoresponsive cleavable linker or a bioorthogonal cleavable linker. In the present application, the linker can be a hydrazone, a disulfide bond, a carbonate, an acetal, a ketal, MC-VC-PABC, PEG8-VA-PABC, MC-VC-PAB, MCC, MC, GGFG (SEQ ID NO: 29) or MC-GGFG, etc. For example, the linker can be MC-VC-PAB or GGFG.

[0175] In the present application, the antibody drug conjugate may further comprise a spacer unit. In the present application, the spacer unit may be embedded between the cleavable linker and the payload, or may itself be part of the cleavable linker. In the present application, the spacer unit can spontaneously rearrange its structure after the cleavable linker is broken under appropriate conditions, thereby releasing the payload connected thereto. In the present application, the spacer unit may be an aminobenzyl alcohol (PAB), a β-glucuronide (β-Glucuronide), a substituted or unsubstituted ethylenediamine, and the like.

[0176] In the present application, the immunoconjugate may comprise the antigen-binding protein described herein, a linker, and a payload. The antigen-binding protein may target PTK7. The linker may be a hydrazone, a disulfide bond, a carbonate, an acetal, a ketal, MC-VC-PABC, PEG8-VA-PABC, MC-VC-PAB, MCC, MC, GGFG (SEQ ID NO: 29), or MC-GGFG, etc. The payload may be MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), DM1, DM4, Tubulysins, PBD, IBD, Dxd, SN-38, a Bcl-xL inhibitor, thailanstatin A, α-amanitin glycoprotein, β-amanitin toxin, carmamycin A, and / or carmamycin B. In the present application, the immunoconjugate may comprise the antigen binding protein, linker and payload described in the present application, the antigen binding protein may be the aforementioned antigen binding protein, the linker may be MC-VC-PAB or GGFG, and the payload may be MMAE or Dxd.

[0177] In the present application, the immunoconjugate may comprise the antigen-binding protein, a linker, and MMAE. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and MMAE. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and MMAE; the antigen-binding protein may comprise HCDR1, HCDR2, and HCDR3; the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and MMAE; the antigen-binding protein may comprise VH; the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 28. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and MMAE; the antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 10 (GAS), and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 11. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and MMAE; the antigen-binding protein may comprise VH and VL; the VH may comprise the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 28, and the VL may comprise the amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 26. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB and MMAE, the antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16.In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and MMAE; the antigen-binding protein may comprise VH and VL; the VH may comprise the amino acid sequence shown in SEQ ID NO: 21; and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and MMAE; the antigen-binding protein may comprise VH and VL; the VH may comprise the amino acid sequence shown in SEQ ID NO: 28; and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0178] In the present application, the immunoconjugate may comprise the antigen-binding protein, a linker, and Dxd. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and Dxd. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and Dxd. The antigen-binding protein may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and Dxd. The antigen-binding protein may comprise VH, wherein the VH may comprise the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 28. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and Dxd. The antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The HCDR1 may comprise the amino acid sequence of SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence of SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence of SEQ ID NO: 3, the LCDR1 may comprise the amino acid sequence of SEQ ID NO: 9, the LCDR2 may comprise the amino acid sequence of SEQ ID NO: 10 (GAS), and the LCDR3 may comprise the amino acid sequence of SEQ ID NO: 11. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and Dxd. The antigen-binding protein may comprise VH and VL. The VH may comprise the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 28, and the VL may comprise the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 26. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB and Dxd, the antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16.In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and Dxd; the antigen-binding protein may comprise VH and VL; the VH may comprise the amino acid sequence shown in SEQ ID NO: 21; and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26. In the present application, the immunoconjugate may comprise the antigen-binding protein, MC-VC-PAB, and Dxd; the antigen-binding protein may comprise VH and VL; the VH may comprise the amino acid sequence shown in SEQ ID NO: 28; and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0179] In the present application, the immunoconjugate may comprise the antigen-binding protein, a linker, and Dxd. In the present application, the immunoconjugate may comprise the antigen-binding protein, GGFG, and Dxd. In the present application, the immunoconjugate may comprise the antigen-binding protein, GGFG, and Dxd, and the antigen-binding protein may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3. In the present application, the immunoconjugate may comprise the antigen-binding protein, GGFG, and Dxd, and the antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 28. In the present application, the immunoconjugate may comprise the antigen-binding protein, GGFG, and Dxd; the antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 (GAS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11. In the present application, the immunoconjugate may comprise the antigen-binding protein, GGFG, and Dxd; the antigen-binding protein may comprise VH and VL; the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 21, or SEQ ID NO: 28, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 26. In the present application, the immunoconjugate may comprise the antigen-binding protein, GGFG, and Dxd, the antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16. In the present application, the immunoconjugate may comprise the antigen-binding protein, GGFG, and Dxd, the antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 21, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.In the present application, the immunoconjugate may comprise the antigen binding protein, GGFG and Dxd, the antigen binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 28, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0180] Antigen recognition receptors

[0181] On the other hand, the present application provides a chimeric antigen receptor (CAR), which may comprise the antigen binding protein.

[0182] In the present application, the antigen recognition receptor may be a chimeric antigen receptor (CAR). In the present application, the antigen recognition receptor may be a T cell receptor (TCR). In the present application, the chimeric antigen receptor may include an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor may further include a hinge region. In the present application, the chimeric antigen receptor may further include an intracellular costimulatory domain.

[0183] In the present application, the antigen binding domain of the chimeric antigen receptor may comprise the antigen binding protein. For example, the antigen binding domain may be an antibody or an antigen binding fragment thereof. In the present application, the antigen binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment.

[0184] In the present application, the antigen-binding domain may comprise a VH, the VH may comprise a HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3. In the present application, the VH may comprise a HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2. In the present application, the VH may comprise a HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1.

[0185] In the present application, the antigen-binding domain may comprise VH, the VH may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

[0186] In the present application, the antigen-binding domain may comprise an antigen-binding protein, which may further comprise a VL, wherein the VL may comprise a LCDR3, and wherein the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11. In the present application, the VL may comprise a LCDR2, and wherein the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 (GAS). In the present application, the VL may comprise a LCDR1, and wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9.

[0187] In the present application, the antigen binding domain may comprise VL, the VL may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 (GAS), and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11.

[0188] In the present application, the antigen-binding domain may comprise an antigen-binding protein, the antigen-binding protein comprising an antibody heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 21 or SEQ ID NO: 28. In the present application, the antigen-binding domain may comprise an antigen-binding protein, the caretaker binding protein may comprise an antibody light chain variable region VL, the VL may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 26. In the present application, the antigen-binding domain may comprise an antigen-binding protein, the antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16. In the present application, the antigen-binding domain may comprise an antigen-binding protein, the antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 21, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26. In the present application, the antigen binding domain may comprise an antigen binding protein, the antigen binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 28, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0189] In the present application, the transmembrane domain may comprise a transmembrane domain derived from any one of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.

[0190] In the present application, the costimulatory domain may comprise a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40 and MyD88.

[0191] In the present application, the intracellular signaling domain may comprise an intracellular signaling domain derived from any one of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and a domain comprising at least one ITAM.

[0192] In the present application, the hinge region may comprise a hinge region derived from any one of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

[0193] In the present application, the chimeric antigen receptor may comprise an antigen binding domain targeting PTK7, a hinge region, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain.

[0194] In the present application, the TCR may comprise the antigen binding protein. In the present application, the TCR may specifically bind to PTK7. In the present application, the TCR may comprise an α chain and a β chain, the α chain comprising an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprising a β chain variable region (TRBV) and a β chain constant region (TRBC). For example, the TRAV and / or TRBV may comprise three hypervariable regions CDR1, CDR2, and CDR3.

[0195] On the other hand, the present application provides a modified immune cell, wherein the immune cell may contain and / or express the chimeric antigen receptor and / or T cell receptor. In the present application, the immune cell may contain and / or express one or more antigen recognition receptors. In the present application, the immune cell may contain and / or express one or more antigen recognition receptors. In the present application, the immune cell may contain and / or express one or more chimeric antigen receptors. In the present application, the immune cell may contain and / or express one or more chimeric antigen receptors.

[0196] In the present application, the immune cells can be T cells, NK cells, NKT cells, dendritic cells, macrophages, TIL cells, iNKT cells, CIK cells, γδT cells and / or DNT cells. For example, the immune cells can be immune effector cells. For example, the immune cells can be T cells. For example, the immune cells can be a mixture, and the mixture can contain different types of immune cells, for example, the mixture can contain one or more immune cells.

[0197] Pharmaceutical composition

[0198] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0199] In the present application, the pharmaceutical composition can include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present application can include liquid, frozen and lyophilized compositions.

[0200] In this application, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0201] In the present application, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0202] Drug combinations

[0203] In another aspect, the present application provides a pharmaceutical combination comprising the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition, and one or more active ingredients. For example, the pharmaceutical combination may further comprise a substance associated with an immune response. For example, the pharmaceutical combination may further comprise a drug associated with an immune response.

[0204] On the other hand, the present application provides a pharmaceutical combination comprising the antigen binding protein, the nucleic acid molecule and / or the cell and a therapeutic agent. In the present application, the therapeutic agent can be selected from one or more of the following groups: an anti-tumor drug, a chemotherapeutic agent, a radioisotope or an immune checkpoint inhibitor.

[0205] On the other hand, the present application also provides a scheme for combining the antigen-binding protein with one or more other active ingredients. For example, the antigen-binding protein is combined with one or more other therapeutic agents. In the present application, the drug combination can be administered separately, simultaneously, or sequentially. In the present application, the drug combination can be administered in the same or different doses or administration routes. For example, the active ingredients in the drug combination are administered to the patient as separate entities in the same / different doses and administration routes. In the present application, the ingredients in the drug combination can be administered to the patient simultaneously in the form of a single entity or dose. For example, the ingredients in the drug combination are administered to the patient simultaneously, jointly, or sequentially as separate entities. In the present application, the specific administration route can be determined according to the type of active ingredient, and the specific dosage can be adjusted according to the severity of the subject's condition, the subject's physical condition, etc.

[0206] In the present application, the different active ingredients in the pharmaceutical combination may be mixed or placed separately. For example, the active ingredients may be placed in the same container. For example, the active ingredients may be placed in different containers.

[0207] Preparation method

[0208] In another aspect, the present application provides methods for preparing the antigen-binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition, and / or the drug combination. For example, the method can include introducing the vector into an immune cell. For example, the method can include coupling the antigen-binding protein to a cytotoxic drug. For example, the method can include culturing the cell under conditions that allow expression of the antigen-binding protein and / or the chimeric antigen receptor.

[0209] In the present application, the method may include linking the antigen binding protein, the linker and the payload by a chemical reaction.

[0210] use

[0211] On the other hand, the present application provides the use of the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug combination in the preparation of a drug, which can be used to prevent, diagnose and / or treat diseases and / or conditions.

[0212] On the other hand, the present application provides a method for preventing, diagnosing and / or treating a disease and / or condition, which may comprise administering the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug combination to a subject in need thereof.

[0213] On the other hand, the present application provides the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug combination, which can be used to prevent, diagnose and / or treat diseases and / or disorders.

[0214] In the present application, the disease and / or condition may be a PTK7-related disease and / or condition. In the present application, the disease and / or condition may be a tumor. In the present application, the tumor may be a solid tumor and / or a hematological tumor. In the present application, the tumor may be a PTK7-positive tumor. In the present application, the tumor may be a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer, prostate cancer, and / or esophageal cancer.

[0215] In the present application, the prevention, diagnosis and / or treatment may be preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith.

[0216] In another aspect, the present application provides use of the antigen binding protein for preparing a diagnostic agent, wherein the diagnostic agent is used to diagnose diseases and / or disorders associated with the expression of the PTK7 protein.

[0217] In another aspect, the present application provides use of the antigen binding protein for preparing a diagnostic agent, wherein the diagnostic agent is used to diagnose diseases and / or disorders associated with the expression of the PTK7 protein.

[0218] In another aspect, the present application provides the antigen binding protein for use in diagnosing diseases and / or disorders associated with the expression of PTK7 protein.

[0219] In the present application, the diagnostic agent can be used alone or in combination with an instrument, apparatus, device, or system. In the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation, and prediction of genetic diseases, the diagnostic agent can be used to perform in vitro detection on human samples (e.g., various body fluids, cells, tissue samples, etc.).

[0220] In the present application, the diagnostic agent can be selected from the following groups: reagents, kits, calibrators and quality control products.

[0221] In the present application, the in vitro detection method can be selected from the following group: Western Blot, ELISA and immunohistochemistry.

[0222] For example, the reagent may include a reagent capable of measuring the expression amount of the PTK7 protein.

[0223] For example, the reagent may be selected from the following group: a reagent for performing Western Blot, a reagent for performing ELISA, and a reagent for performing immunohistochemistry.

[0224] In another aspect, the present application further provides a detection kit, which may include the antigen binding protein, and is used to detect the presence and / or content of PTK7 in a sample or subject. For example, the detection kit can be used to prevent, diagnose and / or treat diseases and / or conditions.

[0225] For example, the present application relates to an immunoassay kit using the antibodies of the present application in combination with immunoassay methods such as ELISA, immunohistochemistry, protein blotting, and flow cytometry.

[0226] In the present application, the kit includes the antigen binding protein of the present application for detecting PTK7-related tumor / cancer cells. Specifically, the immunoassay kit will contain the antibody of the present application as the first antibody binding to PTK7, and optional immunoassay reagents in a suitable container component.

[0227] For example, the antibody can be pre-bound to a solid support, such as a column matrix and / or a microtiter plate well.

[0228] In the present application, the immunodetection reagents in the kit can be in any of a variety of forms, including those detectable labels that are bound or linked to a given antibody. Detectable labels that are bound or linked to a secondary binding ligand can also be included. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0229] In the present application, other immunoassay reagents suitable for use in the kits of the present application include a two-component reagent comprising a secondary antibody having binding affinity for the first antibody, and a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As described above, a variety of exemplary labels are known in the art and all such labels can be used in conjunction with the present application.

[0230] On the other hand, the antigen binding protein, the immunoconjugate, the chimeric antigen receptor, the modified immune cell, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug combination described in the present application are used in the preparation of a detection kit, and the detection kit is used for diagnosis.

[0231] In the present application, the kit may further comprise an appropriate aliquot of a PTK7 composition, whether labeled or unlabeled, which can be used to prepare a standard curve for the detection assay. The kit may contain the antibody-label conjugate in fully conjugated form, in the form of an intermediate, or in separate portions that are conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium or in a lyophilized form.

[0232] In the present application, the container means of the test kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody or preferably an appropriate aliquot of the antibody can be placed. The test kit of the present application will also typically include means for containing the antibody, antigen, and any other reagent containers, which are in a sealed form for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the vials are retained.

[0233] On the other hand, the present application also provides a method for detecting the presence and / or content of PTK7, which may include using the antigen binding protein.

[0234] On the other hand, the present application provides a method for diagnosing a disease and / or condition related to the expression of PTK7 protein in a subject, the method comprising: contacting a sample derived from the subject with the antigen-binding protein, and determining the presence and / or amount of a substance capable of specifically binding to the antigen-binding protein in the sample.

[0235] In another aspect, the present application provides a method for detecting PTK7 in a sample or subject, the method comprising administering the antigen binding protein. In the present application, the administration can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0236] Nucleic acids, vectors, and cells

[0237] In another aspect, the present application provides an isolated nucleic acid molecule, which can encode the antigen binding protein and / or the chimeric antigen receptor.

[0238] In the present application, the nucleic acid molecule may further comprise a sequence encoding a signal peptide.

[0239] In the present application, the nucleic acid molecule can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cleavage and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis.

[0240] In the present application, the nucleic acid molecule may be DNA and / or RNA. In the present application, the nucleic acid molecule may be an artificially synthesized nucleic acid analog. In the present application, the nucleic acid molecule may be a modified nucleic acid molecule.

[0241] On the other hand, the present application provides a vector, which may contain the nucleic acid molecule.

[0242] In the present application, the vector may comprise one or more nucleic acid molecules. In the present application, the vector may comprise one or more nucleic acid molecules.

[0243] In the present application, the vector may be an expression vector or a cloning vector. In the present application, the vector may be a viral vector or a non-viral vector. In the present application, the vector may be a viral vector, a plasmid vector, a phage vector or other vectors commonly used in, for example, genetic engineering. For example, the viral vector may be an adenovirus, an adeno-associated virus, a retrovirus (including a lentivirus). In the present application, the vector may be a fusion vector or a non-fusion vector.

[0244] In the present application, the vector may further comprise other genes. For example, the other genes may be marker genes.

[0245] In the present application, the vector may contain a variety of elements that control expression. For example, the vector may include a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and / or a reporter gene. For example, the vector may also contain a replication initiation site. For example, the vector may include components that assist entry into cells. In order to allow the nucleic acid molecule to replicate in the vector, the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeats.

[0246] In another aspect, the present application provides a cell, which may contain the nucleic acid molecule and / or the vector.

[0247] In the present application, the cell may include the progeny of a single cell. Due to natural, accidental or intentional mutations, the progeny may not necessarily be completely identical to the original parent cell (in the form of total DNA complement or in the genome).

[0248] In the present application, the cell can be a prokaryotic cell (eg, a bacterial cell), a CHO cell, a NS / 0 cell, a HEK293T cell or a HEK293A cell, or other eukaryotic cells, such as a fungal or yeast cell.

[0249] In the present application, the cell may comprise one or more of the nucleic acid molecules and / or one or more vectors. In the present application, the vector may comprise one or more of the nucleic acid molecules and / or one or more vectors.

[0250] In the present application, the vector can be introduced into the cell by methods known in the art. For example, the method can be electroporation, lipofectine transfection or lipofectamin transfection.

[0251] On the other hand, the present application provides a method for preparing the antigen-binding protein, which comprises culturing the cells under conditions allowing the antigen-binding protein to be expressed.

[0252] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0253] Example

[0254] Example 1 Preparation of mouse monoclonal antibodies against PTK7

[0255] The method for preparing mouse monoclonal antibodies utilizes the hybridoma preparation technique invented by Kohler and Milstein in 1975 (Nature, 1975, 256:495-497). Human PTK7 / CCK4-His protein (KACYUS cat no. CCK-HM104) was first used as the immunizing antigen, and immunization was performed using Freund's Adjuvant Complete (FCA, Sigma cat no. F5881) and Freund's Adjuvant Incomplete (FICA, Sigma cat no. F5006). Multiple BalB / C, ICR, and SJL mice were subcutaneously immunized at multiple sites. After three immunizations, serum was collected and titered using ELISA. When the serum titer reached the target value, a pre-fusion boost immunization was performed. Finally, the best mice were selected to obtain spleen cells for fusion with SP2 / 0 myeloma cells. Hybridoma cell lines were screened using HAT assays. Hybridoma cell supernatants were then screened using ELISA to identify hybridoma clones that bind to humans. These human-binding hybridoma clones were then screened for monkey and mouse specific binding using ELISA to identify monoclonal hybridoma cell lines that specifically bind to humans, mice, and monkeys. These selected monoclonal cell lines were then affinity screened (Biacore) to obtain mouse-derived PTK7 antibodies. The CDR sequences (IMGT definition) of the mouse anti-PTK7 antibody are shown below.

[0256] Table 1 CDR sequences of mouse anti-PTK7 antibodies

[0257] Example 2 Cloning and humanization of anti-PTK7 antibody variable region gene sequences

[0258] 2.1 Cloning of variable region genes of antibodies in hybridoma cells

[0259] Based on TAKARA's 5' RACE technology, cDNA sequences of mouse antibody variable regions expressed in hybridoma cell lines were cloned. Total cellular RNA was first extracted according to the RNeasy 96 Kit (Qiagen, Catalog No. 74181). Heavy and light chain variable region gene-specific cDNAs were synthesized using the SMARTer 5' RACE Synthesis Kit (TAKARA, Catalog No. 634859) according to the manufacturer's instructions. The 5' and 3' ends of the cDNA sequences were modified using PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively. This enabled the resulting PCR products to be seamlessly cloned into the existing recombinant antibody expression vectors pHB-Fc and pHB-CK. The pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence; the pHB-CK vector contains the human κ light chain constant region gene sequence. The PCR amplified products of the heavy and light chain variable regions were cloned into an expression vector using an In-fusion cloning reagent (TAKARA, Catalog No. 639650) to generate a human-mouse chimeric antibody expression vector. This vector was then transformed into competent E. coli DH5α cells (Yisheng Biotechnology, Catalog No. FYE607-80VL). Monoclonal colonies were selected and Sanger sequenced to analyze the variable region sequences, yielding the variable region sequence of the anti-PTK7 chimeric antibody (Catalog No. 900868).

[0260] The amino acid sequence of VH of the anti-PTK7 chimeric antibody (No. 900868) is shown in SEQ ID NO: 8, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; the amino acid sequence of 900868 VL is shown in SEQ ID NO: 16, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 (GAS), and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11.

[0261] 2.2 Expression of chimeric antibodies

[0262] The expression vector obtained in 2.1 was amplified in E. coli, and sufficient plasmid was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No. DP117) for transient transfection and expression of the chimeric antibody. The host cells used for expression were CHO-S cells (Thermo Fisher Scientific, Catalog No. R80007). The heavy and light chain vectors were mixed with polyetherimide (PEI, Polysciences, Catalog No. 24765-1) to form a liposome complex, which was then transfected into CHO-S cells and cultured in an incubator for 7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity chromatography column to obtain the human-mouse chimeric antibody.

[0263] 2.3 Humanization of murine anti-PTK7 antibody

[0264] The variable region sequences of the antibodies were compared with the available sequences in the NCBI IgBlast database, and through identification and analysis, the FR regions suitable for constructing CDR-grafted heavy and light chains were finally determined.

[0265] During the modification process, modification sites were designed based on conserved amino acid residues in the human antibody FR region and key amino acid residues within the antibody FR region. Mouse-derived back mutations were designed in the variable regions of the heavy and light chains of the chimeric antibody. Overlap extension PCR primers were synthesized, and a humanized point mutation antibody gene library was constructed using PCR technology and ligated into an antibody expression plasmid. The constructed expression plasmids were expressed in CHO-S cells and purified to obtain humanized antibody proteins. The humanized antibodies were screened for affinity and endocytosis using Biacore and cell bioactivity assays, resulting in two high-performance humanized anti-PTK7 antibodies, 900935 and 900937.

[0266] The amino acid sequence of 900935VH is shown in SEQ ID NO:21, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; the amino acid sequence of 900935VL is shown in SEQ ID NO:26, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10 (GAS), and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.

[0267] The amino acid sequence of 900937VH is shown in SEQ ID NO:28, HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; the amino acid sequence of 900937VL is shown in SEQ ID NO:26, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10 (GAS), and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.

[0268] 2.4 Antigen affinity of anti-PTK7 antibodies

[0269] Antibody affinity was determined by Biacore (SPR) using the following protocol: a Biacore 8k laminar flow cell was set at 25°C. The test antibody was used as the ligand. Cynomolgus / rhesus PTK7, His-tagged (ACRO Biosystems, PT7-C52H3) and human PTK7 / CCK4, His-tagged (ACRO Biosystems, PT7-H52H3) were diluted to 50 nM, followed by six two-fold dilutions for detection. The flow rate was 30 μL / min, with binding times of 120 s and dissociation times of 600 s. Kinetic constants were analyzed using 10 mM glycine, pH 1.5 (Cytiva, BR100354), at a flow rate of 50 μL / min and a regeneration time of 30 s.

[0270] The affinity test results of the anti-PTK7 humanized antibodies are shown in Table 2. 900868, 900935, and 900937 can simultaneously recognize human and cynomolgus monkey PTK7 antigens, and all have higher or equivalent affinities than the control antibodies 900797 and BMS (7C8). After humanization, the chimeric antibody 900868 has a KD value comparable to that of the chimeric antibody, indicating that the antibodies of the present application have good affinity before and after humanization. The experimental results show that the humanized antibodies described in the present application maintain the same antigen affinity as the parent antibody, and the binding activity of the antibodies described in the present application to the PTK7 antigen is better than that of the control antibody, and have a higher affinity.

[0271] Table 2 Affinity determination of anti-PTK7 humanized antibodies

[0272] The control antibodies 900797 (US20150315293A1) and BMS (7C8) (US9505845B2) are anti-PTK7 antibodies from Pfizer and BMS, respectively, and were independently prepared based on the sequences published in the patents.

[0273] 2.5 Detection of endocytosis of anti-PTK7 antibodies in tumor cells PANC-1

[0274] The antibody internalization was detected on the pancreatic cancer cell line PANC-1. PANC-1 cells with good growth (purchased from the Chinese Academy of Sciences) were collected and counted, and resuspended in 1% BSA / PBS working solution to 5×10 5 40 μL / well of cell suspension was plated into a 96-well U-bottom plate. Chimeric antibody 900868, control antibody 900797, humanized antibodies 900935 and 900937 were diluted to 60 μg / mL with 1% BSA / PBS working solution, and then diluted 3-fold to 10 concentrations, for a total of 11 concentration gradients. The diluted antibody was added to the 96-well U-bottom plate with cells at 40 μL / well, 1% BSA was used as NC (negative control), and incubated at 4°C for 30 minutes. Two identical plates need to be set up for subsequent endocytosis detection at different temperatures. After the incubation is completed, 1% BSA / PBS working solution was added to the 96-well U-shaped plate at 200 μL / well, centrifuged at 300g for 3 minutes, and the supernatant was discarded and repeated once. The cells in the 96-well U-shaped plates were then resuspended in 100 μL / well of 1% BSA / PBS working solution. One experimental plate was incubated at 4°C for 3 h, and the other experimental plate was incubated at 37°C for 3 h. After incubation, 1% BSA / PBS working solution was added to the 96-well U-shaped plates, and 200 μL / well of 1% BSA / PBS working solution was added. Centrifugation was repeated at 300g for 3 min, and the supernatant was discarded. After washing, the fluorescent secondary antibody (R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ Fragment, Jackson, Cat. No. 109-115-098) was diluted 1:200 in 1% BSA / PBS working solution. 20 μL / well was added to the sample wells and blank wells (cells + fluorescent secondary antibody). The same volume of 1% BSA / PBS working solution was added to the cell-blank wells, and the cells were incubated at 4°C in the dark for 30 min. After incubation, add 200 μL / well of 1% BSA / PBS working solution to a 96-well U-shaped plate. Centrifuge at 300g for 3 minutes, discard the supernatant, and repeat two times for a total of three washes. After washing, resuspend the cells in 100 μL / well of 1% BSA / PBS working solution and analyze them on a Canto II flow cytometer to read the MFI values. GraphPad Prism 8 software was used to fit the data using a four-parameter equation to generate the antibody-target cell endocytosis curve.

[0275] The target cell endocytosis curves are shown in Figure 1. Antibodies 900868, 900935, and 900937 all exhibited moderate endocytosis activity, with similar or higher endocytosis activity compared to the control antibody 900868. The endocytosis activity of the antibodies was dose-dependent. These experimental results demonstrate that the antibodies described herein possess high endocytosis activity and are suitable for preparation as ADCs.

[0276] Example 3 Anti-PTK7 Antibody Drug Conjugate

[0277] 3.1 Coupling

[0278] Two anti-PTK7 humanized antibodies 900937 and 900935 were conjugated with MMAE using MC-VC-PAB as the linker, with a target DAR value of 4.

[0279] The preparation process of 900935-MMAE is as follows:

[0280] (1) Antibody preparation: The antibody was replaced in coupling buffer (20 mM His-His·HCl, pH 6.0);

[0281] (2) Reduction reaction: Antibody, DTPA (10 mM), and TCEP (5 mM) were added sequentially and placed in a thermomixer to achieve a molar ratio of antibody to TCEP of 1:2.7 and a final DTPA concentration of 1 mM. The mixture was stirred at 15°C and 400 rpm for 1.5 hours.

[0282] (3) Coupling reaction: After the reduction reaction is completed, place the mixture in an ice-water bath, add 20% DMSO to a final ratio, mix thoroughly, add MC-VC-PAB-MMAE at a molar ratio of 1:6.8 between the antibody and MC-VC-PAB-MMAE, mix thoroughly, and place in a thermomixer at 25°C and 400 rpm for 1 hour.

[0283] (4) After the coupling is completed, dialyze repeatedly using an ultrafiltration tube or membrane package (30KD) for several times, and then take an appropriate amount for concentration, HIC-HPLC, SEC-HPLC and free drug residue detection.

[0284] The preparation process of 900937-MMAE is as follows:

[0285] (1) Antibody preparation: The antibody was replaced in coupling buffer (20 mM His-His·HCl, pH 6.0);

[0286] (2) Reduction reaction: Antibody, DTPA (10 mM), and TCEP (5 mM) were added sequentially and placed in a thermomixer to achieve a molar ratio of antibody to TCEP of 1:2.9 and a final DTPA concentration of 1 mM. The mixture was stirred at 25°C and 400 rpm for 1.5 hours.

[0287] (3) Coupling reaction: After the reduction reaction is completed, place the mixture in an ice-water bath, add 20% DMSO to a final ratio, mix thoroughly, add MC-VC-PAB-MMAE at a molar ratio of 1:7 between the antibody and MC-VC-PAB-MMAE, mix thoroughly, and place in a thermomixer at 25°C and 400 rpm for 1 hour.

[0288] (4) After the coupling is completed, dialyze repeatedly using an ultrafiltration tube or membrane package (30KD) for several times, and then take an appropriate amount for concentration, HIC-HPLC, SEC-HPLC and free drug residue detection.

[0289] Positive control antibody 900797 and negative control antibody 900543 were conjugated to MMAE using MC-VC-PAB as the linker. The target DAR value was 4. The preparation process was as follows:

[0290] An appropriate volume of antibody was added to a 20mM His / His-HCl solution (pH 6.0) and mixed. Approximately 2.5 equivalents of TCEP (tris(2-carboxyethyl)phosphine) solution were then added and mixed. The mixture was allowed to react at 25°C for 120 minutes. Subsequently, 8-10 equivalents of MC-VC-PAB-MMAE dissolved in dimethyl sulfoxide were added to the above solution, mixed, and reacted at 25°C for 120 minutes. After completion, 10mM cysteine ​​was added to terminate the reaction, and the mixture was allowed to stand at 25°C for 30 minutes. Finally, a 2mL desalting column with a 40kDa MWCO was used to remove residual small molecule drug from the antibody-drug conjugate, and the ADC product, conjugated to the antibody and MC-VC-PAB-MMAE, was concentrated to the target concentration.

[0291] 3.2 Biacore (SPR) Detection of ADC Affinity

[0292] Biacore (SPR) affinity analysis of ADCs (900935-MMAE and 900937-MMAE) was performed using the following method: a Biacore 8k laminar flow cell was set at 25°C. The detection antibody was used as the ligand. Cynomolgus / rhesus PTK7 with a His tag (ACRO Biosystems, PT7-C52H3) and human PTK7 / CCK4 with a His tag (ACRO Biosystems, PT7-H52H3) were diluted to 50 nM and then tested in a six-fold two-fold dilution series. The flow rate was 30 μL / min, with binding times of 120 s and dissociation times of 300 s. Kinetic constants were analyzed using 10 mM glycine, pH 1.5 (Cytiva, BR100354), at a flow rate of 50 μL / min and a regeneration time of 30 s.

[0293] The affinity test results of the anti-PTK7 ADC are shown in Table 3. 900935-MMAE and 900937-MMAE can simultaneously recognize human and cynomolgus monkey PTK7 antigens, with KD values ​​comparable to those of 900937. The experimental results demonstrate that the antibodies described herein can maintain similar affinity to naked antibodies after being formulated into ADCs, demonstrating strong binding activity.

[0294] Table 3 Affinity determination of anti-PTK7-ADC

[0295] 3.3 ADC Binding Assay on PANC-1 Tumor Cells

[0296] The binding of the ADC prepared with the antibodies described in this application to the pancreatic cancer cell line PANC-1 was tested. PANC-1 cells (purchased from the Chinese Academy of Sciences) in good growth condition were collected and counted. An appropriate amount of cell suspension was transferred to a 15 mL centrifuge tube. After adding 10 mL of PBS, the tube was centrifuged at 500 g for 3 minutes, and the supernatant was discarded. 3 mL of a 1:1000 diluted Zombie series dye (Zombie violet) was added. TM Fixable Viability Kit, BioLegend, Cat. No. 423114), incubate in the dark for 15 min, add 1% BSA / PBS working solution 10 mL for washing after incubation, centrifuge at 500 g for 3 min, discard the supernatant, repeat once, and then resuspend the treated cells in 1% BSA / PBS working solution, count and dilute to 1 × 10 6100 μL / well of 1% BSA / PBS working solution was added to the 96-well U-bottom plate. Antibody-drug conjugates 900937-MMAE, 900797-MMAE, 900935-MMAE, and control antibody 900797 were diluted to 20 μg / mL with 1% BSA / PBS working solution, and then 3-fold serial dilution was performed to 10 concentrations, for a total of 11 concentration gradients. The diluted antibodies were added to the 96-well U-bottom plate with cells at 20 μL / well. The control (cells + fluorescent secondary antibody) and cell blank wells were mixed with the same volume of 1% BSA / PBS and incubated at room temperature for 30 minutes. After incubation, 1% BSA / PBS working solution was added to the 96-well U-shaped plate at 100 μL / well. The cells were centrifuged at 500g for 3 minutes, and the supernatant was discarded. This was repeated twice for a total of three washes. After washing, a fluorescent secondary antibody (R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ Fragment, Jackson, Cat. No. 109-115-098) was diluted 1:200 in 1% BSA / PBS working solution and added to the sample wells and the (cells + fluorescent secondary antibody) wells at 20 μL / well. The same volume of 1% BSA / PBS working solution was added to the cell-free wells and incubated at room temperature for 15 min in the dark. After incubation, 100 μL / well of 1% BSA / PBS working solution was added to the 96-well U-shaped plate. The cells were centrifuged at 500 g for 3 min, and the supernatant was discarded. This was repeated two times for a total of three washes. After washing, the cells were resuspended in 100 μL / well of 1% BSA / PBS working solution and analyzed on a Canto II flow cytometer. MFI values ​​were read. Data were analyzed using GraphPad Prism 8 software using a four-parameter equation to calculate the antibody-target cell binding curve.

[0297] The binding results of ADC and control antibody to target cells are shown in Figure 2. The humanized antibodies 900935 and 900937, after being coupled with MMAE, and the control antibody 900797, after being coupled with MMAE, have comparable binding abilities to PTK7 on PANC-1 target cells, indicating that the antibodies described in the present application still have strong affinity after being prepared as ADCs and can bind to PTK7 on target cells with high binding activity.

[0298] 3.4 Detection of ADC Cytotoxicity on BxPC-3 Tumor Cells

[0299] The cell killing effect of the ADC prepared by the antibody described in this application on the pancreatic cancer cell line BxPC-3 was tested. Day 1 BxPC-3 cells (purchased from ATCC) with good growth status were collected and counted, and resuspended in culture medium to 2×10 4100 μL / well of a cell suspension containing 100 cells / mL was plated into a 96-well cell culture plate. Side wells were sealed with PBS, and 200 μL / well was added. The cells were incubated at 37°C, 5% CO2 for 6 h. 900937-MMAE, 900935-MMAE, 900797-MMAE, and 900543-MMAE (isotype control; 900543 is an IgG1 (Fc silenced) negative control antibody targeting the small molecule TNP) were diluted to 1280 nM in culture medium (RPMI 1640 + 10% FBS). This was followed by a 4-fold serial dilution to two concentrations, and then a 3-fold serial dilution to six concentrations, for a total of nine concentrations. The diluted antibodies were added to the 96-well cell culture plate at 100 μL / well and incubated at 37°C, 5% CO2 for another 6 days. Day 7 After incubation, the 96-well cell culture plate was centrifuged at 300g for 3 minutes, all the culture medium was aspirated, and then equilibrated to room temperature was added. Luminescent Cell Viability Assay Kit (Promega, Cat. No. G7573) was used at 100 μL / well. The cells were shaken at 300 rpm for 10 minutes at room temperature in the dark, and the cells were detected using a microplate reader with full wavelength. GraphPad Prism 8 software was used to fit the data using a four-parameter equation to generate dose-effect curves.

[0300] The results of BxPC-3 target cell killing are shown in Figure 3. Compared with 900543-MMAE, 900935-MMAE and 900937-MMAE showed better killing effects on BxPC-3 target cells and comparable killing effects to 900797-MMAE. This indicates that the humanized antibodies 900935 and 900937 can have a higher killing effect after being conjugated with MMAE. These experimental results demonstrate that the antibodies described herein retain strong target cell binding and killing abilities after being formulated into ADCs.

[0301] 3.5 ADC Cytotoxicity Test on HGC-27 Tumor Cells

[0302] The cell killing effect of the ADC prepared by the antibody described in this application on HGC-27 cells was detected. HGC-27 cells in the logarithmic growth phase were prepared into 1×10 4 900937-MMAE, 900937-MMAE and 900797-MMAE were diluted to 1 μM with HGC-27 complete cell culture medium, then diluted 2-fold to 9 gradient concentrations, 50 μL / well was added to a 96-well plate, and then 1×10 4Place the 96-well plate in a carbon dioxide incubator (3111, Thermo Fisher) at 5% CO2 at 37°C for 4 days, take out the 96-well plate, and add 100 μL of substrate (G7573, Promega) was added and incubated in the dark for 15 minutes. Data were collected using a microplate reader. Dose-effect curves were fitted using GraphPad Prism software.

[0303] The results of HGC-27 target cell killing are shown in Figure 4. Compared with 900797-MMAE, 900935-MMAE and 900937-MMAE showed lower HGC-27 target cell counts at the same dose, indicating that the humanized antibodies 900935 and 900937 have a higher killing effect after being conjugated with MMAE. These experimental results demonstrate that the antibodies described herein retain strong target cell binding and enhanced target cell killing capabilities after being formulated into ADCs.

[0304] 3.6 In vivo efficacy

[0305] 3.6.1 Antitumor Efficacy Testing of ADCs in the PANC-1 CDX Pancreatic Cancer Mouse Model

[0306] A PANC-1 pancreatic cancer mouse model was established using NTG mice and the efficacy of the test antibody-drug conjugates was tested. The human pancreatic cancer cells PANC-1 used in this example were cultured in DMEM containing 10% fetal bovine serum in a 37°C, 5% CO2 cell culture incubator. Every 3 to 4 days, the cells were cultured in flasks after confluence. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo. PANC-1 tumor cells were resuspended in PBS + Matrigel (1:1) at a concentration of 1×10 8 The tumor was inoculated subcutaneously on the right flank of the experimental animals at a rate of 100 μL per animal. 3 The mice were divided into 6 groups (administered via tail vein) with 6 mice in each group. The specific dosing schedule is shown in Table 4.

[0307] The results of anti-tumor efficacy testing in the PANC-1 CDX pancreatic cancer mouse efficacy model are shown in Figure 5. 900543-MMAE serves as a negative control. After the humanized antibodies 900935 and 900937 described in this application were conjugated to MMAE, 900935-MMAE and 900937-MMAE significantly inhibited tumor growth at a dose of 2 mg / kg, with tumor volume gradually decreasing. The inhibitory effect was positively correlated with the dosage administered. These experimental results demonstrate that the antibodies described in this application, when prepared as ADCs, possess high anti-tumor activity and are able to effectively inhibit tumor growth in the PANC-1 CDX pancreatic cancer mouse efficacy model.

[0308] Table 4 Dosing regimen for pancreatic cancer PANC-1 CDX model

[0309] 3.6.2 Antitumor Efficacy Testing of ADCs in PDX Pancreatic Cancer Mouse Model

[0310] The experimental animals used in this example were severely immunodeficient mice, which were Il2rg knockout mice on a NOD / Shi-Scid background strain, meeting the conditions required for the model. 3 ) were subcutaneously inoculated into the right forelimb of each mouse, and the tumor volume (100 mm 3 The animals were randomly divided into groups according to their weight (about 100 mg / kg), with 4 animals in each group. The groups included (1) negative control group: Vehicle group (solvent control group); (2) test drug group: 900937-MMAE group (10 mg / kg). The administration frequency was twice a week, with two weeks of administration and two weeks of observation.

[0311] The anti-tumor efficacy test results in the PDX pancreatic cancer mouse model are shown in Figure 6. After the humanized antibody 900937 described in this application was conjugated to MMAE, tumor volume continued to decrease after one week of administration, eventually approaching zero. 900937-MMAE completely inhibited tumor growth without affecting mouse body weight. These experimental results demonstrate that the antibody described in this application, when formulated as an ADC, exhibits high anti-tumor activity and is able to completely inhibit tumor growth in the PDX pancreatic cancer mouse model.

[0312] 3.6.3 Antitumor Efficacy Testing of ADCs in PDX Lung Cancer Mouse Model

[0313] The experimental animals used in this example were severely immunodeficient mice, which were Il2rg knockout mice on a NOD / Shi-Scid background strain, meeting the conditions required for the model. 3 ) were subcutaneously inoculated into the right forelimb of each mouse, and the tumor volume (100 mm 3 The animals were randomly divided into groups (approximately 200 mg / kg) and body weight, with 4 animals in each group; the groups included (1) negative control group: Vehicle group; (2) test drug group: 900937-MMAE group (10 mg / kg); the administration frequency was twice a week, two weeks of administration, and two weeks of observation.

[0314] The anti-tumor efficacy test results in the PDX lung cancer mouse model are shown in Figure 7. After the humanized antibody 900937 described in this application was conjugated to MMAE, tumor volume continued to decrease after one week of administration, eventually approaching zero. 900937-MMAE completely inhibited tumor growth without affecting mouse body weight. These experimental results demonstrate that the antibody described in this application, when formulated as an ADC, exhibits high anti-tumor activity and is able to completely inhibit tumor growth in the PDX lung cancer mouse model.

[0315] 3.6.4 Antitumor Efficacy Testing of ADCs in a PDX Gastric Cancer Mouse Model

[0316] Female Nu / Nu mice, 6-8 weeks old, weighing 18-22 g, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. When the tumor of the LD1-0017-200710 model-bearing mice grew to 500-800 mm 3 The tumor was removed under sterile conditions and then the tumor tissue was evenly cut into tumor pieces of about 3 mm × 3 mm × 3 mm (about 30 mg) and inoculated subcutaneously on the right side of Nu / Nu mice. 3 The animals were randomly divided into groups (approximately 200 mg / kg) and body weight, with 4 animals in each group; the groups included (1) negative control group: Vehicle group; (2) test drug group: 900937-MMAE group (10 mg / kg); the administration frequency was twice a week, two weeks of administration, and two weeks of observation.

[0317] The anti-tumor efficacy test results in the PDX gastric cancer mouse model are shown in Figure 8. After the humanized antibody 900937 described in this application was conjugated to MMAE, tumor volume continued to decrease one week after administration. 900937-MMAE significantly inhibited tumor growth without affecting mouse body weight. These experimental results demonstrate that the antibody described in this application, when formulated as an ADC, exhibits high anti-tumor activity and can effectively inhibit tumor growth in the PDX lung cancer mouse model.

[0318] 3.6.5 Antitumor Efficacy Testing of ADCs in PDX Colorectal Cancer Mouse Models

[0319] Female Nu / Nu mice, 6-8 weeks old, weighing 18-22 g, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. When the tumor of the LD1-2012-361664 model-bearing mice grew to 500-800 mm 3 The tumor was removed under sterile conditions and then the tumor tissue was evenly cut into tumor pieces of about 3 mm × 3 mm × 3 mm (about 30 mg) and inoculated subcutaneously on the right side of Nu / Nu mice. 3The animals were randomly divided into groups (approximately 200 mg / kg) and body weight, with 4 animals in each group; the groups included (1) negative control group: Vehicle group; (2) test drug group: 900937-MMAE group (10 mg / kg); the administration frequency was twice a week, two weeks of administration, and two weeks of observation.

[0320] The anti-tumor efficacy test results in the PDX colorectal cancer mouse model are shown in Figure 9. After the humanized antibody 900937 described in this application was conjugated to MMAE, tumor volume continued to decrease one week after administration. 900937-MMAE significantly inhibited tumor growth without affecting mouse body weight. These experimental results demonstrate that the antibody described in this application, when prepared as an ADC, possesses high anti-tumor activity and can effectively inhibit tumor growth in the PDX lung cancer mouse model.

[0321] Example 4: Preparation of Anti-PTK7 Antibody Drug Conjugates by Selecting Different Payloads and Linkers

[0322] 4.1 Coupling

[0323] The PTK7 humanized antibody 900937 was Dxd-conjugated with a linker of GGFG (SEQ ID NO: 29) and a target DAR value of 4 or 8.

[0324] The preparation process of 900937-Dxd-DAR4 is as follows:

[0325] An appropriate volume of antibody was added to a 20mM His / His-HCl solution (pH 6.0) and mixed thoroughly. Approximately 2.5 equivalents of 10mM TCEP (tris(2-carboxyethyl)phosphine) solution were then added, mixed thoroughly, and allowed to stand at 25°C for 120 minutes. Eight to ten equivalents of Deruxtecan (Dxd) dissolved in dimethyl sulfoxide were then added to the solution, mixed thoroughly, and allowed to stand at 25°C for 120 minutes. The reaction was terminated by adding 10mM cysteine. Finally, a 2mL spin desalting column (40K) was used to remove any residual small molecule drug from the antibody-drug conjugate, yielding a pure Dxd-antibody conjugated product.

[0326] The preparation process of 900937-Dxd-DAR8 is as follows:

[0327] An appropriate volume of antibody was added to a 20mM His / His-HCl solution (pH 6.0) and mixed thoroughly. Approximately 10 equivalents of 10mM TCEP (tris(2-carboxyethyl)phosphine) solution were then added and mixed thoroughly. The mixture was then incubated at 25°C for 120 minutes. 10-15 equivalents of Dxd dissolved in dimethyl sulfoxide were then added to the above solution, mixed thoroughly, and incubated at 25°C for 120 minutes. The reaction was terminated by adding 10mM cysteine. Finally, a 2mL spin desalting column (40kDa) was used to remove any residual small molecule drug from the antibody-drug conjugate to obtain a pure Dxd-antibody conjugated product.

[0328] Negative control antibody 900543 was Dxd-conjugated with a GGFG linker and a target DAR value of 8. The preparation process was the same as 900937-Dxd-DAR8.

[0329] 4.2 Detection of the Cytotoxicity of 900937-Dxd on NCI-H446 Tumor Cells

[0330] The cell killing effect of the ADC prepared by the antibody described in this application on NCI-H446 cells was tested. NCI-H446 is a human lung cancer cell line with high expression of PTK7 and low expression of HER2. NCI-H446 cells in the logarithmic growth phase were prepared with complete medium to form 3×10 4 Then add 3×10 cells / mL cell suspension to each well. 3 Cell suspension of 100 μL / mL. 900937-DXD-DAR4 and 900937-DXD-DAR8 were diluted to 6000 nM with NCI-H446 complete medium, first diluted 5-fold to 1 concentration, then diluted 15-fold to 7 concentrations. DS8201 (Trastuzumab Deruxtecan, purchased from Daiichi Sankyo) and 900543-Dxd-DAR8 were diluted to 12000 nM with NCI-H446 complete medium, first diluted 2-fold to 2 concentrations, then diluted 4-fold to 6 concentrations, and the above gradient antibodies were added to the 96-well plate with cell suspension at 100 μL / well. The 96-well plate was placed in a carbon dioxide incubator (1272, ESCO) with 5% CO2 and cultured at 37°C for 5 days. The 96-well plate was taken out and added to each well. 100 μL of substrate (G7573, Promega) was added and incubated in the dark for 10 minutes. Data were collected using a microplate reader. Dose-effect curves were fitted using GraphPad Prism software.

[0331] The results of NCI-H446 target cell killing are shown in Figure 10. Compared with 900937-Dxd and 900543-Dxd, the number of NCI-H446 target cells is lower at the same dose, indicating that 900937-Dxd has a better target cell killing effect. 900937-Dxd also has a higher killing effect than DS8201, indicating that the antibodies described in this application, after being prepared as ADCs, have better killing effects on tumor types that are not well-effective with commercially available ADCs, and can exhibit better therapeutic effects. The experimental results show that the antibodies described in this application still have a strong ability to bind to target cells and have a higher ability to kill target cells after being prepared as ADCs using different payloads.

[0332] 4.3 Antitumor Efficacy of 900937-Dxd in the NCI-H446 CDX Lung Cancer Mouse Model

[0333] The NCI-H446 lung cancer mouse model was established using 6-8 week old NTG female mice and the efficacy of the tested antibody drug conjugates was tested. NCI-H446 tumor cells were resuspended in PBS at a concentration of 1×10 8 cells / mL, inoculated subcutaneously in the right flank of the experimental animals, 50 μL / animal, and when the tumor grew to an average volume of 100-150 mm 3 The mice were divided into 7 groups with 6 mice in each group. The specific dosing schedule is shown in Table 5.

[0334] Table 5 Dosage regimen for the lung cancer NCI-H446 model

[0335] The anti-tumor efficacy results in the NCI-H446 lung cancer mouse model are shown in Figure 11. 900543-Dxd-DAR8 serves as a negative control. The humanized antibody 900937 described herein, after being conjugated to Dxd, significantly inhibited tumor growth at a dose of 3 mg / kg, with tumor volume gradually decreasing. This inhibitory effect was positively correlated with the dose administered. These experimental results demonstrate that the antibodies described herein, when formulated as ADCs with various payloads, exhibited strong anti-tumor activity and were able to effectively inhibit tumor growth in the NCI-H446 CDX lung cancer mouse model.

Claims

1. An isolated antigen-binding protein capable of binding to PTK7, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, wherein the VH comprises HCDR3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:

3.

2. The antigen-binding protein according to claim 1, wherein the VH comprises HCDR2, and the amino acid sequence of the HCDR2 is shown in SEQ ID NO:

2.

3. The antigen-binding protein according to any one of claims 1-2, wherein the VH comprises HCDR1, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:

1.

4. The antigen-binding protein according to any one of claims 1 to 3, wherein the VH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:

3. 5 . The antigen-binding protein according to claim 1 , wherein the VH comprises H-FR1, and the amino acid sequence of the H-FR1 is shown in SEQ ID NO: 4 or SEQ ID NO:

17.

6. The antigen-binding protein according to any one of claims 1 to 5, wherein the VH comprises H-FR2, and the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 5 or SEQ ID NO:

18.

7. The antigen-binding protein according to any one of claims 1 to 6, wherein the VH comprises H-FR3, the amino acid sequence of which is shown in SEQ ID NO: 6, SEQ ID NO: 19 or SEQ ID NO:

27.

8. The antigen-binding protein according to any one of claims 1 to 7, wherein the VH comprises H-FR4, and the amino acid sequence of the H-FR4 is shown in SEQ ID NO: 7 or SEQ ID NO:

20.

9. The antigen binding protein according to any one of claims 1 to 8, wherein the VH comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from any one of the following combinations: (1) The amino acid sequence of H-FR1 is shown in SEQ ID NO: 4, the amino acid sequence of H-FR2 is shown in SEQ ID NO: 5, the amino acid sequence of H-FR3 is shown in SEQ ID NO: 6, and the amino acid sequence of H-FR4 is shown in SEQ ID NO: 7; (2) the amino acid sequence of H-FR1 is shown in SEQ ID NO: 17, the amino acid sequence of H-FR2 is shown in SEQ ID NO: 18, the amino acid sequence of H-FR3 is shown in SEQ ID NO: 19, and the amino acid sequence of H-FR4 is shown in SEQ ID NO: 20; and (3) The amino acid sequence of the H-FR1 is shown in SEQ ID NO: 17, the amino acid sequence of the H-FR2 is shown in SEQ ID NO: 18, the amino acid sequence of the H-FR3 is shown in SEQ ID NO: 27, and the amino acid sequence of the H-FR4 is shown in SEQ ID NO:

20.

10. The antigen-binding protein according to any one of claims 1 to 9, wherein the amino acid sequence of the VH is shown in SEQ ID NO: 8, SEQ ID NO: 21 or SEQ ID NO:

28.

11. The antigen-binding protein according to any one of claims 1 to 10, comprising an antibody light chain variable region VL, wherein the VL comprises LCDR3, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

11.

12. The antigen-binding protein of claim 11, wherein the VL comprises LCDR2, the amino acid sequence of which is shown in SEQ ID NO: 10 (GAS).

13. The antigen binding protein according to any one of claims 11-12, wherein the VL comprises LCDR1, the amino acid sequence of which is shown in SEQ ID NO:

9.

14. The antigen-binding protein according to any one of claims 11 to 13, wherein the VL comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 10 (GAS), and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

11. 15 . The antigen-binding protein according to claim 11 , wherein the VL comprises L-FR1, and the amino acid sequence of the L-FR1 is shown in SEQ ID NO: 12 or SEQ ID NO:

22.

16. The antigen-binding protein according to any one of claims 11 to 15, wherein the VL comprises L-FR2, the amino acid sequence of which is shown in SEQ ID NO: 13 or SEQ ID NO:

23.

17. The antigen-binding protein according to any one of claims 11 to 16, wherein the VL comprises an L-FR3, the amino acid sequence of which is shown in SEQ ID NO: 14 or SEQ ID NO:

24.

18. The antigen-binding protein according to any one of claims 11 to 17, wherein the VL comprises an L-FR4, and the amino acid sequence of the L-FR4 is shown in SEQ ID NO: 15 or SEQ ID NO:

25.

19. The antigen binding protein according to any one of claims 11 to 18, wherein the VL comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1, L-FR2, L-FR3 and L-FR4 are selected from any one of the following combinations: (1) the amino acid sequence of L-FR1 is shown in SEQ ID NO: 12, the amino acid sequence of L-FR2 is shown in SEQ ID NO: 13, the amino acid sequence of L-FR3 is shown in SEQ ID NO: 14, and the amino acid sequence of L-FR4 is shown in SEQ ID NO: 15; and (2) The amino acid sequence of the L-FR1 is shown in SEQ ID NO: 22, the amino acid sequence of the L-FR2 is shown in SEQ ID NO: 23, the amino acid sequence of the L-FR3 is shown in SEQ ID NO: 24, and the amino acid sequence of the L-FR4 is shown in SEQ ID NO:

25.

20. The antigen binding protein according to any one of claims 11-19, wherein the amino acid sequence of the VL is shown in SEQ ID NO: 16 or SEQ ID NO:

26.

21. The antigen-binding protein according to any one of claims 1 to 20, comprising an antibody heavy chain variable region VH and an antibody light chain variable region VL, wherein the VH comprises HCDR1-3, the VL comprises LCDR1-3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10 (GAS) and SEQ ID NO: 11, respectively.

22. The antigen-binding protein according to any one of claims 1 to 21, comprising VH and VL, wherein the amino acid sequence of the VH is shown in SEQ ID NO: 8, SEQ ID NO: 21 or SEQ ID NO: 28, and the amino acid sequence of the VL is shown in SEQ ID NO: 16 or SEQ ID NO:

26.

23. The antigen binding protein according to any one of claims 1 to 22, comprising a VH and a VL selected from any one of the following combinations: (1) The amino acid sequence of the VH is shown in SEQ ID NO: 8, and the amino acid sequence of the VL is shown in SEQ ID NO: 16; (2) the amino acid sequence of the VH is shown in SEQ ID NO: 21, and the amino acid sequence of the VL is shown in SEQ ID NO: 26; and (3) The amino acid sequence of the VH is shown in SEQ ID NO: 28, and the amino acid sequence of the VL is shown in SEQ ID NO:

26.

24. The antigen binding protein of any one of claims 1-23, further comprising an immunoglobulin constant region.

25. The antigen binding protein of claim 24, wherein the immunoglobulin constant region is a human immunoglobulin constant region.

26. The antigen binding protein according to any one of claims 24-25, wherein the immunoglobulin constant region is a heavy chain constant region of human IgG and / or a human light chain constant region. The antigen-binding protein according to claim 26 , wherein the heavy chain constant region of human IgG is the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. The antigen-binding protein according to claim 26 , wherein the human light chain constant region is a human kappa (Kappa) or lambda (Lambda) light chain constant region.

29. The antigen binding protein of any one of claims 1-28, wherein the antigen binding protein is an antibody or an antigen binding fragment thereof.

30. The antigen binding protein of claim 29, wherein the antigen binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH and / or dAb fragment of the antibody.

31. The antigen binding protein of any one of claims 1-30, wherein the antigen binding protein is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

32. The antigen binding protein of any one of claims 1-31, wherein the antigen binding protein is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

33. The antigen binding protein of any one of claims 1-32, wherein the antigen binding protein is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

34. An immunoconjugate comprising the antigen binding protein of any one of claims 1-33.

35. The immunoconjugate of claim 34, wherein the immunoconjugate comprises the antigen binding protein, a linker, and a payload.

36. The immunoconjugate of claim 35, wherein the payload is a cytotoxic drug, a polymer, a protein, a radioisotope, a nucleic acid compound, and / or a glucocorticoid.

37. The immunoconjugate of any one of claims 35-36, wherein the payload is a cytotoxic drug.

38. The immunoconjugate of any one of claims 35-37, wherein the payload is a chemotherapeutic drug, a tubulin inhibitor, a DNA damaging agent, and / or a topoisomerase I inhibitor.

39. The immunoconjugate of any one of claims 35-38, wherein the payload is MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), DM1, DM4, Tubulysins, PBD, IBD, Dxd, SN-38, a Bcl-xL inhibitor, thailanstatin A, α-amanitin, β-amanitin, carmamicin A, and / or carmamicin B.

40. The immunoconjugate of any one of claims 35-39, wherein the payload is MMAE.

41. The immunoconjugate of any one of claims 35-39, wherein the payload is Dxd.

42. The immunoconjugate of any one of claims 35-41, wherein the immunoconjugate is an antibody drug conjugate (ADC).

43. The immunoconjugate of claim 42, wherein the ADC is a single-loaded ADC, a dual-loaded ADC, or a multi-loaded ADC.

44. The immunoconjugate of any one of claims 35-43, wherein the linker is a non-cleavable linker, an enzyme-cleavable linker, an acid-cleavable linker, a GSH-cleavable reducing linker, an Fe(II)-cleavable linker, a photoresponsive cleavable linker, and / or a bioorthogonal cleavable linker.

45. The immunoconjugate of any one of claims 35-44, wherein the linker is MC-VC-PAB or GGFG (SEQ ID NO: 29).

46. ​​The immunoconjugate of any one of claims 34-45, comprising an antigen binding protein, MC-VC-PAB, and MMAE, wherein the antigen binding protein comprises VH and VL, wherein the VH comprises HCDR1-3, the VL comprises LCDR1-3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10 (GAS), and SEQ ID NO: 11, respectively.

47. The immunoconjugate of any one of claims 34-45, comprising an antigen binding protein, GGFG, and Dxd, wherein the antigen binding protein comprises VH and VL, wherein the VH comprises HCDR1-3, the VL comprises LCDR1-3, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10 (GAS), and SEQ ID NO: 11, respectively.

48. A chimeric antigen receptor (CAR) comprising the antigen binding protein of any one of claims 1-33.

49. A modified immune cell comprising the chimeric antigen receptor of claim 48.

50. An isolated nucleic acid molecule encoding the antigen binding protein of any one of claims 1-33 and / or the chimeric antigen receptor of claim 48.

51. A vector comprising the nucleic acid molecule of claim 51.

52. A cell comprising the nucleic acid molecule of claim 51 and / or the vector of claim 52.

53. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-33, the immunoconjugate of any one of claims 34-47, the chimeric antigen receptor of claim 48, the modified immune cell of claim 49, the nucleic acid molecule of claim 50, the vector of claim 51 and / or the cell of claim 52, and optionally a pharmaceutically acceptable carrier.

54. A pharmaceutical combination comprising the antigen binding protein of any one of claims 1-33, the immunoconjugate of any one of claims 34-47, the chimeric antigen receptor of claim 48, the modified immune cell of claim 49, the nucleic acid molecule of claim 50, the vector of claim 51, the cell of claim 52 and / or the pharmaceutical composition of claim 53 and a therapeutic agent.

55. A detection kit comprising the antigen binding protein according to any one of claims 1-33, wherein the detection kit is used to detect the presence and / or content of PTK7 in a sample or a subject.

56. A method for detecting the presence and / or amount of PTK7, comprising using the antigen binding protein of any one of claims 1-33.

57. Use of the antigen binding protein of any one of claims 1-33, the immunoconjugate of any one of claims 34-47, the chimeric antigen receptor of claim 48, the modified immune cell of claim 49, the nucleic acid molecule of claim 50, the vector of claim 51, the cell of claim 52, the pharmaceutical composition of claim 53 and / or the pharmaceutical combination of claim 54 in the preparation of a medicament for preventing and / or treating a disease and / or condition.

58. Use according to claim 57, wherein the disease and / or disorder is a tumor.

59. The use according to claim 58, wherein the tumor is a solid tumor and / or a hematological tumor.

60. The use according to any one of claims 58-59, wherein the tumor is a PTK7-positive tumor.

61. The method of any one of claims 58-60, wherein the tumor is a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer, prostate cancer and / or esophageal cancer.

62. A method for preventing and / or treating a disease and / or condition, comprising administering to a subject in need thereof the antigen binding protein of any one of claims 1-33, the immunoconjugate of any one of claims 34-47, the chimeric antigen receptor of claim 48, the modified immune cell of claim 49, the nucleic acid molecule of claim 50, the vector of claim 51, the cell of claim 52, the pharmaceutical composition of claim 53, and / or the pharmaceutical combination of claim 54.

63. The method of claim 62, wherein the disease and / or condition is a tumor.

64. The method of claim 63, wherein the tumor is a solid tumor and / or a hematological tumor.

65. The method of any one of claims 63-64, wherein the tumor is a PTK7-positive tumor.

66. The method of any one of claims 63-65, wherein the tumor is a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer, prostate cancer, and / or esophageal cancer.

67. The antigen binding protein of any one of claims 1-33, the immunoconjugate of any one of claims 34-47, the chimeric antigen receptor of claim 48, the modified immune cell of claim 49, the nucleic acid molecule of claim 50, the vector of claim 51, the cell of claim 52, the pharmaceutical composition of claim 53 and / or the pharmaceutical combination of claim 54, for use in preventing and / or treating a disease and / or condition.

68. The antigen binding protein, immunoconjugate, chimeric antigen receptor, modified immune cell, nucleic acid molecule, vector, cell, pharmaceutical composition and / or pharmaceutical combination of claim 67, wherein the disease and / or disorder is a tumor.

69. The antigen binding protein, immunoconjugate, chimeric antigen receptor, modified immune cell, nucleic acid molecule, vector, cell, pharmaceutical composition and / or drug combination of claim 68, wherein the tumor is a solid tumor and / or a hematological tumor.

70. The antigen binding protein, immunoconjugate, chimeric antigen receptor, modified immune cell, nucleic acid molecule, vector, cell, pharmaceutical composition and / or pharmaceutical combination of any one of claims 68-69, wherein the tumor is a PTK7-positive tumor.

71. The antigen binding protein, immunoconjugate, chimeric antigen receptor, modified immune cell, nucleic acid molecule, vector, cell and / or pharmaceutical composition of any one of claims 68-70, wherein the tumor is a primary breast tumor, lung adenocarcinoma, lung squamous cell carcinoma, lung cancer, non-small cell lung cancer, colon cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, acute myeloid leukemia, kidney cancer, bladder cancer, prostate cancer and / or esophageal cancer.

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