WO2023020551A1 - 抗ptk7单域抗体及其应用 - Google Patents

抗ptk7单域抗体及其应用 Download PDF

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WO2023020551A1
WO2023020551A1 PCT/CN2022/113106 CN2022113106W WO2023020551A1 WO 2023020551 A1 WO2023020551 A1 WO 2023020551A1 CN 2022113106 W CN2022113106 W CN 2022113106W WO 2023020551 A1 WO2023020551 A1 WO 2023020551A1
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ptk7
domain antibody
single domain
antibody
present
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French (fr)
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黄仲廉
丁航海
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Nanomab Technology Ltd
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Nanomab Technology Ltd
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Priority to JP2024510277A priority Critical patent/JP2024531379A/ja
Priority to CN202280055408.1A priority patent/CN118139893A/zh
Priority to AU2022329227A priority patent/AU2022329227A1/en
Priority to EP22857862.1A priority patent/EP4389769A4/en
Priority to US18/684,078 priority patent/US20240360248A1/en
Publication of WO2023020551A1 publication Critical patent/WO2023020551A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1063—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from stomach or intestines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies, in particular to a specific single-domain antibody against PTK7, and more specifically to its coding sequence and its application in disease diagnosis and treatment.
  • PTK7 Protein tyrosine kinase 7
  • CCK4 colon cancer kinase 4
  • PTK7 is overexpressed in a variety of tumor tissues, including breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, and colon cancer, and its overexpression is associated with poor prognosis and increased risk of metastasis.
  • PTK7 has also been confirmed to be highly enriched in tumor-initiating cells (TIC) or cancer stem cells in patient-derived tumor tissues. Therefore, targeting PTK7 not only eliminates general cancer cells, but also eliminates TIC responsible for tumor recurrence, becoming a natural candidate for targeted therapy (Damelin, 2017).
  • PTK7 has no catalytic activity, it is impractical to develop typical tyrosine kinase inhibitors, and multiple drugs targeting it, including different antibody conjugates and different formats, are in clinical development.
  • cofetuzumab pelidotin a new type of antibody-conjugated drug (ADC), using humanized antibody hu6M024 as a targeting carrier and calendulain microtubule inhibitor Aur0101, has been used in metastatic triple-negative breast cancer, platinum-resistant First-in-human clinical trials in ovarian cancer and non-small cell lung cancer.
  • F-18-labeled Aptamer aptamer as an immunoimaging tracer targeting PTK7 enabled specific, selective and high-affinity surveillance of PTK7 in xenograft models of several tumor cell lines (HCT116 and UMG) expression, and may further develop PTK7-targeted therapies (Jacobson, 2015).
  • Single-domain antibody namely camel heavy chain single-domain antibody VHH (variable domain of heavy chain of heavy-chain antibody)
  • VHH variable domain of heavy chain of heavy-chain antibody
  • the molecular weight of single-domain antibodies is 1/10 of that of traditional antibodies. It has the characteristics of high stability, good water solubility, simple humanization, high targeting, and strong penetration.
  • a radioisotope targeting carrier it can quickly and specifically It can penetrate the tumor tissue and bind the target selectively, and the unbound antibody can be quickly cleared from the blood, reducing the radiation dose of the body.
  • D'Huyvetter 2014
  • the purpose of the present invention is to provide an anti-PTK7 single domain antibody with good PTK7 antigen binding property.
  • the first aspect of the present invention provides a complementarity determining region CDR of the VHH chain of an anti-PTK7 single domain antibody, and the complementarity determining region CDR of the VHH chain comprises:
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the complementarity determining region CDR of the VHH chain comprises:
  • the complementarity determining region CDR of the VHH chain comprises CDR1, CDR2 and CDR3 selected from the following group:
  • the second aspect of the present invention provides a VHH chain of an anti-PTK7 single domain antibody, the VHH chain comprising the framework region FR and the complementarity determining region CDR described in the first aspect of the present invention.
  • the framework region consists of FR1, FR2, FR3 and FR4 selected from the group consisting of:
  • the framework region is composed of:
  • the VHH chain of the anti-PTK7 single domain antibody has an amino acid sequence as shown in any one of SEQ ID NO: 1-4.
  • the VHH chain of the anti-PTK7 single domain antibody has the amino acid sequence shown in SEQ ID NO:1.
  • the third aspect of the present invention provides an anti-PTK7 single-domain antibody, the single-domain antibody is a single-domain antibody against the PTK7 protein, and has a VHH of the amino acid sequence shown in any one of SEQ ID NO: 1-4 chain.
  • the single domain antibody has the VHH chain of the amino acid sequence shown in SEQ ID NO:1.
  • the fourth aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of: the CDR region described in the first aspect of the present invention, the anti-PTK7 monoclonal protein described in the second aspect of the present invention The VHH chain of a domain antibody, or the anti-PTK7 single domain antibody described in the third aspect of the present invention.
  • the polynucleotide has a nucleotide sequence as shown in any one of SEQ ID NO.:5-8.
  • the polynucleotide includes DNA, cDNA or RNA.
  • the fifth aspect of the present invention provides an expression vector containing the polynucleotide described in the fourth aspect of the present invention.
  • the expression vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the sixth aspect of the present invention provides a host cell containing the expression vector of the fifth aspect of the present invention, or the polynucleotide of the fourth aspect of the present invention integrated in its genome.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of Escherichia coli and yeast cells.
  • the seventh aspect of the present invention provides a method for producing an anti-PTK7 single domain antibody, comprising the steps of:
  • the anti-PTK7 single domain antibody has an amino acid sequence as shown in any one of SEQ ID NO: 1-4.
  • the eighth aspect of the present invention provides an immunoconjugate comprising:
  • VHH chain of the anti-PTK7 single domain antibody according to the second aspect of the present invention, or the anti-PTK7 single domain antibody according to the third aspect of the present invention and (b) a coupling moiety selected from the group consisting of: A marker, drug, toxin, cytokine, radionuclide, or enzyme can be detected.
  • the coupling moiety is a drug or a toxin.
  • the coupling moiety is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer X-ray tomography) contrast agents, or capable of producing detectable Products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, precursors Drug activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or nanoparticles in any form, etc.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • chemotherapeutic agents eg, cisplatin
  • the immunoconjugate contains: the multivalent (such as bivalent) VHH chain of the anti-PTK7 single domain antibody described in the second aspect of the present invention, the anti-PTK7 single domain antibody described in the third aspect of the present invention Anti-PTK7 single domain antibody.
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeats of the VHH chain of the anti-PTK7 single domain antibody described in the second aspect of the present invention, the present invention The anti-PTK7 single domain antibody according to the third aspect.
  • the ninth aspect of the present invention provides the VHH chain as described in the second aspect of the present invention, the anti-PTK7 single domain antibody as described in the third aspect of the present invention, or the immunoconjugate as described in the eighth aspect of the present invention
  • the purposes of the method are used for preparing (a) reagents for detecting PTK7 molecules; (b) medicines for treating tumors.
  • the detection includes flow detection and cell immunofluorescence detection.
  • the tumor is a tumor with high expression of PTK7.
  • 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.
  • the tenth aspect of the present invention provides a pharmaceutical composition comprising: (i) the single domain antibody as described in the third aspect of the present invention or the immunoconjugated antibody as described in the eighth aspect of the present invention substance; and (ii) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare a drug for treating tumors, and the tumors are selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, colon cancer, or a combination thereof .
  • a method for in vitro (diagnostic and non-diagnostic) detection of PTK7 protein in a sample comprising the steps of:
  • the twelfth aspect of the present invention provides a method for treating tumors, comprising administering the single domain antibody as described in the third aspect of the present invention, the immunoconjugate as described in the eighth aspect of the present invention to a subject in need, Or the pharmaceutical composition as described in the tenth aspect of the present invention.
  • the tumor is a tumor with high expression of PTK7.
  • 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.
  • the subject includes a human or a non-human mammal.
  • the thirteenth aspect of the present invention provides a radioimmunoimaging method, comprising using the single domain antibody as described in the third aspect of the present invention, or the immunoconjugate as described in the eighth aspect of the present invention for imaging.
  • Fig. 1 is the detection result of human PTK7-Fc protein antigen. The results showed that the purity of the protein reached more than 90% on SDS-PAGE.
  • Figure 2 is the construction and quality inspection results of two anti-PTK7 single domain antibody phage display libraries.
  • Figure A is the PCR identification diagram of the first and second PCR amplification products of the two libraries. The results show that a VHH gene fragment with a size of about 400bp is finally obtained;
  • Figure B is the storage capacity detection diagram of the two libraries.
  • Fig. 3 is the result of flow cytometry detection of the binding activity of anti-PTK7 single domain antibody to PTK7-positive HCT116 cells. The results showed that all four single-domain antibodies could effectively bind to PTK7 protein on the surface of HCT116 cells.
  • the present invention utilizes human-derived PTK7 extracellular segment antigen protein to immunize camels to obtain a high-quality immune single-domain antibody gene library. Then the PTK7 protein molecule is coupled to the microtiter plate to display the correct spatial structure of the PTK7 protein, and the antigen in this form is screened by the phage display technology for the immune single-domain antibody gene library (camel heavy chain antibody phage display gene library) to obtain PTK7-specific single domain antibody gene. The gene was then transferred to E. coli to obtain a single-domain antibody strain that can be highly expressed in E. coli and has high specificity.
  • human-derived PTK7 extracellular segment antigen protein to immunize camels to obtain a high-quality immune single-domain antibody gene library. Then the PTK7 protein molecule is coupled to the microtiter plate to display the correct spatial structure of the PTK7 protein, and the antigen in this form is screened by the phage display technology for the immune single-domain antibody gene library (camel heavy chain
  • single domain antibody of the present invention 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 used interchangeably, and all refer to specific recognition and binding to PTK7 (including human, Single domain antibodies to mouse and monkey PTK7).
  • the present invention includes the single-domain antibody whose amino acid sequence is as described in any one of SEQ ID NO: 1-4, particularly preferably the single-domain antibody whose VHH chain amino acid sequence is as shown in SEQ ID NO: 1.
  • single domain antibody As used herein, the terms “single domain antibody”, “VHH”, and “nanobody” have the same meaning and are used interchangeably, and refer to the variable region of the heavy chain of a cloned antibody, constructed from only one heavy chain variable region.
  • a single-domain antibody composed of domains, which is the smallest fully functional antigen-binding fragment.
  • CH1 light chain and heavy chain constant region 1
  • the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
  • antibody or "immunoglobulin” is a heterotetrameric protein of about 150,000 Daltons with identical structural features, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain .
  • VL variable region
  • Specific amino acid residues form the interface between the variable domains of the light and heavy chains.
  • variable means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each contain four FR regions that are roughly in a ⁇ -sheet configuration connected by three CDRs that form connecting loops, in some cases forming partial ⁇ -sheet structures.
  • the CDRs in each chain are brought into close proximity by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example involved in the antibody-dependent cytotoxicity of the antibody.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to polypeptides that specifically bind to the PTK7 protein, such as proteins or polypeptides with heavy chain variable regions . They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), and the amino acid sequences of the four FRs It is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a ring structure, and the ⁇ sheets formed by the FRs in between are close to each other in the spatial structure.
  • the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • the antibody of the present invention refers to a polypeptide that has PTK7 protein binding activity and includes the above CDR region.
  • the term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): deletion, insertion and/or substitution of one or more amino acids, and addition of one or several amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof.
  • the invention also includes fragments of the single domain antibodies of the invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention.
  • An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • a polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
  • biomolecules nucleic acid, protein, etc.
  • the biomolecules involved in the present invention include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl2 . Transformation can also be performed by electroporation, if desired.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses 6. Liposomes; 7. Nanomagnetic particles; 8. Drug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 9. Therapeutic agents (eg, , cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the invention can be directly used for binding PTK7 protein molecules, and thus can be used for treating tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single domain antibody (or its conjugate) of the present invention and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the invention can also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the single domain antibody bears a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
  • Colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the anti-PTK7 single-domain antibody is labeled with colloidal gold to obtain a colloidal gold-labeled single-domain antibody.
  • the anti-PTK7 single-domain antibody is labeled with a radioactive isotope to obtain an isotope-labeled single-domain antibody.
  • the anti-PTK7 single domain antibody of the invention has good specificity and high titer, and can be used for clinical diagnosis against PTK7.
  • the present invention also relates to methods for detecting PTK7 protein.
  • the steps of the method are roughly as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of PTK7 protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or a fragment thereof) or a detection plate of the present invention.
  • the kit further includes a container, an instruction manual, a buffer and the like.
  • the present invention also provides a detection kit for detecting the level of PTK7, which includes an antibody that recognizes PTK7 protein, a lysis medium for dissolving samples, general reagents and buffers required for detection, such as various buffers, detection labeling, detection substrates, etc.
  • the test kit may be an in vitro diagnostic device.
  • the single domain antibody of the present invention has a wide range of biological and clinical application values, and its application involves the diagnosis and treatment of diseases related to PTK7, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy against PTK7.
  • the present invention provides the application of anti-PTK7 single domain antibody in the diagnosis and treatment of tumors, which are tumors with abnormally high expression of PTK7 (including mRNA and/or protein levels), specifically, the tumors include but not limited to: breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, colon cancer, etc.
  • the present invention has developed a single-domain antibody against the PTK7 target for the first time
  • the single domain antibody of the present invention has good specificity and can bind to human, mouse and rhesus monkey PTK7;
  • the single-domain antibody of the present invention can effectively accumulate in PTK7 high-expression tumor models, and can be applied to PTK7-targeted cancer diagnosis and curative effect evaluation, and can be used to develop a new generation of PTK7-targeted therapy;
  • Embodiment 1 Human PTK7-Fc protein antigen expression
  • Transient expression of human PTK7 extracellular domain protein in mammalian cells HEK293F Cloning human PTK7 extracellular domain gene into pFUSE-IgG recombinant plasmid, mixed with transfection reagent PEI 1:3, and then transfected into HEK293F cells; 37°C, 6 Culture in a %CO2 shaker incubator for 6 days; then collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; wash the beads with phosphate buffer solution pH 7.0, and elute the protein with 0.1M pH 3.0 glycine solution ; Then the eluted protein was ultrafiltered into PBS solution, and after measuring the yield, samples were taken for SDS-PAGE detection.
  • the purified PTK7-Fc protein was used to immunize two Xinjiang Bactrian camels. After seven immunizations, the total RNA was isolated from the peripheral blood of the camels, and the VHH gene was amplified by reverse transcription and PCR (Fig. 2A). Cloned into the phage vector pMECS, transformed into TG1 host cells to construct a phage display library. The two constructed libraries were serially diluted and plated to determine the library capacity. At the same time, 24 clones were randomly selected from each constructed library for colony PCR detection.
  • each plasmid was electrotransformed into Escherichia coli WK6, spread on the culture plate of LA+glucose, and cultivated overnight at 37°C; a single colony was selected and inoculated on Cultivate in 5 mL of LB culture medium containing ampicillin overnight at 37°C on a shaker; inoculate 1 mL of the overnight strain into 330 mL of TB culture liquid, and culture on a shaker at 37°C. Cultivate overnight on a shaking table; collect bacteria by centrifugation, and obtain crude antibody extract by osmosis method; prepare purified single domain antibody by nickel column ion affinity chromatography. After testing, the purity of the expressed and purified single domain antibody was greater than 90%, which was used for candidate functional activity research, and finally obtained the following 4 leading single domain antibody strains.
  • Antibody number Amino acid sequence number Nucleotide sequence number MY2132-4-22 SEQ ID NO: 1 SEQ ID NO: 5 MY2132-4-55 SEQ ID NO: 2 SEQ ID NO: 6 MY2132-4-79 SEQ ID NO: 3 SEQ ID NO: 7 MY2132-5-51 SEQ ID NO: 4 SEQ ID NO: 8
  • sequences of the four single domain antibodies are as follows, and the three CDR regions are underlined respectively.
  • Example 4 Enzyme-linked immunoassay (ELISA) detection of anti-PTK7 single domain antibody and different species of PTK7
  • PTK7-Fc of human or mouse species or PTK7-His antigen protein of rhesus monkey species to prepare the microtiter plate, overnight at 4°C; wash 4 times with PBS-T the next day, add 1% BSA, and keep at room temperature Block for 2 hours; wash 4 times with PBS-T, add anti-PTK7 single domain antibody diluted according to gradient, place at 37°C for 1 hour; wash 4 times with PBS-T, add mouse anti-His-HRP antibody or mouse anti-HA-HRP Antibody, placed at 37°C for 1 hour; washed 4 times with PBS-T, added TMB soluble substrate, and left at room temperature for 10 minutes; read the absorbance value at 450nm wavelength, and calculated the Kd value of each single domain antibody according to the absorbance value.
  • the test results are shown in Table 2.
  • the anti-PTK7 single domain antibody of the present invention can bind to human, mouse or rhesus monkey PTK7.
  • the stationary phase human PTK7-Fc antigen protein was immobilized on the surface of the CM-5 sensor chip by carboxyl amino reaction; the anti-PTK7 single domain antibody was diluted to different concentrations with HBS buffer, and the anti-PTK7 single domain antibody was added at a flow rate of 30ul/min. Combining for 90s and dissociation for 600s, observe the binding process of anti-PTK7 single domain antibody and antigen; wash with 10mM Glycine for chip regeneration, and proceed to the next anti-PTK7 single domain antibody determination.
  • Example 6 ELISA detection of anti-PTK7 single domain antibody and human PTK7 epitope identification group
  • the test results are shown in Table 5.
  • the anti-PTK7 single domain antibody of the present invention can be divided into two groups according to the binding epitope, wherein MY2132-4-22 is group 1, MY2132-4-55, MY2132-4-79 and MY2132-5 -51 for group 2.
  • Antibody binding function verification was carried out using the tumor cell HCT116 with high expression of PTK7: the cultured BxPc3 cells were digested with trypsin and neutralized with complete medium, and the cells were washed once with PBS, and then the cells were collected; the cells were evenly distributed into 96-well plates, Add human Fc blocking antibody, incubate at 4°C for 15 minutes; wash cells once with PBS after centrifugation, add anti-PTK7 single domain antibody or non-targeting single domain antibody (negative control), incubate at 4°C for 30 minutes; wash with PBS after centrifugation Cells once, then add anti-HA-Alexa Fluor488 or mouse anti-PTK7-FITC antibody (positive control), incubate at 4°C for 20 minutes; wash the cells once with PBS, centrifuge at 4°C for 5 minutes, discard the supernatant, add 200ul PBS to re-incubate Suspend the cells, and detect the Alexa Fluor488 signal of each sample by flow
  • the anti-PTK7 single domain antibody of the present invention can effectively bind to the PTK7 protein on the surface of tumor cells.
  • Example 8 SPECT/CT Imaging of Anti-PTK7 Single Domain Antibody in Mouse Tumor Xenograft Model
  • the anti-PTK7 single domain antibody of the present invention can effectively accumulate in PTK7 high-expression tumor models, and is applied to PTK7-targeted cancer diagnosis and curative effect evaluation, and at the same time, develops a new generation of PTK7-targeted therapy.

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Abstract

提供了一组针对PTK7的特异性单域抗体编码序列及其应用。提供的抗PTK7单域抗体能有效与PTK7抗原结合，为靶向PTK7的药物包括核素偶联药物，抗体偶联药物及多特异性抗体药物等研发提供了基础。

Description

抗PTK7单域抗体及其应用 技术领域
本发明涉及抗体领域，具体地涉及针对PTK7的特异性单域抗体，更具体地涉及其编码序列和其在疾病诊断和治疗中的应用。
背景技术
蛋白酪氨酸激酶7(PTK7)，又名结肠癌激酶4(CCK4)，是一种高度保守、无催化活性的跨膜蛋白酪氨酸激酶(PTK)，参与造血细胞，体细胞祖细胞以及干细胞发育过程的非标准Wnt信号转导收体(Peradziryi，2012)。
研究表明，PTK7在多种肿瘤组织中过度表达，包括乳腺癌、肺癌、胰腺癌、宫颈癌、卵巢癌、结肠癌，其过度表达与预后不良和转移风险增加有关。另外，PTK7也被证实病人源性肿瘤组织中的肿瘤启动细胞(TIC)或肿瘤干细胞高度富集。因此，靶向PTK7不单消除一般癌细胞，更能消除负责肿瘤复发的TIC，成为靶向治疗的一个天然候选靶点(Damelin，2017)。然而，由于PTK7没有催化活性，因此对此开发典型酪氨酸激酶抑制剂是不切实际的，以其为靶点的不同抗体偶联物以及不用型式的等多种药物正处于临床研发中。其中新型抗体偶联药物(ADC)，利用人源化抗体hu6M024作为靶向载体与金盏花素微管抑制剂Aur0101偶联而成的cofetuzumab pelidotin，已于转移性三阴性乳腺癌，铂耐药卵巢癌和非小细胞肺癌进行首次人体临床试验。其中早期研究数据证明，每3周一次2.8mg/kg cofetuzumab pelidotin是安全的，并在接受治疗的患者中观察到抗肿瘤活性，总体客观有效率分別为铂耐药卵巢癌27％(n＝63)，非小细胞肺癌19％(n＝31)和三阴性乳腺癌21％(n＝29)(Maitland,2021)。此外，利用F-18标记的Aptamer核酸适体作为靶向PTK7的免疫显像示踪剂能够在种肿瘤细胞系(HCT116和UMG)的异种移植模型中特异性、选择性和高亲和力地监察PTK7表达，并可能进一步开发PTK7靶向治疗(Jacobson,2015)。
截至目前，市场上尚未有针对PTK7靶点的单域抗体药物。单域抗体即骆驼重链单域抗体VHH(variable domain of heavy chain of heavy-chain antibody)，是目前可以得到的具有完整功能的稳定的可结合抗原的最小单位。单域抗体其分子量是传统抗体的1/10,具有稳定性高、水溶性好、人源化简单、靶向性高、穿透性强等特点，作为放射性同位素靶向载体，能够快速、特异性地穿透肿瘤组织结合靶标，而 无结合抗体能很快从血液清除，减低身体放射性剂量，对比传统抗体用于开发放射免疫成像及放射免疫疗法具有众多明显优势(D’Huyvetter,2014)。
因此，本领域需要开发一种抗PTK7单域抗体，尤其是能够单一有效地结合PTK7的特异性单域抗体，开发新一代放射免疫成像及放射免疫疗法。
发明内容
本发明的目的在于提供一种具备良好的PTK7抗原结合性的抗PTK7单域抗体。
本发明的第一方面，提供了一种抗PTK7单域抗体VHH链的互补决定区CDR，所述VHH链的互补决定区CDR包含：
SEQ ID NO:9-11所示的CDR1；
SEQ ID NO:12-14所示的CDR2；和
SEQ ID NO:15-18所示的CDR3。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述VHH链的互补决定区CDR包含：
SEQ ID NO:9所示的CDR1；
SEQ ID NO:12所示的CDR2；和
SEQ ID NO:15所示的CDR3。
在另一优选例中，所述VHH链的互补决定区CDR包含选自下组的CDR1、CDR2和CDR3：
组 CDR1序列编号 CDR2序列编号 CDR3序列编号
1 9 12 15
2 10 13 16
3 10 13 17
4 11 14 18。.
本发明的第二方面，提供了一种抗PTK7单域抗体的VHH链，所述VHH链包括框架区FR和本发明第一方面所述的互补决定区CDR。
在另一优选例中，所述框架区由选自下组的FR1、FR2、FR3和FR4组成：
组 FR1序列编号 FR2序列编号 FR3序列编号 FR4序列编号
1 19 22 25 29
2 20 23 26 30
3 20 23 27 29
4 21 24 28 31。
在另一优选例中，所述的框架区由：
SEQ ID NO:19所示的FR1，SEQ ID NO:22所示的FR2，SEQ ID NO:25所示的FR3，和SEQ ID NO:29所示的FR4组成。
在另一优选例中，所述的抗PTK7单域抗体的VHH链具有如SEQ ID NO:1-4任一所示的氨基酸序列。
在另一优选例中，所述的抗PTK7单域抗体的VHH链具有如SEQ ID NO:1所示的氨基酸序列。
本发明的第三方面，提供了一种抗PTK7单域抗体，所述单域抗体是针对PTK7蛋白的单域抗体，并且具有如SEQ ID NO:1-4任一所示的氨基酸序列的VHH链。
在另一优选例中，所述单域抗体具有如SEQ ID NO:1所示的氨基酸序列的VHH链。
本发明的第四方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：本发明第一方面所述的CDR区、本发明第二方面所述的抗PTK7单域抗体的VHH链、或本发明第三方面所述的抗PTK7单域抗体。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.:5-8任一所示的核苷酸序列。
在另一优选例中，所述的多核苷酸包括DNA、cDNA或RNA。
本发明的第五方面，提供了一种表达载体，所述表达载体含有本发明第四方面所述的多核苷酸。
在另一优选例中，所述表达载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第六方面，提供了一种宿主细胞，所述宿主细胞含有本发明的第五方面所述的表达载体，或其基因组中整合有本发明第四方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞。
本发明的第七方面，提供了一种产生抗PTK7单域抗体的方法，包括步骤：
(a)在适合产生单域抗体的条件下，培养本发明第六方面所述的宿主细胞，从而获得含所述抗PTK7单域抗体的培养物；以及(b)从所述培养物中分离或回收所述的抗PTK7单域抗体。
在另一优选例中，所述的抗PTK7单域抗体具有如SEQ ID NO:1-4任一所示的氨基酸序列。
本发明的第八方面，提供了一种免疫偶联物，所述免疫偶联物含有：
(a)如本发明第二方面所述的抗PTK7单域抗体的VHH链、或如本发明第三方面所述的抗PTK7单域抗体；和(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第二方面所述的抗PTK7单域抗体的VHH链、如本发明第三方面所述的抗PTK7单域抗体。
在另一优选例中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第二方面所述的抗PTK7单域抗体的VHH链、本发明第三 方面所述的抗PTK7单域抗体。
本发明的第九方面，提供了如本发明第二方面所述的VHH链，如本发明第三方面所述的抗PTK7单域抗体，或如本发明第八方面所述的免疫偶联物的用途，用于制备(a)用于检测PTK7分子的试剂；(b)用于治疗肿瘤的药物。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
在另一优选例中，所述肿瘤为PTK7高表达的肿瘤。
在另一优选例中，所述肿瘤选自下组：乳腺癌、肺癌、胰腺癌、宫颈癌、卵巢癌、结肠癌，或其组合。
本发明的第十方面，提供了一种药物组合物，所述药物组合物包含：(i)如本发明第三方面所述的单域抗体或如本发明第八方面所述的免疫偶联物；和(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备治疗肿瘤的药物，所述的肿瘤选自下组：乳腺癌、肺癌、胰腺癌、宫颈癌、卵巢癌、结肠癌，或其组合。
本发明的第十一方面，提供了一种体外(诊断和非诊断性)检测样品中PTK7蛋白的方法，包括步骤：
(1)将样品与本发明第三方面所述的单域抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在PTK7蛋白。
本发明的第十二方面，提供了一种治疗肿瘤的方法，包括向需要的对象施用如本发明第三方面所述的单域抗体，如本发明第八方面所述的免疫偶联物，或如本发明第十方面所述的药物组合物。
在另一优选例中，所述肿瘤为PTK7高表达的肿瘤。
在另一优选例中，所述肿瘤选自下组：乳腺癌、肺癌、胰腺癌、宫颈癌、卵巢癌、结肠癌，或其组合。
在另一优选例中，所述对象包括人或非人类哺乳动物。
本发明的第十三方面，提供了一种放射性免疫成像方法，包括使用如本发明第三方面所述的单域抗体，或如本发明第八方面所述的免疫偶联物进行成像。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是人PTK7-Fc蛋白抗原的检测结果。结果表明，该蛋白在SDS-PAGE纯度达到90％以上。
图2是两个抗PTK7单域抗体噬菌体展示文库的构建和质量检测结果。图A为两个文库第一次和第二次PCR扩增产物的PCR鉴定图，结果表明，最终获得大小约为400bp的VHH基因片段；图B为两个文库的库容检测图，结果表明，两个文库库容分別为2.2x10 9和3.4x10 9CFU；图C为两个文库的插入率检测图，结果表明，两个文库的VHH插入率分別为96％和100％；图D为两个文库的富集检测图，结果表明，两个文库的特异性噬菌体经三轮筛选后分別达到60和84倍富集。
图3是抗PTK7单域抗体与PTK7阳性表达HCT116细胞结合活性的流式细胞术检测结果。结果表明，4株单域抗体均能够有效结合HCT116细胞表面的PTK7蛋白。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次成功地研发出了一组抗PTK7单域抗体。实验结果表明，本发明获得的4株PTK7单域抗体均能以高亲和力特异性结合人或猴PTK7蛋白。
具体地，本发明利用人源的PTK7胞外段抗原蛋白免疫骆驼，获得高质量的免疫单域抗体基因文库。然后将PTK7蛋白分子偶联在酶标板上，展示PTK7蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫单域抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了PTK7特异性的单域 抗体基因。再将此基因转至大肠杆菌中，从而获得了能在大肠杆菌中高效表达的、且特异性高的单域抗体株。
术语
如本文所用，术语“本发明单域抗体”、“本发明的抗PTK7单域抗体”、“本发明PTK7单域抗体”可互换使用，均指特异性识别和结合于PTK7(包括人、小鼠和猴PTK7)的单域抗体。本发明包括氨基酸序列如SEQ ID NO:1-4任一项所述的的单域抗体，特别优选的是VHH链的氨基酸序列如SEQ ID NO:1所示的单域抗体。
如本文所用，术语“单域抗体”、“VHH”、“纳米抗体”(nanobody)具有相同的含义并可互换使用，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“重链可变区”与“V H”可互换使用。
如本文所用，术语“可变区”与“互补决定区(Complementarity Determining Region，CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PTK7蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与 另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PTK7蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含单域抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通 常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔，脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；9.疗剂(例如，顺铂)或任何形式的纳米颗粒等。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PTK7蛋白分子，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的单域抗体
在本发明的一个优选例中，所述单域抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，抗PTK7的单域抗体用胶体金标记，得到胶体金标记的单域抗体。在本发明的另一个优选的方案中，抗PTK7的单域抗体用放射性同位素标记，得到同位素标记的单域抗体。
本发明的抗PTK7单域抗体具有很好的特异性，很高的效价，可用于针对PTK7的临床诊断。
检测方法
本发明还涉及检测PTK7蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中PTK7蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测PTK7水平的检测试剂盒，该试剂盒包括识别PTK7蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的单域抗体有广泛生物应用价值和临床应用价值，其应用涉及到与PTK7相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对PTK7的临床诊断和靶向治疗。
本发明提供了抗PTK7单域抗体在诊断和治疗肿瘤中的应用，所述肿瘤为PTK7异常高表达(包括mRNA和/或蛋白水平)的肿瘤，具体地，所述肿瘤包括但不限于：乳腺癌、肺癌、胰腺癌、宫颈癌、卵巢癌、结肠癌等。
本发明的主要优点包括：
(a)本发明首次研发了针对PTK7靶点的单域抗体；
(b)本发明的单域抗体与PTK7蛋白结合的亲和力高；
(c)本发明的单域抗体特异性好，可以与人、鼠和恒河猴PTK7结合；
(d)本发明的单域抗体能够于PTK7高表达肿瘤模型有效积聚，可应用于PTK7靶向的癌症诊断及疗效评估，同时用于开发新一代PTK7靶向治疗；
(e)本发明的单域抗体制备方法简单，易于大量生产。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1：人PTK7-Fc蛋白抗原表达
利用哺乳动物细胞HEK293F瞬转表达人PTK7胞外段蛋白：将人PTK7胞外段基因克隆至pFUSE-IgG重组质粒，与转染试剂PEI 1:3混合后转染至HEK293F细胞；37℃，6％CO2摇床培养箱中培养6天；随后收集细胞上清，与Protein A珠子在室温下结合1h；再用磷酸盐缓冲液pH 7.0洗涤珠子后，用0.1M pH3.0甘氨酸溶液洗脱蛋白；再将洗脱的蛋白超滤至PBS溶液中，测定产量后取样进行SDS-PAGE检测。
检测结果如图1所示，PTK7-Fc蛋白抗原的纯度大于90％，可用于骆驼免疫及抗体筛选。
实施例2：人PTK7胞外段蛋白免疫文库构建及筛选
将纯化的PTK7-Fc蛋白蛋白免疫2只新疆双峰驼，7次免疫结束后从骆驼外周血中分离total RNA，经反转录和PCR扩增出VHH基因(图2A)，再将VHH基因克隆至噬菌体载体pMECS上，转化至TG1宿主细胞中构建成噬菌体展示文库。两个构建文库被梯度稀释后涂板并测定库容，同时每个构建文库随 机挑取24颗克隆进行菌落PCR检测。
结果表明，两个构建文库库容分別为2.2x10 9和3.4x10 9CFU(图2B)，文库插入率为分別为96％和100％(图2C)。
随后利用噬菌体展示技术进行3轮“吸附-洗涤-富集”，最终两个文库的特异性噬菌体分別达到60和84倍富集(图2D)。从以上富集的噬菌体克隆中随机挑选400颗针对人PTK7-Fc进行PE-ELISA鉴定，将获得的阳性克隆(比值＞3)全部进行测序鉴定，将所有序列不同的单域抗体(107株)均作为候选对象。
实施例3：抗PTK7单域抗体的表达和純化
从实施例2测序分析所获得的抗体克隆挑选出40株，将各质粒电转至大肠杆菌WK6中，并将其涂布在LA+glucose的培养平板上，37℃培养过夜；挑选单个菌落接种在5mL含有氨苄青霉素的LB培养液中，37℃摇床培养过夜；接种1mL的过夜菌种至330mL TB培养液中，37℃摇床培养，培养到OD0.6-1时，加入IPTG，28℃摇床培养过夜；离心收菌，并利用渗透法获得抗体粗提液；经镍柱离子亲和层析制备纯化的单域抗体。经检测表达纯化的单域抗体纯度大于90％，用于候选功能活性研究，最终获得下列4株先导单域抗体。
表1 PTK7单域抗体序列编号
抗体编号 氨基酸序列编号 核苷酸序列编号
MY2132-4-22 SEQ ID NO：1 SEQ ID NO：5
MY2132-4-55 SEQ ID NO：2 SEQ ID NO：6
MY2132-4-79 SEQ ID NO：3 SEQ ID NO：7
MY2132-5-51 SEQ ID NO：4 SEQ ID NO：8
4株单域抗体的序列如下，三个CDR区分别用下划线标出。
Figure PCTCN2022113106-appb-000001
Figure PCTCN2022113106-appb-000002
Figure PCTCN2022113106-appb-000003
实施例4：抗PTK7单域抗体与不同种属PTK7的酶联免疫法(ELISA)检测
利用人或鼠种属的PTK7-Fc或恒河猴种属的PTK7-His抗原蛋白包备酶标板，4℃过夜；第二天用PBS-T洗涤4次，加入1％BSA，室温下封闭2小时；用PBS-T洗涤4次，加入按梯度稀释的抗PTK7单域抗体，37℃放置1小时；用PBS-T洗涤4次，加入鼠抗His-HRP抗体或鼠抗HA-HRP抗体，37℃放置1小时；用PBS-T洗涤4次，加入TMB可溶性底物，室温下放置10分钟；在450nm波长读取吸收值，根据吸收值计算各单域抗体Kd值。
检测结果如表2所示，本发明的抗PTK7单域抗体可与人,鼠或恒河猴PTK7结合。
表2抗PTK7单域抗体ELISA结果
Figure PCTCN2022113106-appb-000004
Figure PCTCN2022113106-appb-000005
实施例5：抗PTK7单域抗体与人PTK7的蛋白结合动力学(Biacore 8K)检测
利用羧基氨基反应将固定相人PTK7-Fc抗原蛋白固定在CM-5传感芯片表面；将抗PTK7单域抗体用HBS缓冲液稀释成不同浓度，以30ul/分钟流速加入抗PTK7单域抗体，结合90s和解离600s，观察抗PTK7单域抗体与抗原结合过程；用10mM Glycine冲洗进行芯片再生，进行下一个抗PTK7单域抗体测定时。
检测结果如表3所示，本发明的抗PTK7单域抗体与PTK7-Fc蛋白的结合能够达到0.18～3.45nM。
表3抗PTK7单域抗体Biacore 8K结果
抗体编号 K on(1/Ms) K off(1/s) Kd(nM)
MY2132-4-22 1.51E+07 3.22E-02 2.13
MY2132-4-55 1.76E+06 3.14E-04 0.18
MY2132-4-79 1.64E+06 3.58E-04 0.22
MY2132-5-51 4.82E+05 1.66E-03 3.45
实施例6：抗PTK7单域抗体与人PTK7表位鉴定分组的ELISA检测
利用不同抗PTK7单域抗体包备酶标板，4℃过夜；第二天用PBS-T洗涤4次，加入13％BSA，室温下封闭1小时；用PBS-T洗涤5次，加入人PTK7-Fc和抗PTK7单域抗体，37℃放置1小时；加入抗人Fc-HRP抗体，37℃放置1小时；用PBS-T洗涤5次，加入TMB可溶性底物，室温下放置2分钟；在450nm波长读取吸收值，根据吸收值计算各单域抗体竞争结果。
检测结果如表5所示，本发明的抗PTK7单域抗体按结合表位能分成2组，其中MY2132-4-22为组1,MY2132-4-55、MY2132-4-79和MY2132-5-51为组2。
表4抗PTK7单域抗体表位鉴定ELISA结果
抗体编号 MY2132-4-22 MY2132-4-55 MY2132-4-79 MY2132-5-51 结论
MY2132-4-22 竞争 非竞争 非竞争 非竞争 1
MY2132-4-55 非竞争 竞争 竞争 竞争 2
MY2132-4-79 非竞争 竞争 竞争 竞争 2
MY2132-5-51 非竞争 竞争 竞争 竞争 2
实施例7：抗PTK7单域抗体与肿瘤细胞的流式细胞分析术(FACS)检测
利用高表达PTK7的肿瘤细胞HCT116进行抗体结合功能验证：将培养的BxPc3细胞用胰酶消化并用完全培养基中和后，用PBS洗涤细胞一次，然后收集细胞；平均分装至96孔板中，加入人Fc阻断抗体，4℃孵育15分钟；离心后用PBS洗涤细胞一次，加入抗PTK7单域抗体或非靶向单域抗体(阴性对照)，4℃孵育30分钟；离心后用PBS洗涤细胞一次，再加入anti-HA-Alexa Fluor488或鼠抗PTK7-FITC抗体(阳性对照)，4℃孵育20分钟；用PBS洗涤一次细胞后，4℃离心5分钟，弃上清，加入200ul PBS重悬细胞，流式检测每个样本Alexa Fluor488信号。
结果如图3所示，本发明的抗PTK7单域抗体能够有效结合肿瘤细胞表面的PTK7蛋白。
实施例8：抗PTK7单域抗体在小鼠肿瘤异种移植模型的SPECT/CT显像
在三羰基试剂盒，加入高锝酸盐，99℃孵育20分钟；冷却试剂瓶至室温，加入盐酸，中和三羰基锝至pH7-7.5；加入抗PTK7单域抗体，37℃孵育1小时；用薄层色谱法对锝标记抗PTK7单域抗体进行放射纯度鉴定。
在祼鼠右侧背部皮下接种1×10 7个PTK7高表达(HCT116)细胞，待肿瘤长至150-200mm 3用于正式实验研究；通过异氟烷麻醉荷瘤鼠，尾静脉注射锝标记抗PTK7单域抗体(～10ug,37MBq)；给药后90分钟进行扫描，采集方式为静态15分钟SPECT、中分辨率全身CT。
本发明的抗PTK7单域抗体能够于PTK7高表达肿瘤模型有效积聚，应用于PTK7靶向的癌症诊断及疗效评估，同时开发新一代PTK7靶向治疗。
本发明的氨基酸序列
Figure PCTCN2022113106-appb-000006
Figure PCTCN2022113106-appb-000007
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种抗PTK7单域抗体VHH链的互补决定区CDR，其特征在于，所述VHH链的互补决定区CDR包含：
    SEQ ID NO:9-11任一项所示的CDR1；
    SEQ ID NO:12-14任一项所示的CDR2；和
    SEQ ID NO:15-18任一项所示的CDR3。
  2. 如权利要求1所述的互补决定区CDR，其特征在于，包含选自下组的CDR1、CDR2和CDR3：
    组 CDR1序列编号 CDR2序列编号 CDR3序列编号 1 9 12 15 2 10 13 16 3 10 13 17 4 11 14 18。.
  3. 一种抗PTK7单域抗体的VHH链，所述VHH链包括框架区FR和权利要求1所述的互补决定区CDR。
  4. 一种抗PTK7单域抗体，其特征在于，所述单域抗体是针对PTK7蛋白的单域抗体，并且具有如SEQ ID NO:1-4任一所示的氨基酸序列的VHH链。
  5. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白质：权利要求1所述的CDR区、权利要求2所述的抗PTK7单域抗体的VHH链、或权利要求3所述的抗PTK7单域抗体。
  6. 如权利要求5所述的多核苷酸，其特征在于，所述多核苷酸具有如SEQ ID NO.:5-8任一所示的核苷酸序列。
  7. 一种表达载体，其特征在于，所述表达载体含有权利要求5所述的多核苷酸。
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的表达载体，或其基因组中整合有权利要求5所述的多核苷酸。
  9. 一种产生抗PTK7单域抗体的方法，其特征在于，包括步骤：
    (a)在适合产生单域抗体的条件下，培养权利要求8所述的宿主细胞，从而获得含所述抗PTK7单域抗体的培养物；以及(b)从所述培养物中分离或回收所述的抗PTK7单域抗体。
  10. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求3所述的抗PTK7单域抗体的VHH链、或如权利要求3所述的抗PTK7单域抗体；和(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  11. 如权利要求3所述的VHH链，如权利要求4所述的抗PTK7单域抗体，或如权利要求10所述的免疫偶联物的用途，其特征在于，用于制备(a)用于检测PTK7分子的试剂；(b)用于治疗肿瘤的药物。
  12. 如权利要求11所述的用途，其特征在于，所述肿瘤为PTK7高表达的肿瘤。
  13. 一种药物组合物，其特征在于，所述药物组合物包含：(i)如权利要求4所述的单域抗体或如权利要求10所述的免疫偶联物；和(ii)药学上可接受的载体。
  14. 一种体外(诊断和非诊断性)检测样品中PTK7蛋白的方法，包括步骤：
    (1)将样品与权利要求4所述的单域抗体接触；
    (2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在PTK7蛋白。
  15. 一种治疗肿瘤的方法，包括向需要的对象施用如权利要求4所述的单域抗体，如权利要求10所述的免疫偶联物，或如权利要求13所述的药物组合物。
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