WO2022121415A1 - Anticorps anti-ca19-9, son application et kit de détection de ca19-9 - Google Patents

Anticorps anti-ca19-9, son application et kit de détection de ca19-9 Download PDF

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WO2022121415A1
WO2022121415A1 PCT/CN2021/117804 CN2021117804W WO2022121415A1 WO 2022121415 A1 WO2022121415 A1 WO 2022121415A1 CN 2021117804 W CN2021117804 W CN 2021117804W WO 2022121415 A1 WO2022121415 A1 WO 2022121415A1
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cdr
antibody
functional fragment
cancer
derivatives
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崔鹏
何志强
孟媛
钟冬梅
娄文娟
覃婷
范凌云
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东莞市朋志生物科技有限公司
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    • 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
    • A—HUMAN NECESSITIES
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    • 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
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
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    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • 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
    • G01N33/57492—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 involving compounds localized on the membrane of tumor or cancer cells
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    • 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
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    • 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; monoclonal antibodies per se are classified with their corresponding antigens
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • 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]
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    • 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]
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to the technical field of antibodies, and in particular, to an anti-CA19-9 antibody, its application and a kit for detecting CA19-9.
  • Tumor markers are chemical substances that reflect the presence of tumors. They do not exist in normal adult tissues but only in embryonic tissues, or the content in tumor tissues greatly exceeds that in normal tissues. Their presence or quantitative changes can indicate the nature of tumors, so as to understand tumor histogenesis, cell differentiation and Cell function to help tumor diagnosis, classification, prognosis and treatment guidance.
  • CA glycoprotein antigens
  • CA glycoprotein antigens
  • Commonly used CA series are: CA 125 (ovarian cancer-related antigen); CA 19-9 (pancreatic, intestinal cancer-related antigen); CA 15-3 (breast cancer-related antigen).
  • Limdholm et al. immunized mice with human colorectal cancer cell COLD205 to obtain a series of antibodies and subsequently found a series of antigens corresponding to these antibodies, including CA19-9, CA50, CA242 and so on. This series of antigens appear on the surface of the same mucin, and the antigenic determinants are all sugar chain structures, but have different tumor specificities, so they can be used as different tumor markers.
  • the tumor marker carbohydrate antigen 199 is a mucin-type carbohydrate protein, which is a glycolipid on the cell membrane with a molecular weight greater than 1000KD. It is named because it is recognized by the mouse monoclonal antibody 116NS19-9. The most sensitive tumor marker for pancreatic cancer reported to date.
  • the increase of CA19-9 indicates the possibility of pancreatitis, liver cirrhosis, diabetes, and gastrointestinal tumors. It exists in the form of salivary mucin in serum and is distributed in normal fetal pancreas, gallbladder, liver, intestine and normal adult pancreas, bile duct epithelium, etc. It is a gastrointestinal tumor-related antigen present in the blood circulation.
  • CA19-9 can be expressed in normal pancreas, bile duct cells, stomach, colon and salivary gland epithelial cells, and the normal serum value is less than 37U/ml.
  • the epitope structure of CA19-9 is very similar to that of CA50, with only one more fucose structure (a structure unique to Lewsi-positive people), which can be distributed in the pancreas and bile duct epithelium of normal people.
  • fucose structure a structure unique to Lewsi-positive people
  • the epitope structure of CA19-9 is very similar to that of CA50, with only one more fucose structure (a structure unique to Lewsi-positive people), which can be distributed in the pancreas and bile duct epithelium of normal people.
  • fucose structure a structure unique to Lewsi-positive people
  • Carbohydrate antigens CA19-9 and carbohydrate antigens CA242 are relatively sensitive tumor markers in digestive system, and have the best sensitivity to pancreatic cancer. At present, it is believed that CA19-9 has important clinical value in the diagnosis of pancreatic cancer, the evaluation of the efficacy of radiotherapy and chemotherapy, the evaluation of prognosis, and the judgment of overall survival. In addition, CA19-9 also has high diagnostic value for cholangiocarcinoma, colorectal cancer and other digestive system tumors.
  • the methods for detecting CA19-9 in clinical laboratories are mainly radioimmunoassay (RIA), immunoturbidimetric assay, enzyme-linked immunosorbent assay (ELISA), time-resolved fluorescence immunoassay and chemiluminescence technology, among which RIA must use radioactive elements Labeling, the detection equipment is complex and expensive, the half-life of its radioactive elements is short, it cannot be stored for a long time, the detection results are unstable, and the existence of radioactive contamination also brings harm to the experimental operators; the enzyme immunoassay has low sensitivity, many influencing factors, easy to cause false negatives and false positives.
  • chemiluminescence technology emerged in the 1980s and is an emerging technology developed after enzyme-linked immunosorbent technology and radioimmunoassay technology. Due to its high sensitivity, high specificity, simple and rapid method, and stable labeling conjugates, relatively Time-resolved fluorescence immunoassay analysis has the characteristics of low cost, simple operation, no radioisotope damage and pollution, and has been developed rapidly.
  • the domestic monoclonal antibodies used to detect CA19-9 are basically purchased from foreign countries, and there are defects in activity and clinical specificity.
  • the present disclosure provides an anti-CA19-9 antibody or a functional fragment thereof, characterized in that the antibody or functional fragment thereof has the following complementarity determining regions:
  • CDR-VH1 G-F-X1-F-S-X2-A-W-M-X3; where: X1 is T or S; X2 is N or D; X3 is D or E;
  • CDR-VH2 E-X1-G-N-K-X2-N-N-H-A-T-Y-Y-A-X3-S-X4-K-G; wherein: X1 is I or L; X2 is A or G; X3 is E or D; X4 is L, V or I;
  • CDR-VH3 X1-T-X2-F-A-Y; wherein: X1 is S or T; X2 is N, Q or R;
  • CDR-VL1 K-A-S-Q-D-X1-N-X2-Y-X3-S; wherein: X1 is I, V or L; X2 is S or T; X3 is I, V or L;
  • CDR-VL2 R-X1-N-R-X2-X3-D; wherein: X1 is G or A; X2 is L, V or I; X3 is L, V or I;
  • CDR-VL3 X1-Q-Y-D-E-X2-P-R; wherein: X1 is L, V or I; X2 is Y or F.
  • X2 is D
  • X1 is I
  • X1 is T
  • X2 is S
  • X1 is A
  • X2 is F.
  • X1 is T.
  • X1 is S.
  • X3 is D.
  • X3 is E.
  • X2 is A in the CDR-VH2.
  • X2 is G.
  • X3 is E.
  • X3 is D in the CDR-VH2.
  • X4 is L.
  • X4 is V.
  • X4 is 1.
  • X2 is N.
  • X2 is Q.
  • X2 is R.
  • X1 is 1.
  • X1 is V.
  • X1 is L.
  • X3 is 1.
  • X3 is V.
  • X3 is L.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X3 is L.
  • X3 is V.
  • X3 is T.
  • X1 is L.
  • X1 is V.
  • X1 is 1.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-65:
  • X2 in CDR-VH1, X2 is N; in CDR-VH2, X1 is L;
  • X1 is S
  • X2 is T
  • X1 is G
  • X2 is Y.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 66-72:
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L, and FR4-L whose sequence is shown in SEQ ID NO: 1-4, and/or, whose sequence is shown in sequence as Heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NOs: 5-8.
  • the antibody further comprises a constant region.
  • the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • the species source of the constant region is sheep, goat, bovine, horse, dairy cow, pig, rat, dog, cat, rabbit, camel, donkey, mouse, deer, mink, duck, goose , turkey, cockfight, chicken or man.
  • the constant region is derived from a mouse.
  • the light chain constant region sequence of the constant region is set forth in SEQ ID NO:9
  • the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO:10.
  • the functional fragment is selected from any one of F(ab')2, Fab, scFv, Fab' and Fv of the antibody.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3- having at least 80% homology with sequences SEQ ID NO: 1, 2, 3, 4, respectively, in order L and FR4-L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H, and FR4-H.
  • the present disclosure provides the use of the anti-CA19-9 antibody or its functional fragment in the preparation of a reagent or kit for tumor diagnosis or auxiliary diagnosis, characterized in that the tumor marker includes CA19-9.
  • the tumor is selected from pancreatic cancer, bile duct cancer, colon cancer, and rectal cancer.
  • the present disclosure provides a reagent or kit for the diagnosis or auxiliary diagnosis of a related tumor with CA19-9 as a marker, characterized in that it comprises the antibody or its function according to any one of claims 1-6 Sexual Fragments.
  • the present disclosure provides a reagent or kit for detecting CA19-9, characterized in that it comprises the anti-CA19-9 antibody or a functional fragment thereof according to any one of claims 1-6.
  • the antibody or functional fragment thereof is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
  • the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, and protein dyes and its derivatives.
  • the enzyme that catalyzes coloration of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6- Phosphoglucose deoxygenase.
  • the radioisotope is selected from212Bi , 131I , 111In , 90Y , 186Re , 211At , 125I , 188Re , 153Sm , 213Bi , 32P , 94mTc ,99mTc , 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu, and 18 F.
  • the chemiluminescent reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, Oxetane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives.
  • the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metal is selected from colloidal gold, colloidal silver, and colloidal selenium.
  • the present disclosure provides a nucleic acid molecule encoding the anti-CA19-9 antibody or a functional fragment thereof according to any one of the above.
  • the present disclosure provides a vector containing a nucleic acid fragment encoding the anti-CA19-9 antibody or a functional fragment thereof according to any one of the above.
  • the present disclosure provides a recombinant cell containing the vector.
  • the present disclosure provides the use of any one of the anti-CA19-9 antibodies or functional fragments thereof described above, and the use of any one of the above-described reagents or kits in detecting CA19-9.
  • the present disclosure provides the use of any of the above-mentioned anti-CA19-9 antibodies or functional fragments thereof, and any of the above-mentioned reagents or kits for detecting CA19-9.
  • the present disclosure provides a method for detecting CA19-9, comprising:
  • the present disclosure provides the use of the anti-CA19-9 antibody or its functional fragment, the reagent or the kit for the diagnosis or auxiliary diagnosis of tumors, the markers of which include CA19-9.
  • the tumor is selected from pancreatic cancer, colorectal cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, gallbladder cancer, and gastrointestinal cancer.
  • the present disclosure provides a method of diagnosing a CA19-9-related disease in a subject, comprising:
  • the disease associated with CA19-9 includes pancreatic cancer, colorectal cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, gallbladder cancer, and gastrointestinal cancer.
  • the present disclosure provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing a recombinant cell capable of recombinantly expressing the anti-CA19-9 antibody or functional fragment thereof described in any one of the above, and separating and purifying the product from the culture The antibody or functional fragment thereof is obtained.
  • FIG. 1 shows the results of reducing SDS-PAGE of the anti-CA19-9 antibody of Example 1.
  • the term "complementarity determining regions” generally refers to: an intact or complete antibody comprises two heavy chains and two light chains; each heavy chain comprises a variable region (VH) and a constant region (CH); Each light chain contains a variable region (VL) and a constant region (CL); the antibody has a "Y" shape, and the stem of the Y consists of the second and the third of the two heavy chains held together by disulfide bonds Composed of three constant regions; each arm of Y includes a variable region and a first constant region of a single heavy chain combined with the variable and constant regions of a single light chain; the variable and heavy regions of the light chain The variable regions of the chains are responsible for antigen binding; the variable regions in both chains typically contain three hypervariable regions, called complementarity determining regions.
  • the term "functional fragment” is intended to refer to a portion of an antibody comprising a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • These functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single chain Fv (scFv), diabodies, triple chain Antibodies (triabodies), tetrabodies (tetrabodies) and minibodies (minibodies).
  • Other functional fragments may include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof, so long as these functional fragments retain binding activity.
  • constant region refers to a relatively stable region of the light and heavy chains of an antibody molecule near the C-terminal amino acid sequence.
  • variable region refers to the region of the light and heavy chains of an antibody molecule that varies greatly in amino acid sequence near the N-terminus.
  • naked anti-stable refers to an antibody or functional fragment thereof that is not labeled, eg, an antibody or functional fragment thereof that is not labeled with a detectable label.
  • Some embodiments of the present disclosure provide anti-CA19-9 antibodies, uses and detection kits thereof.
  • the antibody has good detection specificity and binding activity to CA19-9, can be used to detect sample CA19-9, and has good sensitivity and specificity.
  • the present disclosure is the detection of CA19-9 and the use of CA19-9 as a marker
  • the diagnosis or auxiliary diagnosis of tumor-related tumors provides a richer selection of antibodies.
  • One embodiment of the present disclosure provides an anti-CA19-9 antibody or a functional fragment thereof, the antibody or functional fragment thereof has the following complementarity determining regions:
  • CDR-VH1 G-F-X1-F-S-X2-A-W-M-X3; where: X1 is T or S; X2 is N or D; X3 is D or E;
  • CDR-VH2 E-X1-G-N-K-X2-N-N-H-A-T-Y-Y-A-X3-S-X4-K-G; wherein: X1 is I or L; X2 is A or G; X3 is E or D; X4 is L, V or I;
  • CDR-VH3 X1-T-X2-F-A-Y; wherein: X1 is S or T; X2 is N, Q or R;
  • CDR-VL1 K-A-S-Q-D-X1-N-X2-Y-X3-S; wherein: X1 is I, V or L; X2 is S or T; X3 is I, V or L;
  • CDR-VL2 R-X1-N-R-X2-X3-D; wherein: X1 is G or A; X2 is L, V or I; X3 is L, V or I;
  • CDR-VL3 X1-Q-Y-D-E-X2-P-R; wherein: X1 is L, V or I; X2 is Y or F.
  • the anti-CA19-9 antibody provided by the present disclosure has the above-mentioned complementarity determining region structure, can specifically bind to CA19-9, has high activity on CA19-9, is used for detecting CA19-9, has good sensitivity and specificity.
  • the antibody can be used to diagnose or assist in the diagnosis of related tumors marked by CA19-9, such as pancreatic cancer, bile duct cancer, colon cancer and rectal cancer.
  • the present disclosure provides more abundant antibody selections for the detection of CA19-9 and the diagnosis or auxiliary diagnosis of related tumors with CA19-9 as a marker.
  • X2 in CDR-VH1, X2 is N; in CDR-VH2, X1 is L; in CDR-VH3, X1 is S; in CDR-VL1, X2 is T; in CDR-VL2, X1 is G; in CDR-VL3, X2 is Y.
  • X1 is T.
  • X1 is S.
  • X3 is D in CDR-VH1.
  • X3 is E.
  • X2 is A in CDR-VH2.
  • X2 is G.
  • X3 is E.
  • X3 is D.
  • X4 is L.
  • X4 is V.
  • X4 is 1.
  • X2 is N in CDR-VH3.
  • X2 is Q.
  • X2 is R.
  • X1 is I.
  • X1 is V.
  • X1 is L.
  • X3 is 1.
  • X3 is V.
  • X3 is L.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X3 is L.
  • X3 is V.
  • X3 is T.
  • X1 is L.
  • X1 is V.
  • X1 is 1.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-65:
  • X2 in CDR-VH1, X2 is N; in CDR-VH2, X1 is L; in CDR-VH3, X1 is S; in CDR-VL1, X2 is T; in CDR-VL2, X1 is G; in CDR-VL3, X2 is Y.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 66-72:
  • the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, and/or the sequences of the light chain framework regions shown in SEQ ID NOs: 1-4 in order
  • the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are shown in sequence as SEQ ID NOs: 5-8.
  • variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by linking the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, FR2-L, FR3-L and FR4- L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L, and FR4-L that are at least 80% identical to the sequences of SEQ ID NOs: 1, 2, 3, 4, respectively, in order , and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% identity to the sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
  • each framework region of the antibody or its functional fragment provided by the present disclosure may be the same as the above-mentioned corresponding framework region (SEQ ID NO: 1, 2, 3, 4, 5, 6 , 7 or 8) with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98% or 99% homology.
  • the antibody further comprises a constant region.
  • the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
  • the species source of the constant region is mammalian or poultry.
  • mammals include sheep, goats, cows, horses, dairy cows, pigs, rats, dogs, cats, rabbits, camels, donkeys, mice, deer, mink, or humans.
  • poultry animals include ducks, geese, turkeys, fighting cocks or chickens.
  • the species source of the constant region is sheep, goat, bovine, horse, dairy cow, pig, rat, dog, cat, rabbit, camel, donkey, mouse, deer, mink, duck , goose, turkey, cockfight, chicken or man.
  • the constant region is derived from a mouse.
  • the light chain constant region sequence of the constant region is shown in SEQ ID NO:9
  • the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.
  • the functional fragment is selected from any one of F(ab')2, Fab, scFv, Fab' and Fv of the antibody.
  • Functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived.
  • the functional fragments of the above-mentioned antibodies can be cleaved by methods including but not limited to enzymatic digestion (including but not limited to pepsin or papain) and/or by chemical reduction Sulfur bond method.
  • enzymatic digestion including but not limited to pepsin or papain
  • chemical reduction Sulfur bond method On the basis of the structure of the complete antibody provided in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.
  • Functional fragments of the above-described antibodies can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by, for example, automated peptide synthesizers such as those sold by including, but not limited to, Applied BioSystems and the like.
  • One embodiment of the present disclosure provides the use of the anti-CA19-9 antibody or its functional fragment according to any one of the above in the preparation of a reagent or kit for diagnosis or auxiliary diagnosis of tumors, wherein the tumor markers include CA19-9.
  • the antibody provided by the present disclosure can be used to detect CA19-9, therefore, it can be used to detect the diagnosis or auxiliary diagnosis of related tumors with CA19-9 as a marker.
  • tumors include, but are not limited to, pancreatic cancer, bile duct cancer, colon cancer, and rectal cancer.
  • one of the markers of pancreatic cancer, bile duct cancer, colon cancer and rectal cancer is CA19-9, but based on the common knowledge of those skilled in the art and the specific binding ability of the antibodies of the present disclosure to CA19-9, the present disclosure
  • the antibody can be used to detect any tumor marked by CA19-9. Therefore, the application of the antibodies provided by the present disclosure to other related tumors with CA19-9 as one of the markers also falls within the protection scope of the present disclosure.
  • One embodiment of the present disclosure provides a reagent or kit for the diagnosis or auxiliary diagnosis of related tumors with CA19-9 as a marker, which comprises the anti-CA19-9 antibody or a functional fragment thereof as described in any one of the above.
  • One embodiment of the present disclosure provides a reagent or kit for detecting CA19-9, which includes the anti-CA19-9 antibody or a functional fragment thereof as described in any one of the above.
  • the antibody or functional fragment thereof in the above reagent or kit is labeled with a detectable label.
  • Detectable markers refer to a class of substances with properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which the qualitative or quantitative of the corresponding target can be achieved. detection.
  • the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
  • the fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs), rhodamine dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or the like compounds), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc.
  • fluorescein dyes and derivatives thereof for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs
  • Alexa series dyes and their derivatives such as including But not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
  • protein dyes and their derivatives for example, including but Not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc.
  • the enzymes that catalyze the coloration of the substrate include but are not limited to horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase enzyme and glucose 6-phosphate deoxygenase.
  • the radioisotopes include but are not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.
  • the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives Derivatives, Dioxetane and its Derivatives, Lopine and its Derivatives and Peroxyoxalate and its Derivatives.
  • the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
  • An embodiment of the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.
  • One embodiment of the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.
  • One embodiment of the present disclosure provides a recombinant cell containing the above-mentioned vector.
  • One embodiment of the present disclosure provides the use of the above-mentioned antibody or functional fragment thereof, and the above-mentioned reagent or kit in detecting CA19-9.
  • One embodiment of the present disclosure provides the use of the above-mentioned antibody or its functional fragment, the above-mentioned reagent or kit for detecting CA19-9.
  • An embodiment of the present disclosure provides a method for detecting CA19-9, including:
  • An embodiment of the present disclosure provides the use of the above-mentioned antibody or functional fragment thereof, the above-mentioned reagent or kit in the diagnosis or auxiliary diagnosis of tumor, and the above-mentioned tumor marker includes CA19-9.
  • the tumor is selected from pancreatic cancer, colorectal cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, gallbladder cancer, and gastrointestinal cancer.
  • One embodiment of the present disclosure provides a method for diagnosing a CA19-9-related disease in a subject, comprising:
  • the above CA19-9-related diseases include pancreatic cancer, colorectal cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, gallbladder cancer, and gastrointestinal cancer.
  • One embodiment of the present disclosure provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing a recombinant cell capable of recombinantly expressing the anti-CA19-9 antibody or functional fragment thereof as described in any one of the above, and extracting from the cultured product The antibody or its functional fragment is obtained by separation and purification.
  • restriction endonuclease and rTaq DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the mRNA was extracted from the hybridoma cell line secreting anti-CA19-9 antibody, and the DNA product was obtained by RT-PCR.
  • the product was added to the pMD-18T vector with rTaq DNA polymerase and then inserted into the pMD-18T vector, and transformed into DH5 ⁇ competent In the cells, 4 clones of the heavy chain (Heavy Chain) and the 4 light chain (Light Chain) gene clones were taken respectively after the colony was grown, and sent to a gene sequencing company for sequencing.
  • the gene sequences obtained by the above sequencing were placed in the IMGT antibody database (IMGT antibody database from: http://www.imgt.org) for analysis, and the VNTI11.5 software was used for analysis to determine the amplification of heavy chain and light chain primer pairs.
  • the added genes are all correct.
  • the VL gene sequence is 324bp, belonging to the VkII gene family, and there is a 57bp leader peptide sequence in front of it;
  • the gene fragment amplified by the heavy chain primer pair Among them, the VH gene sequence is 345 bp, belonging to the VH1 gene family, and there is a 57 bp leader peptide sequence in front of it.
  • pcDNATM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector, the expression vector has introduced polyclonal restriction sites such as HindIII, BamHI, EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the above pMD-18T
  • 3.4A expression vector pcDNA3.4A expression vector
  • the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases on both ends, respectively, and the light chain of 0.73KB was amplified by PCR amplification method. Gene fragment and 1.4kb heavy chain gene fragment.
  • the heavy chain and light chain gene fragments were digested with HindIII/EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were connected to the 3.4A expression vector to obtain the heavy chain. Chain and light chain recombinant expression plasmids.
  • the recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid
  • plasmid obtained in step 1-(3) Dilute the plasmid obtained in step 1-(3) to 40 ⁇ g/100 ⁇ L with ultrapure water, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, The samples were counted on the 3rd, 5th, and 7th days, and the samples were collected and tested on the 7th day.
  • the coating solution (the main component NaHCO 3 ) was diluted with goat anti-mouse IgG 1ug/ml for microplate coating, 100 ⁇ L per well, overnight at 4°C; the next day, the washing solution (the main component Na 2 HPO 4 +NaCl) was washed twice , pat dry; add blocking solution (20%BSA+80%PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; add diluted CHO cell supernatant, 100 ⁇ L/well, 37°C, 60min; shake off the plate Inner liquid, pat dry; add 20% mouse negative blood to block, 120 ⁇ L per well, 37 °C, 1h; shake off the liquid in the plate, pat dry, add diluted CA199 human ascites, 100 uL per well, 37 °C, 40min; washing solution Wash 5 times and pat dry; add HRP (horseradish peroxidase)-labeled CA199 monoclonal antibody, 100uL per well, 37°C, 30min; add
  • step 2-(2) Dilute the plasmid obtained in step 2-(2) with ultrapure water to 40 ⁇ g/100 ⁇ l, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, Counting the next day; 25 ⁇ mol/L MSX 96-well pressurized culture for about 25 days.
  • the cells obtained in step 2-(3) were recovered by passage, they were first cultured in a 125ml shake flask, the inoculation volume was 30ml, and the medium was 100% Dynamis medium, placed at a rotating speed of 120r/min and a temperature of 37 °C in a shaker with 8% carbon dioxide. After culturing for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/ml, and the expansion volume is calculated according to the production demand, and the medium is 100% Dynamis medium. After that, the culture was expanded every 72h. When the cell volume meets the production requirements, the seeding density is strictly controlled to be about 500,000 cells/ml for production.
  • Shaking flask parameters rotating speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Feed feeding start feeding every day after culturing in the shake flask for 72 hours.
  • HyClone Cell Boost Feed 7a is fed 3% of the initial culture volume every day, and Feed 7b is fed daily at 1/1000 of the initial culture volume. Supplement until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3 g/L on the sixth day. Samples were collected on the 13th day.
  • Affinity purification was performed using a protein A affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g foreign control antibody as control.
  • the electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, and one Mr is 50KD (heavy chain, SEQ ID NO: 14), the other Mr is 28KD (light chain, SEQ ID NO: 13).
  • Example 2 The antibody (WT) of Example 1 was analyzed, and its heavy chain variable region amino acid sequence is shown in SEQ ID NO: 12, wherein the amino acid sequence of each complementarity determining region is as follows:
  • CDR-VH1 G-F-S(X1)-F-S-N(X2)-A-W-M-E(X3);
  • CDR-VH2 E-L(X1)-G-N-K-A(X2)-N-N-H-A-T-Y-Y-A-D(X3)-S-I(X4)-K-G;
  • CDR-VH3 S(X1)-T-R(X2)-F-A-Y;
  • CDR-VL1 K-A-S-Q-D-L(X1)-N-T(X2)-Y-L(X3)-S;
  • CDR-VL2 R-G(X1)-N-R-L(X2)-I(X3)-D;
  • CDR-VL3 I(X1)-Q-Y-D-E-Y(X2)-P-R.
  • the coating solution (the main component NaHCO 3 ) was diluted with goat anti-mouse IgG 1 ⁇ g/ml for microplate coating, 100 ⁇ l per well, overnight at 4°C; the next day, the washing solution (the main component Na 2 HPO4 + NaCl) was washed twice, Pat dry; add blocking solution (20%BSA+80%PBS), 120 ⁇ l per well, 37°C, 1h, pat dry; add the diluted purified antibodies in Table 1, 100 ⁇ l/well, 37°C, 60min; shake off The liquid in the plate, patted dry, add 20% mouse negative blood to block, 120 ⁇ l per well, 37°C, 1 h; shake off the liquid in the plate, pat dry, add diluted CA19-9 human ascites (from clinical samples), 100 ⁇ l per well , 37°C, 40min; wash 5 times with washing solution, pat dry; add HRP-labeled CA19-9 monoclonal antibody (same as the above purified antibody), 100 ⁇ l per well, 37°C,
  • WT 1 1.846 0.867 0.391 0.198 0.094 0.054
  • WT 2 1.877 0.774 0.398 0.202 0.097 0.053
  • WT 3 1.822 0.836 0.377 0.191 0.073 0.051
  • WT 4 1.898 0.733 0.355 0.204 0.069 0.050
  • WT 5 1.852 0.706 0.319 0.169 0.061 0.051
  • WT 6 1.894 0.733 0.392 0.199 0.103 0.052
  • the above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for state observation, and the 21-day sample was tested for activity. , the results showed that there was no obvious change in the protein status of the antibodies under the three test conditions for 21 days, and the activity did not show a significant downward trend with the increase of the test temperature, indicating that the above antibodies were stable.
  • Table 7 below shows the OD results of the enzyme immunoassay activity detection of the mutant 1 antibody for 21 days.
  • the antibodies provided by the embodiments of the present disclosure have high correlation in actual detection, and can obtain relatively accurate detection results.
  • the present disclosure provides an antibody against CA19-9, its application and a kit for detecting CA19-9.
  • the antibody has good detection specificity and binding activity to CA19-9, can be used to detect sample CA19-9, and has good sensitivity and specificity.
  • the present disclosure is the detection of CA19-9 and the use of CA19-9 as a marker
  • the diagnosis or auxiliary diagnosis of tumor-related tumors provides a richer selection of antibodies.
  • the reagents or kits prepared by the present disclosure also have the same technical effects as the above-mentioned antibodies, such as better detection sensitivity and specificity, and have higher industrial practicability and application value, as well as broader market application prospects.

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Abstract

L'invention concerne un anticorps anti-CA19-9, une application de celui-ci et un kit de détection de CA19-9. L'anticorps ou le fragment fonctionnel de celui-ci a une région de détermination de complémentarité de chaîne lourde et une région de détermination de complémentarité de chaîne légère, peut se lier spécifiquement à CA19-9, et est utilisé pour détecter CA19-9 et diagnostiquer des tumeurs associées.
PCT/CN2021/117804 2020-12-08 2021-09-10 Anticorps anti-ca19-9, son application et kit de détection de ca19-9 WO2022121415A1 (fr)

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