Anti-PTK7 single-domain antibody and its applications

Abstract

Provided is a specific single domain antibody coding sequence for PTK7 and its application. The provided anti-PTK7 single domain antibody can effectively bind to PTK7 antigen, providing a basis for developing drugs targeting PTK7, including nuclide-coupled drugs, antibody-drug conjugates and multispecific antibody drugs, etc.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, specifically, to specific single-domain antibodies against PTK7, more specifically to its coding sequence and its applications in the diagnosis and treatment of diseases.

Background Art

[0002] Protein tyrosine kinase 7 (PTK7), also known as colon cancer kinase 4 (CCK4), is a highly conserved, catalytically inactive transmembrane protein tyrosine kinase (PTK) that is involved in non-canonical Wnt signaling receptor during the development of hematopoietic cells, somatic progenitor cells, and stem cells (Peradziryi, 2012).

[0003] Studies have shown that PTK7 is overexpressed in various tumor tissues, including breast, lung, pancreatic, cervical, ovarian, and colon cancers, and that its overexpression is associated with poor prognosis and an increased risk of metastasis. Furthermore, PTK7 has been confirmed to be highly enriched in tumor initiation cells (TICs) or tumor stem cells in patient-derived tumor tissue. Therefore, targeting PTK7 is a natural candidate target point for targeted therapy, as it eliminates not only common cancer cells but also TICs that cause tumor recurrence (Damelin, 2017). However, because PTK7 lacks catalytic activity, the development of typical tyrosine kinase inhibitors is not feasible, and various antibody conjugates and different forms of drugs targeting it are currently in clinical development. Here, the novel antibody-drug conjugate (ADC) cofetuzumab pelidotin, targeting the humanized antibody hu6M024 as a vector and coupled with the calendula microtubule inhibitor Aur0101, will undergo a first-in-human clinical trial in metastatic triple-negative breast cancer, platinum-resistant ovarian cancer, and non-small cell lung cancer. Initial study data show that cofetuzumab pelidotin at a dose of 2.8 mg / kg every three weeks is safe, exhibiting antitumor activity in treated patients, with overall objective response rates of 27% (n=63) for platinum-resistant ovarian cancer, 19% (n=31) for non-small cell lung cancer, and 21% (n=29) for triple-negative breast cancer, respectively (Maitland, 2021). Furthermore, using F-18 labeled aptamer nucleic acid aptamers as PTK7-targeting immunoimaging tracers allows for specific, selective, and high-affinity monitoring of PTK7 expression in xenograft models of tumor cell lines (HCT116 and UMG), enabling further development of PTK7-targeting therapies (Jacobson, 2015).

[0004] Currently, no single-domain antibody drugs targeting the PTK7 target point are available on the market. Single-domain antibodies, specifically camel heavy-chain single-domain antibodies (VHH, variable domain of heavy chain of heavy-chain antibody), are the smallest currently available units that are fully functional, stable, and capable of binding to antigens. Single-domain antibodies have a molecular weight that is 1 / 10th that of conventional antibodies, and are characterized by high stability, good water solubility, ease of humanization, high targeting ability, and strong permeability. As a vector targeting radioisotopes, they can rapidly and specifically penetrate tumor tissue and bind to the target, and unbound antibodies are rapidly removed from the blood, reducing the body's radiation dose. Compared to conventional antibodies, they offer many clear advantages in the development of radioimmunoimaging and radioimmunotherapy (D'Huyvetter, 2014).

[0005] Therefore, in this field, there is a need to develop anti-PTK7 single-domain antibodies, especially specific single-domain antibodies that can effectively bind only to PTK7, in order to develop a new generation of radioimmunoimaging and radioimmunotherapy. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide an anti-PTK7 single-domain antibody having good PTK7 antigen-binding ability. [Means for solving the problem]

[0007] A first aspect of the present invention provides a complementarity-determining region (CDR) of an anti-PTK7 single-domain antibody VHH chain, wherein the complementarity-determining region (CDR) of the VHH chain is CDR1 shown in SEQ ID NO:9~11, CDR2 shown in SEQ ID NO:12~14, and Includes CDR3 as shown in SEQ ID NO: 15~18.

[0008] In another preferred example, CDR1, CDR2, and CDR3 are isolated by framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

[0009] In another preferred example, the complementarity-determining region (CDR) of the VHH chain is: CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:12, and Includes CDR3 as indicated by SEQ ID NO:15.

[0010] In another preferred example, the complementarity-determining region (CDR) of the VHH chain includes CDR1, CDR2, and CDR3 selected from the group consisting of the following: [Table 1]

[0011] A second aspect of the present invention provides a VHH chain of an anti-PTK7 single-domain antibody, the VHH chain comprising a framework region (FR) and a complementarity-determining region (CDR) as described in the first aspect of the present invention.

[0012] In another preferred example, the framework region consists of FR1, FR2, FR3, and FR4 selected from the group consisting of the following: [Table 2]

[0013] In another preferred example, the framework region comprises FR1, indicated by SEQ ID NO:19, FR2, indicated by SEQ ID NO:22, FR3, indicated by SEQ ID NO:25, and FR4, indicated by SEQ ID NO:29.

[0014] In another preferred example, the VHH chain of the anti-PTK7 single-domain antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 4. In another preferred example, the VHH chain of the anti-PTK7 single-domain antibody has an amino acid sequence as shown in SEQ ID NO: 1.

[0015] A third aspect of the present invention provides an anti-PTK7 single-domain antibody, wherein the single-domain antibody is a single-domain antibody against the PTK7 protein and has a VHH chain with an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 4. In another preferred example, the single-domain antibody has a VHH chain with an amino acid sequence as shown in SEQ ID NO: 1.

[0016] A fourth aspect of the present invention provides a polynucleotide, wherein the polynucleotide encodes a protein selected from the group consisting of the CDR region described in the first aspect of the present invention, the VHH chain of the anti-PTK7 single-domain antibody described in the second aspect of the present invention, or the anti-PTK7 single-domain antibody described in the third aspect of the present invention.

[0017] In another preferred example, the polynucleotide has a nucleotide sequence as shown in any one of SEQ ID NOs: 5 to 8. In another preferred example, the polynucleotide contains DNA, cDNA or RNA.

[0018] A fifth aspect of the present invention provides an expression vector, wherein the expression vector contains the polynucleotide described in the fourth aspect of the present invention. In another preferred example, the expression vector includes a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0019] The sixth aspect of the present invention provides a host cell, which contains the expression vector described in the fifth aspect of the present invention or has the polynucleotide described in the fourth aspect of the present invention integrated into its genome. In another preferred example, the host cell includes a prokaryotic cell or a eukaryotic cell. In another preferred example, the host cell is selected from the group consisting of Escherichia coli and yeast cells.

[0020] The seventh aspect of the present invention is (a) culturing the host cell described in the sixth aspect of the present invention under conditions suitable for the production of a single-domain antibody, thereby obtaining a culture containing the anti-PTK7 single-domain antibody; and (b) separating or recovering the anti-PTK7 single-domain antibody from the culture, and providing a method for producing an anti-PTK7 single-domain antibody.

[0021] In another preferred example, the anti-PTK7 single-domain antibody has an amino acid sequence as shown in any one of SEQ ID NO: 1 to 4.

[0022] The eighth aspect of the present invention provides an immunoconjugate, and the immunoconjugate is (a) the VHH chain of the anti-PTK7 single-domain antibody described in the second aspect of the present invention, or the anti-PTK7 single-domain antibody described in the third aspect of the present invention, and (b) a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0023] In another preferred example, the coupling moiety is a drug or a toxin. In another preferred example, the coupling moiety is a detectable marker.

[0024] In another preferred example, the conjugate is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or nanoparticles of any form.

[0025] In another preferred example, the immunoconjugate comprises a polyvalent (e.g., bivalent) VHH chain of the anti-PTK7 single-domain antibody according to the second aspect of the present invention and the anti-PTK7 single-domain antibody according to the third aspect of the present invention.

[0026] In another preferred example, polyvalent means that the amino acid sequence of the immunoconjugate comprises a plurality of repeating VHH chains of the anti-PTK7 single-domain antibody described in a second aspect of the present invention and the anti-PTK7 single-domain antibody described in a third aspect of the present invention.

[0027] A ninth aspect of the present invention provides an application for a VHH chain according to the second aspect of the present invention, an anti-PTK7 single-domain antibody according to the third aspect of the present invention, or an immunoconjugate according to the eighth aspect of the present invention, used for the preparation of (a) a reagent for detecting PTK7 molecules and (b) a drug for treating tumors.

[0028] In another preferred example, the detection includes flow cytometry detection and cellular immunofluorescence detection. In another preferred example, the tumor is a PTK7-highly expressing tumor. In another preferred example, the tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, colon cancer, or a combination thereof.

[0029] A tenth aspect of the present invention provides a pharmaceutical composition comprising (i) a single-domain antibody as described in a third aspect of the present invention or an immunoconjugate as described in an eighth aspect of the present invention, and (ii) a pharmaceutically acceptable carrier.

[0030] In another preferred example, the pharmaceutical composition is in the form of an injectable dosage. In another preferred example, the pharmaceutical composition is used to prepare a drug for treating a tumor, the tumor being selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, colon cancer, or a combination thereof.

[0031] An eleventh aspect of the present invention is: (1) The step of bringing a sample into contact with a single-domain antibody according to the third aspect of the present invention, (2) A method for in vitro (diagnostic and non-diagnostic) detection of PTK7 protein in a sample is provided, comprising the step of detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PTK7 protein in the sample.

[0032] A twelfth aspect of the present invention provides a method for treating a tumor, comprising administering to a target subject a single-domain antibody according to the third aspect of the present invention, an immune conjugate according to the eighth aspect of the present invention, or a pharmaceutical composition according to the tenth aspect of the present invention.

[0033] In another preferred example, the tumor is a PTK7-highly expressing tumor. In another preferred example, the tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, colon cancer, or a combination thereof. In another preferred example, the subject includes humans or non-human mammals.

[0034] A thirteenth aspect of the present invention provides a radioimmunoimaging method comprising imaging with a single-domain antibody described in the third aspect of the present invention or an immunoconjugate described in the eighth aspect of the present invention. [Effects of the Invention]

[0035] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0036] [Figure 1] This is the detection result for the human PTK7-Fc protein antigen. The results indicate that the purity of the protein reaches over 90% by SDS-PAGE. [Figure 2] The results of construction and mass detection of two anti-PTK7 single-domain antibody phage display libraries are shown. Figure A is the PCR identification figure of the first and second PCR amplification products of the two libraries, showing that the results ultimately yield a VHH gene fragment of approximately 400 bp in size. Figure B is the volume detection figure of the two libraries, showing that the volumes of the two libraries are 2.2 × 10⁹ and 3.4 × 10⁹ CFU, respectively. Figure C is the insertion rate detection figure of the two libraries, showing that the VHH insertion rates of the two libraries are 96% and 100%, respectively. Figure D is the enrichment detection figure of the two libraries, showing that the specific phages in the two libraries were enriched 60-fold and 84-fold, respectively, after three rounds of screening. [Figure 3] This is a result of detecting the binding activity of anti-PTK7 single-domain antibodies to PTK7-positive expressing HCT116 cells by flow cytometry. The results show that all four strains of single-domain antibodies can effectively bind to the PTK7 protein on the surface of HCT116 cells. [Modes for carrying out the invention]

[0037] After extensive and detailed research and a large-scale screening, the inventors have developed the first anti-PTK7 single-domain antibodies. Experimental results show that all four strains of PTK7 single-domain antibodies obtained by this invention can bind specifically to human or monkey PTK7 proteins with high affinity.

[0038] Specifically, the present invention involves immunizing camels with human PTK7 extracellular segment antigen protein to obtain a high-quality immune single-domain antibody gene library. The PTK7 protein molecule is then coupled to an ELISA plate to display the precise spatial structure of the PTK7 protein. This antigen is then screened against the immune single-domain antibody gene library (camel heavy-chain antibody phage display gene library) using phage display technology to obtain a PTK7-specific single-domain antibody gene. This gene is then introduced into E. coli to obtain a highly specific single-domain antibody strain that can be efficiently expressed in E. coli.

[0039] term As used herein, the terms “single-domain antibody of the present invention,” “anti-PTK7 single-domain antibody of the present invention,” and “PTK7 single-domain antibody of the present invention” are interchangeable and all refer to single-domain antibodies that specifically identify and bind to PTK7 (human, mouse, and monkey PTK7). The present invention comprises single-domain antibodies having the amino acid sequence described in any one of SEQ ID NO: 1 to 4, and particularly preferably single-domain antibodies such that the amino acid sequence of the VHH chain is shown in SEQ ID NO: 1.

[0040] As used herein, the terms “single-domain antibody,” “VHH,” and “nanobody” are synonymous and interchangeable, referring to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting solely of the heavy chain variable region, which is the smallest antigen-binding fragment with full functionality. Typically, after obtaining an antibody that naturally lacks the constant region 1 (CH1) of both the light and heavy chains, the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of the heavy chain variable region.

[0041] As used herein, the terms “antibody” or “immunoglobulin” refer to a heterotetrameric glycoprotein of approximately 150,000 daltons having the same structural characteristics, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes differs. Each heavy and light chain also has intrachain disulfide bonds arranged at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the constant region of the light chain facing the first constant region of the heavy chain, and the variable region of the light chain facing the variable region of the heavy chain. Certain amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0042] As used herein, the term “variable” refers to a difference in the arrangement of specific portions of the variable region in an antibody, which forms the binding and specificity of different particular antibodies to a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called complementarity-determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved portion of the variable region is called the framework region (FR). The natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folding configuration, connected by three CDRs that form a connecting ring, and can sometimes form a partial β-folding structure. The CDRs of each chain are closely adjacent by FR regions and, together with the CDRs of another chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). While the constant region is directly involved in antibody binding to antigens, it also exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity.

[0043] As used herein, “heavy chain variable region” and “V H The term "..." is used interchangeably. As used herein, the terms “variable region” and “complementarity determining region (CDR)” are interchangeable.

[0044] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3. In a preferred embodiment of the present invention, the heavy chain of the antibody includes the heavy chain variable region and the heavy chain constant region.

[0045] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are interchangeable and refer to polypeptides that specifically bind to PTK7 proteins, such as proteins or polypeptides that shake the heavy chain variable region. These may or may not contain starting methionine.

[0046] The present invention further provides other proteins or fusion expression products having the antibody of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) of a heavy chain containing a variable region, provided that the variable region is identical to, or at least 90% homologous to, the heavy chain variable region of the antibody of the present invention, or at least 95% homologous.

[0047] Generally, the antigen-binding properties of an antibody can be explained by three specific regions located within the heavy chain variable region, called the variable region (CDR). This section is divided into four framework regions (FRs), and the amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a cyclic structure, and the β-folds formed by the FRs between them are spatially close. The heavy chain CDR and its corresponding light chain CDR constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of similar antibodies, the amino acids that make up the FR or CDR region can be determined.

[0048] The heavy chain variable region of the antibody of the present invention is of particular interest because at least a portion of it is involved in binding to the antigen. Accordingly, the present invention includes molecules having an antibody heavy chain variable region having a CDR, insofar as its CDR has homology of 90% or more (preferably 95% or more, most preferably 98% or more) with the CDRs identified herein.

[0049] The present invention includes not only complete antibodies but also antibody fragments having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention further includes the antibody fragments, derivatives, and analogs.

[0050] As used herein, the terms “fragment,” “derivative,” and “analog” refer to polypeptides that possess essentially the same biological function or activity as the antibodies of the present invention. Polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted (the substituted amino acid residues may or may not be encoded by the genetic code), or (ii) polypeptides having substituents on one or more amino acid residues, or (iii) polypeptides formed by the fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by the fusion of an additional amino acid sequence with a polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence or protein sequence for purifying the polypeptide or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0051] The antibody of the present invention refers to a polypeptide having binding activity to the PTK7 protein and containing the above-mentioned CDR region. The term further includes variant forms of polypeptides having the same function as the antibody of the present invention and containing the above-mentioned CDR region. These variant forms include (but are not limited to) the deletion, insertion, and / or substitution of one or more amino acids, and the addition of one or more amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids having similar or similar properties usually does not alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and / or N-terminus usually does not alter the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention.

[0052] The variant forms of the polypeptide include homologous sequences, conserved variants, allelic variants, native mutants, induced mutants, proteins encoded by DNA that can hybridize to the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0053] The present invention further provides other polypeptides, such as fusion proteins, that include a single-domain antibody or a fragment thereof. In addition to most full-length polypeptides, the present invention further includes fragments of the single-domain antibody of the present invention. Typically, such fragments have at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0054] In the present invention, "conservative variant of the antibody of the present invention" refers to a polypeptide formed by the substitution of up to 10 amino acids, preferably up to 8, more preferably up to 5, and most preferably up to 3, amino acids with similar or identical amino acids compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by substitution with amino acids as shown in Table A. [Table 3]

[0055] The present invention further provides polynucleotide molecules encoding the antibody or its fragment or its fusion protein. The polynucleotides of the present invention may be in DNA form or RNA form. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0056] The polynucleotides encoding mature polypeptides of the present invention include coding sequences that encode only mature polypeptides, coding sequences for mature polypeptides and various additional coding sequences, coding sequences for mature polypeptides (and any additional coding sequences) and non-coding sequences.

[0057] The term “polynucleotide encoding polypeptide” includes a polynucleotide that encodes this polypeptide, or further includes a polynucleotide that includes additional coding and / or non-coding sequences.

[0058] The present invention further relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the aforementioned polynucleotides under stringent conditions. In the present invention, “stringent conditions” means (1) hybridization and elution at lower ionic strength and higher temperature, for example, 0.2 × SSC, 0.1% SDS, 60°C, or (2) addition of a denaturing agent during hybridization, for example, 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, or (3) hybridization that occurs only when the homology between the two sequences is at least 90%, more preferably 95%. Furthermore, polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as mature polypeptides.

[0059] The full-length nucleotide sequence or fragments of the antibody of the present invention can typically be obtained by PCR amplification, recombination, or artificial synthesis. Especially when the fragment length is short, a viable method is to synthesize the relevant sequence using artificial synthesis. Generally, several small fragments are first synthesized and then joined together to obtain a very long sequence fragment. Furthermore, the heavy chain coding sequence and expression tag (e.g., 6His) can be fused to form a fusion protein.

[0060] Once a relevant sequence is obtained, it can be obtained in large quantities using recombination. Typically, this is achieved by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from host cells grown by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules that exist in isolated forms.

[0061] Currently, DNA sequences encoding the protein of the present invention (or its fragments or derivatives thereof) can be obtained entirely by chemical synthesis. These DNA sequences can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0062] The present invention further relates to vectors comprising the aforementioned appropriate DNA sequence and an appropriate promoter or regulatory sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0063] The host cell may be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Typical examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella tiphimuria, fungal cells such as yeast, insect cells such as Drosophila S2 or Sf9, and animal cells such as CHO, COS7, and 293 cells.

[0064] The transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of DNA absorption are obtained by treatment with the CaCl2 method after the exponential growth phase, a step well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation if necessary. When the host is a eukaryote, conventional mechanical methods such as calcium phosphate coprecipitation, microinjection, and electroporation, as well as DNA transfection methods such as liposome packaging, can be selected.

[0065] The resulting transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium can be selected from a variety of conventional media. The host cells are cultured under conditions suitable for proliferation. After the host cells have proliferated to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature change or chemical induction), and the cells are cultured further for a certain period.

[0066] The recombinant polypeptides in the above method can be expressed intracellularly or at the cell membrane, or secreted extracellularly. Recombinant proteins can be isolated and purified by various isolation methods using physical, chemical, and other properties as needed. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional regeneration processes, treatment with protein precipitants (salting-out), centrifugation, osmotic sterilization, superprocessing, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid-phase chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations thereof.

[0067] The antibodies of the present invention can be used alone and can be conjugated or coupled with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified moieties, or any combination thereof.

[0068] Diagnostic detectable markers include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0069] Therapeutic agents that can conjugate or couple with the antibodies of the present invention include, but are not limited to, 1. radionuclides, 2. biological toxins, 3. cytokines such as IL-2, 4. gold nanoparticles / nanorods, 5. viral particles, 6. liposomes, 7. nanomagnetic particles, 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), 9. therapeutic agents (e.g., cisplatin), or nanoparticles in any form.

[0070] Pharmaceutical composition The present invention further provides compositions. Preferably, the composition is a pharmaceutical composition comprising the antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium, where the pH is typically about 5 to 8, preferably about 6 to 8, and the pH value varies depending on the properties of the formulated substance and the disease being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration.

[0071] The pharmaceutical composition of the present invention can be used for the treatment of tumors because it can be used directly to bind to PTK7 protein molecules. Furthermore, it can be used in conjunction with other therapeutic agents.

[0072] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%) of the single-domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation must be consistent with the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection by conventional methods, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions must be prepared under sterile conditions. The dose of the active ingredient is a therapeutically effective dose, for example, about 10 μg / kg body weight to about 50 mg / kg body weight daily. Furthermore, the polypeptide of the present invention can also be used in combination with other therapeutic agents.

[0073] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where this safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage must take into account factors such as the route of administration and the patient's health condition, and these are all within the scope of the skills of a skilled physician.

[0074] Labeled single-domain antibody In a preferred example of the present invention, the single-domain antibody has a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, gold colloid markers, colored markers, or fluorescent markers.

[0075] Gold colloid labeling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, an anti-PTK7 single-domain antibody is labeled with gold colloid to obtain a gold colloid-labeled single-domain antibody. In another preferred embodiment of the present invention, an anti-PTK7 single-domain antibody is labeled with a radioisotope to obtain an isotope-labeled single-domain antibody. The anti-PTK7 single-domain antibody of the present invention has good specificity and high titer, and can be used for clinical diagnosis of PTK7.

[0076] Detection method The present invention also relates to a method for detecting the PTK7 protein. The steps of the method are broadly as follows: obtain a cell and / or tissue sample, dissolve the sample in a medium, and detect the level of PTK7 protein in the dissolved sample. In the detection method of the present invention, the sample used is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.

[0077] kit The present invention further provides a kit comprising the antibody (or fragment thereof) or a detection plate of the present invention, wherein in a preferred example of the present invention, the kit further comprises a container, instructions for use, buffer, etc. The present invention further provides a detection kit for detecting PTK7 levels, the kit comprising an antibody that identifies the PTK7 protein, a lysis medium for dissolving the sample, general-purpose reagents necessary for detection, and buffers such as various buffers, detection labels, and detection substrates. The detection kit may be an in vitro diagnostic device.

[0078] application As described above, the single-domain antibody of the present invention has broad biological and clinical application value, and its applications relate to multiple fields such as the diagnosis and treatment of PTK7-related diseases, basic medical research, and biological research. Preferred applications are clinical diagnosis and targeted therapy for PTK7. The present invention provides the application of an anti-PTK7 single-domain antibody in the diagnosis and treatment of tumors, wherein the tumors are those having abnormally high expression of PTK7 (including mRNA and / or protein levels), and specifically include, but are not limited to, breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, colon cancer, and the like.

[0079] The main advantages of this invention are as follows: (a) The present invention is the first to develop a single-domain antibody against the PTK7 target point. (b) The single-domain antibody of the present invention has high affinity for binding to the PTK7 protein. (c) The single-domain antibody of the present invention has good specificity and can bind to human, mouse, and rhesus monkey PTK7. (d) The single-domain antibody of the present invention can be effectively accumulated in PTK7-highly expressing tumor models and can be applied to the diagnosis and efficacy evaluation of cancers targeting PTK7, and can also be used in the development of new generation PTK7-targeting therapies. (e) The method for preparing single-domain antibodies of the present invention is simple and facilitates mass production.

[0080] The present invention will be described in more detail below in conjunction with specific examples. It should be understood that these examples are used solely to illustrate the present invention and do not limit its scope. In the following examples, experimental methods that do not specify detailed conditions typically follow conventional conditions, such as those described in, for example, Sambrook et al., "Molecular Cloning: An Experimental Manual" (translated by Huang Peitang, Beijing, Science Press, 2002), or conditions suggested by the manufacturer. Unless otherwise specified, percentages and quantities are calculated by weight. The experimental materials and reagents used in the following examples are available through commercial channels unless otherwise specified.

[0081] Example 1: Expression of human PTK7-Fc protein antigen Human PTK7 extracellular segment protein is primarily expressed using mammalian cells HEK293F. The human PTK7 extracellular segment gene is cloned into a pFUSE-IgG recombinant plasmid, mixed with transfection reagent PEI1:3, and then transfected into HEK293F cells. The cells are cultured for 6 days at 37°C in a 6% CO2 shaker incubator. The cell supernatant is then collected and conjugated with protein A beads at room temperature for 1 hour. After washing the beads with phosphate buffer pH 7.0, the protein is eluted using 0.1M glycine solution pH 3.0. The eluted protein is ultrafiltered into PBS solution, the yield is measured, and samples are taken for SDS-PAGE detection. The detection results are shown in Figure 1, and the purity of the PTK7-Fc protein antigen exceeds 90%, making it suitable for camel immunization and antibody screening.

[0082] Example 2: Construction and screening of a human PTK7 extracellular segment protein immunolibrary. Two Xinjiang Bactrian camels were immunized with purified PTK7-Fc protein. After 7 days of immunization, total RNA was isolated from the camels' peripheral blood, and the VHH gene was amplified by reverse transcription and PCR (Figure 2A). The VHH gene was cloned into the phage vector pMECS, and TG1 host cells were transformed to construct a phage display library. The two constructed libraries were serially diluted, plated, and measured in volume. Simultaneously, 24 clones were randomly selected from each constructed library and colony PCR detection was performed. According to the results, the capacity of the two constructed libraries was 2.2 × 10⁶ each. 9 and 3.4 × 10 9 It is a CFU (Figure 2B), and the library insertion rates are 96% and 100%, respectively (Figure 2C).

[0083] Next, using phage display technology, three rounds of "adsorption-wash-concentration" were performed, and finally, the specific phages in the two libraries reached enrichment 60-fold and 84-fold, respectively (Figure 2D). From these enriched phage clones, 400 were randomly selected for PE-ELISA identification of human PTK7-Fc. All resulting positive clones (ratio >3) were sequenced and identified, and all single-domain antibodies (107 strains) with different sequences were used as candidate targets.

[0084] Example 3: Expression and purification of an anti-PTK7 single-domain antibody From the antibody clones obtained by sequencing analysis in Example 2, 40 strains were selected. Each plasmid was electroporated into E. coli WK6, spread on an LA+glucose culture plate, and incubated overnight at 37°C. A single colony was collected and inoculated into 5 mL of LB culture medium containing ampicillin. Incubated overnight in a shaker at 37°C. 1 mL of the final solution strain was inoculated into 330 mL of TB culture medium and incubated in a shaker at 37°C. When culturing to an OD of 0.6-1, IPTG was added, and the culture was incubated overnight in a shaker at 28°C. Bacteria were collected by centrifugation, and crude antibody extract was obtained using Shintoho. Purified single-domain antibodies were prepared by nickel column ion affinity chromatography. Detection revealed that the purity of the expressed and purified single-domain antibodies exceeded 90%, and they were used to study candidate functional activity. Finally, the following four major single-domain antibodies were obtained. [Table 4]

[0085] The sequences of the four single-domain antibodies are as follows, with the three CDR regions underlined. SEQ ID NO:1 QVQLQESGGGSVQAGGSLRLSCTAS GFTFDDYDMV WYRQAPGNEYEWLS TVSRGGN LYYADSVKGRFTISRDNAKNTVYLNMSRVKPEDTAVYYC AADLRSVPSTYRARTCRGRYCRDYWGQGTQVTVSS

[0086] SEQ ID NO:2 QVQLQESGGGSVQAGGSLRLSCAAS GYIWSRYCMG WFRQAPGKAREGVA RFNSDGN TRYVDSVKGRFTISRDNAKNTLYLQMNNLQPEDTARYYC AADPRFDCGYDAVFNPTHFPY WGRGTQVTVSS

[0087] SEQ ID NO:3 QVQLQESGGGSVQAGGSLRLSCAAS GYIWSRYCMG WFRQAPGKAREGVA RFNSDGN TRYVDSVKGRFTISRDNAKNTLYLQMNNLQPEDTAR YYCAADPRFDCGYDAVFNPTHFPY WGQGTQVTVSS

[0088] SEQ ID NO:4 QVQLQESGGGLVQPGGSLTLACAAS GFAFSSYAMR WVRQAPGKGLEGVS SISAGGDE TYYADFAKGRFTISRDNAKNTLYLQLNSLKTEDTAMYYC AKGDRNSPRYSSWPITP VGQGTQVTVSS

[0089] SEQ ID NO:5 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTACAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTTTGATGATTATGACATGGTCTGGTACCGCCAGGCTCCAGGGAATGAGTACGAGTGGCTCTCAACTGTTAGTCGTGGTGGTAATTTATACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAACACGGTGTATCTTAATATGAGCAGAGTGAAACCTGAGGACACGGCCGTCTATTACTGCGCGGCAGACCTTCGCTCGGTTCCCTCGACGTATCGGGCGAGAACTTGTCGCGGTCGTTACTGCCGCGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0090] SEQ ID NO:6 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTGGAGTAGGTACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGCGCGCGAGGGGGTCGCGCGCTTTAATAGTGATGGTAACACAAGGTACGTAGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAACACTCTGTATCTGCAAATGAACAACCTGCAACCTGAGGACACTGCCAGGTACTACTGTGCGGCAGATCCTCGCTTCGATTGCGGCTATGACGCCGTGTTTAACCCGACGCACTTTCCTTACTGGGGCCGGGGGACCCAGGTCACCGTCTCCTCA

[0091] SEQ ID NO:7 CAGGTGCAGCTGCAGGATCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTGGAGTAGGTACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGCCGCGGAGGGGTCGCCGGCTTTAATAGTGATGGTAACAAGGTACGTAGACTCCGTGA AGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAACACTCTGTATCTGCAAATGAACAACCTGCAACCTGAGGACACTGCCAGGTACTGTGCGGCAGATCCTCGCTTCGATTGCGGCTATGACGCCGTGTTTAACCCGACGCACTTTCCTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0092] SEQ ID NO:8 CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGACACTCGCCTGTGCAGCCTCTGGATTCGCCTTCAGTAGCTATGCCATGCGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTCGAGGGGGTCTCAAGTATTAGTGCTGGTGGTGAATGAAACATACTATGCAGAC TTCGCGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAATACGCTGTATTTGCAGCTGAACAGCCTGAAAACTGAGGACAGGCCATGTATTACTGCAAAGGGGATCGTAACTCCCCCAGATACAGTAGTTGGCCCATCACACCCGTAGGTCAGGGGACCCAGGTCACCGTCTCCTCA

[0093] Example 4: anti-PTK7 single domain antibody and different types of PTK7 antibody detection ELISA plates are coated with human or mouse PTK7-Fc or rhesus monkey PTK7-His antigen protein, left overnight at 4°C, washed four times with PBS-T the next day, 1% BSA is added, blocked at room temperature for 2 hours, washed four times with PBS-T, gradient diluted anti-PTK7 single-domain antibody is added, left at 37°C for 1 hour, washed four times with PBS-T, mouse anti-His-HRP antibody or mouse anti-HA-HRP antibody is added, left at 37°C for 1 hour, washed four times with PBS-T, TMB-soluble substrate is added, left at room temperature for 10 minutes, absorption values ​​are read at a wavelength of 450 nm, and the Kd value of each single-domain antibody is calculated based on the absorption values. The detection results are shown in Table 2, demonstrating that the anti-PTK7 single-domain antibody of the present invention can bind to human, mouse, or rhesus monkey PTK7. [Table 5]

[0094] Example 5: Detection of protein binding kinetics (Biacore 8K) of anti-PTK7 single-domain antibody and human PTK7 Using a carboxyamino group reaction, the stationary phase human PTK7-Fc antigen protein is immobilized on the surface of the CM-5 sensor tip. Anti-PTK7 single-domain antibodies are diluted to various concentrations in HBS buffer, bound to the anti-PTK7 single-domain antibodies at a flow rate of 30 ul / min, bound for 90 seconds, separated for 600 seconds, and the binding process between the anti-PTK7 single-domain antibody and the antigen is observed. When measuring the next anti-PTK7 single-domain antibody, the tip is regenerated by rinsing with 10 mM glycine. The detection results are shown in Table 3, and the binding of the anti-PTK7 single-domain antibody of the present invention to the PTK7-Fc protein can reach 0.18 to 3.45 nM. [Table 6]

[0095] Example 6: ELISA detection of anti-PTK7 single-domain antibody and human PTK7 epitope identification and grouping. ELISA plates were coated with different anti-PTK7 single-domain antibodies, left overnight at 4°C, washed four times with PBS-T the following day, 13% BSA was added, left at room temperature for 1 hour, washed five times with PBS-T, human PTK7-Fc and anti-PTK7 single-domain antibodies were added, left at 37°C for 1 hour, anti-human Fc-HRP antibody was added, left at 37°C for 1 hour, washed five times with PBS-T, TMB-soluble substrate was added, left two plates at room temperature, absorption values ​​were read at a wavelength of 450 nm, and the competitive results for each single-domain antibody were calculated based on the absorption values. The detection results are shown in Table 5. The anti-PTK7 single-domain antibodies of the present invention can be divided into groups according to their expression epitopes, where MY2132-4-22 is group 1, and MY2132-4-55, MY2132-4-79, and MY2132-5-51 are group 2. [Table 7]

[0096] Example 7: Flow cytometry (FACS) detection of tumor cells with anti-PTK7 single-domain antibody Antibody binding function will be verified using HCT116 tumor cells that highly express PTK7. Cultured B×Pc3 cells will be digested with trypsin, neutralized with complete medium, washed once with PBS, then collected and evenly distributed into 96-well plates, human Fc blocking antibody will be added, incubated at 4°C for 15 minutes, and centrifuged. Wash cells once with PBS, add anti-PTK7 single-domain antibody or non-targeted single-domain antibody (negative control), incubated at 4°C for 30 minutes, washed once with PBS after centrifugation, add anti-HA-Alexa Fluor488 or mouse anti-PTK7-FITC antibody (positive control), incubated at 4°C for 20 minutes, washed cells once with PBS, centrifuged at 4°C for 5 minutes, discarded the supernatant, resuspended cells in 200 μl of PBS, and the Alexa Fluor488 signal for each sample will be detected by flow cytometry. The results are shown in Figure 3, demonstrating that the anti-PTK7 single-domain antibody of the present invention can effectively bind to the PTK7 protein on the surface of tumor cells.

[0097] Example 8: SPECT / CT imaging of anti-PTK7 single-domain antibody in a mouse tumor xenograft model Pertechnetium acid is added to the tricarboxyl group kit, incubated at 99°C for 20 minutes, the reagent bottle is cooled to room temperature, hydrochloric acid is added to neutralize the technetium tricarboxyl group to pH 7-7.5, anti-PTK7 single-domain antibody is added, incubated at 37°C for 1 hour, and the radioactive purity of the technetium-labeled anti-PTK7 single-domain antibody is identified by thin-layer chromatography. 1 x 10 subcutaneous tissue on the right dorsal side of a nude mouse 7 Individual high-expression PTK7 (HCT116) cells were inoculated, and tumors measuring 150-200 mm were used for formal experimental research. 3 After waiting until the tumors had grown, the tumor-bearing mice were anesthetized with isoflurane, and technetium-labeled anti-PTK7 single-domain antibody (~10ug, 37MBq) was injected into the tail vein. 90 minutes after administration, the mice were scanned using static 15-minute SPECT and medium-resolution whole-body CT. The anti-PTK7 single-domain antibody of the present invention can be effectively accumulated in PTK7-high-expression tumor models and can be applied to the diagnosis and efficacy evaluation of PTK7-targeted cancers, as well as to the development of new generation PTK7-targeted therapies.

[0098] [Table 8-1] [Table 8-2]

[0099] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. The VHH chain of an anti-PTK7 single-domain antibody containing the following complementarity-determining region (CDR): CDR1, indicated by SEQ ID NO: 9, CDR2 shown in SEQ ID NO: 12, and CDR3 as shown in SEQ ID NO: 15; or CDR1, indicated by SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 13, and CDR3 as shown in SEQ ID NO: 16; or CDR1, indicated by SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 13, and CDR3 as shown in SEQ ID NO: 17; or CDR1, indicated by SEQ ID NO: 11, CDR2, shown in SEQ ID NO: 14, and CDR3, indicated by SEQ ID NO:

18.

2. The VHH chain of an anti-PTK7 single-domain antibody, The VHH chain of the anti-PTK7 single-domain antibody according to claim 1, characterized in that the VHH chain includes a framework region (FR).

3. It is an anti-PTK7 single-domain antibody, The anti-PTK7 single-domain antibody is characterized by being a single-domain antibody against the PTK7 protein and having a VHH chain of the amino acid sequence shown in any one of SEQ ID NO: 1 to 4.

4. It is a polynucleotide, The polynucleotide is characterized in that it encodes a protein selected from the group consisting of the VHH chain of the anti-PTK7 single-domain antibody described in claim 1, or the anti-PTK7 single-domain antibody described in claim 3.

5. The polynucleotide is characterized by having a nucleotide sequence represented by one of SEQ ID NO. 5 to 8. The polynucleotide according to claim 4.

6. An expression vector, The expression vector is characterized by comprising the polynucleotide described in claim 4.

7. It is a host cell, The host cell is characterized in that it contains the expression vector described in claim 6, or the polynucleotide described in claim 4 is incorporated into its genome.

8. A method for producing an anti-PTK7 single-domain antibody, (a) A step of culturing the host cells described in claim 7 under conditions suitable for the production of a single-domain antibody, thereby obtaining a culture containing the anti-PTK7 single-domain antibody, and (b) The step of separating or recovering the anti-PTK7 single-domain antibody from the culture. A method for producing the anti-PTK7 single-domain antibody, characterized by including the following:

9. It is an immune conjugate, This immune conjugate is, (a) The VHH chain of the anti-PTK7 single-domain antibody described in claim 2, and (b) A coupling portion selected from the group consisting of a detectable marker, drug, toxin, cytokine, radionuclide, or enzyme. The immunoconjugate characterized by containing

10. It is an immune conjugate, This immune conjugate is, (a) The anti-PTK7 single-domain antibody according to claim 3, and (b) A coupling portion selected from the group consisting of a detectable marker, drug, toxin, cytokine, radionuclide, or enzyme. The immunoconjugate characterized by containing

11. The use of the VHH chain described in claim 2, the anti-PTK7 single-domain antibody described in claim 3, or the immunoconjugate described in claim 9 or 10, The use is characterized by being used in the preparation of (a) a reagent for detecting PTK7 molecules, or (b) a drug for treating tumors.

12. The tumor is characterized by being a tumor with high expression of PTK7. The use described in claim 11.

13. A pharmaceutical composition, The pharmaceutical composition is characterized by comprising (i) a single-domain antibody as described in claim 3, or an immunoconjugate as described in claim 9 or 10, and (ii) a pharmaceutically acceptable carrier.

14. A pharmaceutical composition according to claim 13 for treating a tumor.

15. A method for in vitro detection of PTK7 protein in a sample, (1) The step of bringing the sample into contact with the single-domain antibody described in claim 3. (2) A method for in vitro detection of PTK7 protein in a sample, comprising the step of detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PTK7 protein in the sample.

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