WO2023035825A1 - Anticorps anti-ca724 ou fragment de liaison à l'antigène correspondant, et son procédé de préparation et son utilisation - Google Patents

Anticorps anti-ca724 ou fragment de liaison à l'antigène correspondant, et son procédé de préparation et son utilisation Download PDF

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WO2023035825A1
WO2023035825A1 PCT/CN2022/110212 CN2022110212W WO2023035825A1 WO 2023035825 A1 WO2023035825 A1 WO 2023035825A1 CN 2022110212 W CN2022110212 W CN 2022110212W WO 2023035825 A1 WO2023035825 A1 WO 2023035825A1
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antibody
seq
antigen
binding fragment
amino acid
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PCT/CN2022/110212
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廉倩倩
徐海伟
常立峻
简·马克·玖塞夫·杜加
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苏州长光华医生物医学工程有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • 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
    • C07K2317/567—Framework region [FR]
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the application belongs to the technical field of immunization, and in particular relates to an anti-CA724 antibody or an antigen-binding fragment thereof and a preparation method and application thereof.
  • CA724 is a tumor marker commonly used clinically in recent years. It is mainly used for early screening of gastrointestinal tumors and is a non-specific tumor marker. Its essence is a high-molecular glycoprotein of cell surface mucin, widely distributed in the cytoplasm of epithelial cells and malignant tumors, with a relative molecular mass of (220-400) ⁇ 10 3 , and can be cloned by cc49 and B72.3 Antibody recognition.
  • CA724 is an important molecular structure that constitutes the skeleton of tumor cells. The molecular structure contains phosphorylated amino acid sequences. With the abnormal proliferation of tumor cells, it can be released into the blood circulation through exocrine. In normal human serum, CA724 ⁇ 6U/mL, When the tissue becomes cancerous, it can quickly enter the blood, and the serum detection level rises sharply. Therefore, CA724 belongs to the tumor marker of carcinoembryonic antigen nature.
  • the detection method of CA724 provided in the research is mainly electrochemiluminescence method, and the detection instruments are mostly derived from Roche Elecsys 1010, 2010, 170 and 601 immunoassay analyzers.
  • the detection instruments are mostly derived from Roche Elecsys 1010, 2010, 170 and 601 immunoassay analyzers.
  • two monoclonal antibodies, cc49 and B72.3, are mostly used to detect CA724.
  • the latter is a specific antibody to CA724.
  • the level of CA724 is detected by using the principle of antigen-antibody sandwich method.
  • CA724 is an indicator with the highest correlation with gastric cancer.
  • the serum level of CA724 in patients with gastric cancer is much higher than that in patients with benign gastric lesions and healthy people, and it has a great diagnostic reference value for gastric cancer.
  • the specificity of CA724 in the diagnosis of gastric cancer is better than other indicators, such as CA199 and CEA.
  • the level of CA724 in the serum of patients with ovarian cancer and non-small cell lung cancer is also significantly increased, and it has a great reference value for the graded diagnosis of liver cancer and colorectal cancer. Therefore, the detection of CA724 has greater clinical significance.
  • the domestic monoclonal antibodies used to detect CA724 are basically purchased from foreign countries, and there are defects in sensitivity and specificity. Therefore, it is necessary to provide more CA724 monoclonal antibodies.
  • the present invention provides an anti-CA724 antibody or an antigen-binding fragment thereof, characterized in that: the antibody or its The antigen-binding fragment has at least one heavy chain CDR domain selected from SEQ ID NO: 1, 2, 3 and/or at least one light chain CDR domain selected from SEQ ID NO: 4, 5, 6.
  • the anti-CA724 antibody or antigen-binding fragment thereof of the present invention is characterized in that the heavy chain variable region of the antibody includes the following three complementarity determining region CDRs: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; the light chain variable region of the antibody includes the following three complementarity determining region CDRs: CDR1' shown in SEQ ID NO:4, CDR2' set forth in SEQ ID NO:5, and CDR3' set forth in SEQ ID NO:6.
  • the anti-CA724 antibody or antigen-binding fragment thereof of the present invention is characterized in that the heavy chain of the antibody comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has such as SEQ ID NO: The amino acid sequence shown in 7, 8, 9 or 10 or having at least 80%, 85%, 90%, 95%, 98% or 99% sequence homology to SEQ ID NO: 7, 8, 9 or 10 Sequence; the light chain of the antibody comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown in SEQ ID NO: 11, 12, 13 or 14 or the same as SEQ ID NO: 11, 12, 13 or 14 have sequences with at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity.
  • the anti-CA724 antibody or antigen-binding fragment thereof of the present invention is characterized in that, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15; the light chain of the antibody The variable region has the amino acid sequence shown in SEQ ID NO: 16.
  • the binding protein further comprises an antibody constant region sequence; preferably, the constant region sequence is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, the constant region sequence; preferably, the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, Goose, turkey, fighting cock or human; preferably, the constant region is derived from mouse.
  • the anti-CA724 antibody or antigen-binding fragment thereof of the present invention is characterized in that the heavy chain amino acid sequence of the antibody is SEQ ID NO: 19.
  • the anti-CA724 antibody or antigen-binding fragment thereof of the present invention is characterized in that the light chain amino acid sequence of the antibody is SEQ ID NO:20.
  • Another aspect of the present invention provides an isolated nucleic acid encoding the anti-CA724 antibody or antigen-binding fragment thereof according to any one of the foregoing.
  • Another aspect of the invention provides an isolated nucleic acid encoding the antibody heavy chain variable region of any one of the preceding items.
  • Another aspect of the invention provides an isolated nucleic acid encoding the antibody light chain variable region of any one of the foregoing.
  • Another aspect of the present invention provides a kind of isolated nucleic acid, described nucleic acid has the nucleotide sequence shown in SEQ ID NO:17.
  • Another aspect of the present invention provides an isolated nucleic acid, said nucleic acid having a nucleotide sequence shown in SEQ ID NO:18.
  • Another aspect of the present invention provides a vector comprising the nucleic acid described in the present invention, preferably the vector is an expression vector.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • Another aspect of the present invention provides a host cell comprising the vector or nucleic acid of the present invention, preferably, the host cell is prokaryotic or eukaryotic, more preferably yeast cells or mammalian cells.
  • Another aspect of the present invention provides a method for preparing the anti-CA724 antibody or an antigen-binding fragment thereof described in any one of the foregoing, the method comprising being suitable for expressing a nucleic acid encoding the anti-CA724 antibody or an antigen-binding fragment thereof culturing the host cell described in any one of the above conditions, optionally isolating the antibody or an antigen-binding fragment thereof, optionally the method further comprises recovering the anti-724 antibody or an antigen-binding fragment thereof from the host cell .
  • Another aspect of the present invention provides a chemically or biologically labeled antibody or antigen-binding fragment thereof of any one of the foregoing.
  • Another aspect of the present invention provides a conjugate obtained by coupling any one of the aforementioned antibodies or antigen-binding fragments thereof to a solid medium or a semi-solid medium.
  • Another aspect of the present invention provides any of the aforementioned antibodies or antigen-binding fragments thereof, any of the aforementioned antibodies or antigen-binding fragments thereof that have been chemically or biologically labeled, and any of the aforementioned conjugates in preparation for diagnosis Application in a diagnostic agent for cancer or a product for detecting CA724.
  • kits for detecting CA724 protein which is characterized by comprising the anti-CA724 antibody or antigen-binding fragment thereof or the conjugate described in any one of the foregoing.
  • the present invention provides a novel anti-CA724 antibody, which can well recognize CA724 and has high affinity and specificity.
  • the antibody has good stability and batch-to-batch variation, can be used to prepare diagnostic agents for diagnosing cancer or products for detecting CA724, and provides a new option for CA724 detection.
  • Fig. 1 is an electropherogram of an anti-CA724 antibody in an embodiment of the present invention.
  • antibody includes polyclonal antibodies and monoclonal antibodies, as well as antigenic compound-binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, and antibody minimal recognition units, as well as these antibody and single-chain derivatives of fragments.
  • the type of antibody can choose IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
  • antibody includes naturally occurring antibodies as well as non-naturally occurring antibodies including, for example, chimeric, bifunctional, and humanized antibodies, and related synthetic isomeric forms (isoforms).
  • antibody is used interchangeably with "immunoglobulin”.
  • VH variable domain of the heavy chain of an antibody
  • VL variable domain of a light chain
  • CDRs complementarity determining regions
  • the framework regions and the extent of the CDRs have been precisely defined, for example in Kabat (see Sequences of Proteins of Immunological Interest), E. Kabat et al., U.S. Department of Health and Human Services Services), (1983)) and Chothia.
  • the framework regions of an antibody, the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs that are primarily responsible for binding to the antigen.
  • frame or "FR” regions mean the regions of an antibody variable domain excluding those regions defined as CDRs.
  • Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3 and FR4) separated by CDRs.
  • variable region VL/VH of the heavy chain and light chain can be obtained by connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the terms “purified” or “isolated” in relation to a polypeptide or nucleic acid mean that the polypeptide or nucleic acid is not in its native medium or in its native form.
  • the term “isolated” includes a polypeptide or nucleic acid that has been removed from its original environment, eg, its natural environment if it occurs in nature.
  • an isolated polypeptide typically is free of at least some proteins or other cellular components with which it is normally associated or is normally mixed or in solution.
  • Isolated polypeptides include such polypeptides as they are naturally produced contained in cell lysates, in purified or partially purified form, recombinant polypeptides, expressed or secreted by cells, and in heterologous host cells or cultures. of the polypeptide.
  • the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (eg, in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
  • One aspect of the present invention provides an anti-CA724 antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof has at least one selected from SEQ ID NO: 1, 2, 3 or a combination of SEQ ID NO: 1, 2, 3
  • the heavy chain variable region of the antibody comprises the following three complementarity determining region CDRs: CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and SEQ ID NO:3 CDR3 shown; the light chain variable region of the antibody includes the following three complementarity determining region CDRs: CDR1' shown in SEQ ID NO:4, CDR2' shown in SEQ ID NO:5, and SEQ ID NO: CDR3' shown in 6.
  • the heavy chain of the antibody comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown in SEQ ID NO: 7, 8, 9 or 10 or the same as SEQ ID NO: 7, 8, 9 or 10 sequences having at least 80%, 85%, 90%, 95%, 98% or 99% sequence homology;
  • the light chain of the antibody comprises 4 FR regions, at least one of which is FR
  • the amino acid sequence of the region has an amino acid sequence as shown in SEQ ID NO: 11, 12, 13 or 14 or has at least 80%, 85%, 90%, 95%, 98% of SEQ ID NO: 11, 12, 13 or 14 Sequences with % or 99% sequence identity, wherein the sequences of SEQ ID NO: 7-14 are as follows:
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 16,
  • the SEQ ID NO: 15, 16 sequences are as follows:
  • the antibodies of the present invention also include 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): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) 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.
  • Another aspect of the present invention provides an isolated nucleic acid encoding the anti-CA724 antibody or antigen-binding fragment thereof of the present invention.
  • Another aspect of the present invention provides an isolated nucleic acid encoding the antibody heavy chain variable region of the present invention.
  • Another aspect of the present invention provides an isolated nucleic acid encoding the antibody light chain variable region of the present invention.
  • Another aspect of the present invention provides a kind of isolated nucleic acid, described nucleic acid has the nucleotide sequence shown in SEQ ID NO:17.
  • the sequence of SEQ ID NO: 17 is shown below:
  • Another aspect of the present invention provides an isolated nucleic acid, said nucleic acid having a nucleotide sequence shown in SEQ ID NO:18.
  • the sequence of SEQ ID NO: 18 is shown below:
  • Another aspect of the present invention provides a host cell comprising the vector or nucleic acid described in any one of the foregoing, preferably, the host cell is prokaryotic or eukaryotic, more preferably yeast cells, mammalian cells.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains in which glycosylation pathways have been "humanized” result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted for growth in suspension can be used.
  • useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293HEK or 293 cells, as e.g. described in ), etc.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • Another aspect of the present invention provides a method for preparing the anti-CA724 antibody or an antigen-binding fragment thereof described in any one of the foregoing, the method comprising being suitable for expressing a nucleic acid encoding the anti-CA724 antibody or an antigen-binding fragment thereof culturing the host cell described in any one of the above conditions, optionally isolating the antibody or an antigen-binding fragment thereof, optionally the method further comprises recovering the anti-724 antibody or an antigen-binding fragment thereof from the host cell .
  • Another aspect of the present invention provides a chemically or biologically labeled antibody or antigen-binding fragment thereof of any one of the foregoing.
  • 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 products capable of producing detectable enzymes.
  • Therapeutic agents that can be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2.
  • Gold nanoparticles /Nanorods (Lapotko et al., 2005, Cancer letters (Cancer letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society (Journal of the American Chemical Society) 128, 2115); 5. Virus particles (Peng et al., 2004 , Gene therapy (Gene therapy) 11, 1234); 6. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 7. Nanomagnetic particles; 8. Prodrug activating enzymes (for example, DT - diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
  • DTD DT - diaphorase
  • BPHL biphenylhydrolase-like protein
  • Another aspect of the present invention provides a conjugate obtained by coupling any one of the aforementioned antibodies or antigen-binding fragments thereof to a solid medium or a semi-solid medium.
  • Another aspect of the present invention provides any of the aforementioned antibodies or antigen-binding fragments thereof, any of the aforementioned antibodies or antigen-binding fragments thereof that have been chemically or biologically labeled, and any of the aforementioned conjugates in preparation for diagnosis Application in a diagnostic agent for cancer or a product for detecting CA724.
  • kits which is characterized by comprising the anti-CA724 antibody or antigen-binding fragment thereof or the conjugate described in any one of the foregoing.
  • the kit further includes a container, an instruction manual, a buffer and the like.
  • the present invention further designs a detection kit for detecting the CA724 protein, which includes an antibody that recognizes the CA724 protein, general reagents and buffers required for detection, such as various buffers, enzyme-linked secondary antibodies, detection labels, detection substrate etc.
  • the test kit may be an in vitro diagnostic device.
  • pcDNA3.4 vector Expi293TM expression system including Expi293 cells, transfection reagents, culture medium, etc. were purchased from ThermoFisher; protein A affinity column was purchased from GE, and other reagents were of analytical grade.
  • mice were immunized by subcutaneous injection with CA724 antigen mixed with an equal amount of immune adjuvant. After the initial immunization, mice were boosted every two weeks for a total of 6 immunizations. Three days after the last immunization, mouse splenic lymphocytes were fused with mouse myeloma SP2/0 using the polyethylene glycol method, cultured in HAT selection medium, and anti-CA724 positive clones were screened out. The screened anti-CA724 positive hybridoma cells were expanded and cultured, the anti-CA724 monoclonal antibody was isolated and purified, and the amino acid sequence of the antibody was carried out by LCMS/MS technology to obtain the antibody variable region sequence and deduce the gene sequence.
  • the heavy chain variable region gene and the light chain variable region gene of the anti-CA724 antibody were respectively cloned into the pcDNA3.4 vector by standard gene cloning techniques to construct heavy chain and light chain expression vectors.
  • the heavy chain plasmid contains the heavy chain variable region and mouse IgG2 ⁇ Fc region; the light chain plasmid contains the light chain variable region and IgG ⁇ Fc region.
  • the corresponding antibody heavy chain amino acid sequence is SEQ ID NO: 19
  • the corresponding antibody light chain amino acid sequence is: SEQ ID NO:20
  • the heavy chain vector and the light chain vector were co-transfected into Expi293F cells for expression. Cultivate and expand cells with Expi293 medium.
  • Expi293F cells Cultivate and expand cells with Expi293 medium.
  • inoculate Expi293F cells at a density of 2.5 ⁇ 10 6 mL in a 125 mL shaker flask, culture medium volume 30 mL, 125 ⁇ 5 rpm, 37°C, 8% CO 2 culture overnight.
  • the cells can be transfected when the cells reach 4.5–5.5 ⁇ 10 6 viable cells/mL, and the cell viability reaches 95–99%. Adjust the cell concentration to 23 ⁇ 10 6 mL with fresh medium for use.
  • the culture medium was collected, centrifuged at 3000 g for 20 min, and the supernatant was filtered at 0.45 ⁇ m, passed through a protein A affinity column for antibody separation and purification. Slowly flow the filtered culture supernatant through the protein A column, wash the column with 20mM, pH7.0 sodium phosphate buffer to wash away the components that do not specifically bind to protein A, and wash the column with 0.1M, pH2.
  • the adsorbed antibody was eluted with 7 glycine-hydrochloric acid buffer solution, and the antibody component was adjusted to neutrality with pH 9.0, 1M Tris-HCl buffer solution.
  • the collected antibodies were analyzed by 8% SDS-PAGE, and the results are shown in FIG. 1 .
  • the anti-CA724 recombinant antibody labeled with biotin or acridinium ester prepared in Example 2 was paired with the anti-CA724 monoclonal antibody (labeled with acridinium ester or biotin) to detect different concentrations of CA724 (S0-S5).
  • the AMQ biosensor is selected to capture the recombinant antibody or monoclonal antibody prepared in Example 1, and then bind or dissociate with the antigen respectively.
  • the kinetic analysis method is as follows:
  • Baseline 1 60s Loading 100-180s Baseline 2 120s Association 300s Dissociation 200s Regeneration 30s
  • the experimental results are shown in the following table.
  • the KD value of the recombinant antibody prepared in Example 1 is close to that of the monoclonal antibody, and the affinity of the two antibodies is similar.

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Abstract

La présente demande concerne un nouvel anticorps anti-CA724, qui est capable d'identifier un puits CA724 et a une affinité et une spécificité élevées. L'anticorps a une bonne stabilité, peut être utilisé pour préparer un agent de diagnostic pour diagnostiquer un cancer ou un produit pour détecter CA724, et fournit un nouveau choix pour la détection de CA724.
PCT/CN2022/110212 2021-09-13 2022-08-22 Anticorps anti-ca724 ou fragment de liaison à l'antigène correspondant, et son procédé de préparation et son utilisation WO2023035825A1 (fr)

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CN202111069682.8A CN113880948B (zh) 2021-09-13 2021-09-13 抗ca724抗体或其抗原结合片段及其制备方法和应用
CN202111069682.8 2021-09-13

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