CA15-3 antigen binding protein and application thereof

Abstract

The invention provides a CA15-3 antigen binding protein and application thereof. The CA15-3 antigen binding protein comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises a CDR3 with a sequence as shown in SEQ ID NO.7 or SEQ ID NO.13; and the light chain variable region comprises a CDR3 with a sequence as shown in SEQ ID NO. 10 or SEQ ID NO. 16. The CA15-3 antigen binding protein is a rabbit monoclonal antibody, compared with a mouse antibody and a polyclonal antibody, the rabbit antibody naturally has higher affinity and higher specificity, the antibody pair obtained through combination is high in specificity and accuracy, and the obtained CA15-3 antigen binding protein can be applied to diagnosis of breast cancer in a paired mode.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a CA15-3 antigen binding protein and an application thereof. Background Art

[0002] CA15-3 (cancer antigen 15-3, also known as Mucin1, MUC1, EMA, MCD, PEM, PUM, KL-6 and MAM6) is a member of the mucin family and a type I transmembrane protein with a highly glycosylated extracellular domain that extends 200-500 nanometers from the cell surface. CA15-3 consists of two subunits, the N-terminal subunit of the extracellular domain and the C-terminal subunit of the cytoplasmic domain. The core protein has a molecular weight of 120 to 220 kDa, and the glycosylated part is greater than 400 kDa.

[0003] CA15-3 is the first breast cancer-related antigen discovered. It is located on the surface of tumor cells. When cells become cancerous, the activity of proteases and salivary enzymes on the cell membrane increases, and the cytoskeleton is destroyed, which leads to the detachment of cell antigens and their release into the blood, increasing the serum CA15-3 level.

[0004] CA15-3 is elevated in breast cancer patients, with a sensitivity of 60% in the early stage of breast cancer and 80% in the late stage of breast cancer. CA15-3 is of great value in the observation of the efficacy of breast cancer, prognosis judgment, and diagnosis of recurrence and metastasis. CA15-3 also has a certain positive rate for other malignant tumors, such as lung cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer or primary liver cancer.

[0005] The development process of CA15-3 detection antibodies mainly includes the following steps: (1) Preparation of the main immunogen: generally using natural antigens, peptides or cells expressing CA15-3; (2) Immunization of immunized animals: selecting suitable immunized animals, usually mice, rabbits, etc.; (3) Injecting the immunogen into the immunized animal to stimulate it to produce CA15-3-specific antibodies; (4) Screening and identification: through phage display, single cell sorting and other screening methods, screen out CA15-3-specific antibodies with specificity and affinity; (5) Further identification and evaluation of candidate antibodies, including affinity determination, flow cytometric analysis, cloning, antibody optimization and improvement.

[0006] However, there are few anti-CA15-3 protein antibodies at present, and the price of purchasing imported brands is high. On the other hand, CA15-3 requires a paired antibody pair with high sensitivity. Therefore, providing a monoclonal antibody that can specifically bind to CA15-3 protein has important application value in the diagnosis of breast cancer. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a CA15-3 antigen binding protein and its application. The CA15-3 antigen binding protein described in the present invention is a rabbit monoclonal antibody. Compared with mouse antibodies and polyclonal antibodies, rabbit antibodies naturally have higher affinity and stronger specificity, and the antibody pair obtained by combination has strong specificity and high accuracy. The obtained antibodies can be paired and applied to the diagnosis of breast cancer.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a CA15-3 antigen binding protein, characterized in that the CA15-3 antigen binding protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR3 having a sequence as shown in SEQ ID NO.7 or SEQ ID NO.13;

[0010] The light chain variable region includes CDR3 having a sequence as shown in SEQ ID NO.10 or SEQ ID NO.16.

[0011] Preferably, the heavy chain variable region comprises CDR1 having a sequence as shown in SEQ ID NO.5 or SEQ ID NO.11.

[0012] Preferably, the heavy chain variable region comprises CDR2 having a sequence as shown in SEQ ID NO.6 or SEQ ID NO.12.

[0013] Preferably, the light chain variable region comprises CDR1 having a sequence as shown in SEQ ID NO.8 or SEQ ID NO.14.

[0014] Preferably, the light chain variable region comprises CDR2 having a sequence as shown in SEQ ID NO.9 or SEQ ID NO.15.

[0015] Preferably, the heavy chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.5, a CDR2 having a sequence as shown in SEQ ID NO.6, and a CDR3 having a sequence as shown in SEQ ID NO.7; and the light chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.8, a CDR2 having a sequence as shown in SEQ ID NO.9, and a CDR3 having a sequence as shown in SEQ ID NO.10; or,

[0016] The heavy chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.11, a CDR2 having a sequence as shown in SEQ ID NO.12, and a CDR3 having a sequence as shown in SEQ ID NO.13; and the light chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.14, a CDR2 having a sequence as shown in SEQ ID NO.15, and a CDR3 having a sequence as shown in SEQ ID NO.16.

[0017] Preferably, the heavy chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.1 or SEQ ID NO.3; and / or,

[0018] The light chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.2 or SEQ ID NO.4.

[0019] Preferably, the sequence of the heavy chain variable region includes SEQ ID NO.1 or SEQ ID NO.3.

[0020] Preferably, the sequence of the light chain variable region includes SEQ ID NO.2 or SEQ ID NO.4.

[0021] Preferably, the sequence of the heavy chain variable region comprises the sequence shown in SEQ ID NO.1, and the sequence of the light chain variable region comprises the sequence shown in SEQ ID NO.2; or,

[0022] The sequence of the heavy chain variable region includes that shown in SEQ ID NO.3, and the sequence of the light chain variable region includes that shown in SEQ ID NO.4.

[0023] Preferably, the antigen binding protein has a strength greater than or equal to 10 -11 It specifically binds to human CA15-3 with an affinity of nmol / L.

[0024] Preferably, the antigen binding protein is an antibody.

[0025] Preferably, the antibody is a monoclonal antibody.

[0026] Preferably, the constant region of the monoclonal antibody is a rabbit-derived constant region.

[0027] In the present invention, CA15-3 natural antigen is used to immunize New Zealand white rabbits. After 4 immunizations, the antiserum titer is detected by enzyme-linked immunosorbent assay (ELISA). After the serum titer reaches a certain dilution, impact immunization is performed, serum is collected for polyclonal antibody purification, and spleen is collected for grinding. Total RNA is extracted from the spleen, and first-chain cDNA is synthesized by reverse transcription. The light and heavy chain variable region fragments (VH / Vκ) of the immunized rabbit are amplified by a set of primers matching the light and heavy chain variable regions FR1 and FR4 of the rabbit antibody, and secondary amplification is performed by another set of primers, and a linker sequence with a complementary overlapping region is added to the antibody light and heavy chain variable fragments, and the VH fragment and the Vκ fragment are assembled into a single-chain antibody fragment (scFv) by overlap extension PCR (SOE PCR).

[0028] After digesting the scFv fragment and phage display vector with the same restriction endonuclease, gel recovery and recombinant vector connection are performed, and after cleaning the connection product, it is electrotransferred to Escherichia coli TG1 to obtain the scFv phage display bacterial library. By culturing the bacterial library, adding the helper phage M13K07 for superinfection, assisting in packaging and rescuing the phage library, and displaying the scFv fragment on the phage surface. The CA15-3 natural antigen is coated in the immunoplate, and the phage library is added after blocking with the blocking liquid. The phage displaying the specific scFv fragment is captured because it can bind to the CA15-3 protein, and the unbound phage is removed by rinsing. The phage is eluted from the immunoplate and infected with the TG1 host in the logarithmic growth phase. After culturing, the helper phage can be used again for superinfection and packaging rescue. After several rounds of screening, the TG1 bacterial solution infected with the screened phage was gradiently diluted and spread on the plate, and then a single clone was picked and cultured in a 96-well plate. Helper phage was added to package the monoclonal phage, and the binding activity of the phage to CA15-3 in the monoclonal supernatant was detected by ELISA to obtain a positive monoclonal clone that specifically binds to CA15-3.

[0029] The candidate positive clones were sequenced and analyzed, and constructed into the eukaryotic expression vector pcDNA3.1(+) containing the constant regions of the heavy and light chains of rabbit antibodies to obtain complete light and heavy chain expression vectors. The light and heavy chain vector plasmids were co-transfected into HEK293E cells for expression, and the positive clone antibody protein was purified from the culture supernatant using Protein A. The binding specificity of the positive clone antibody protein to CA15-3 was verified by ELISA and the affinity was determined, and finally two antibodies that specifically bind to CA15-3 were obtained.

[0030] In the present invention, two antibodies that specifically bind to CA15-3 protein were screened and obtained, which are denoted as CF24 and CF128. The amino acid sequences are shown below, and the underlined sequences in the sequences are the antibody CDR regions.

[0031] SEQ ID NO.1 (amino acid sequence of the heavy chain variable region of CF24)

[0032] QSVEESGGRLVTPGTPLTLTCTVFGFSLS DFGVS WVRQAPGKGLEWIG YIWIDGNTDY ASWAKG RFTISKASTTVDLKITSPTTEDTATYFCAR MRDIRTYAYAGYNI WGPGTLVTVSS.

[0033] SEQ ID NO. 5 (heavy chain CDR1 of CF24): DFGVS.

[0034] SEQ ID NO. 6 (heavy chain CDR2 of CF24): YIWIDGNTDYASWAKG.

[0035] SEQ ID NO. 7 (heavy chain CDR3 of CF24): MRDIRTYAYAGYNI.

[0036] SEQ ID NO.2 (amino acid sequence of the light chain variable region of CF24)

[0037] YVMMTQTPASVSEPVGGTVTIKC QASQSIYSYLA WYQQKPGQPPKLLIY KASTLAS GV PSRFKGSGSGTQFTLTISDLECDDAATYYC QSTYDSEDNT FGGGTKVVVE.

[0038] SEQ ID NO.8 (light chain CDR1 of CF24): QASQSIYSYLA.

[0039] SEQ ID NO.9 (light chain CDR2 of CF24): KASTLAS.

[0040] SEQ ID NO. 10 (light chain CDR3 of CF24): QSTYDSEDNT.

[0041] SEQ ID NO.3 (amino acid sequence of the heavy chain variable region of CF128)

[0042] QSVKESEGGLFKPTDTLTLTCTVSGFSLS SNAIY WVRQAPGNGLEWIG AIYSYGGTYYA NWAKS RSTITRNTNLNTATLKMTSLTAADTATYFCAR GGVGDSDSAINI WGPGTLVTVSS.

[0043] SEQ ID NO.11 (heavy chain CDR1 of CF128): SNAIY.

[0044] SEQ ID NO. 12 (heavy chain CDR2 of CF128): AIYSYGGTYYANWAKS.

[0045] SEQ ID NO. 13 (heavy chain CDR3 of CF128): GGVGDSDSAINI.

[0046] SEQ ID NO.4 (amino acid sequence of the light chain variable region of CF128)

[0047] ALVMTQTPASVSEPVGGTVTIKC QASQSIGSNLA WYQQKPGQRPKLLIY YASTLES GVS SRFKGSRSGTEFTLTISDLECDDAATYYC QQGYSSGGVDNV FGGGTKLEIK.

[0048] SEQ ID NO. 14 (light chain CDR1 of CF128): QASQSIGSNLA.

[0049] SEQ ID NO. 15 (light chain CDR2 of CF128): YASTLES.

[0050] SEQ ID NO. 16 (light chain CDR3 of CF128): QQGYSSGGVDNV.

[0051] In a second aspect, the present invention provides a nucleic acid molecule encoding the CA15-3 antigen binding protein described in the first aspect.

[0052] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0053] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains at least one copy of the expression vector described in the third aspect; or the nucleic acid molecule described in the second aspect is integrated into the genome of the host cell.

[0054] In a fifth aspect, the present invention provides a reagent for detecting CA15-3 protein in a sample, wherein the reagent comprises the CA15-3 antigen binding protein described in the first aspect.

[0055] In a sixth aspect, the present invention provides a kit, wherein the kit comprises the CA15-3 antigen binding protein described in the first aspect; or the kit comprises the reagent for detecting CA15-3 protein in a sample described in the fifth aspect.

[0056] In a seventh aspect, the present invention provides use of the CA15-3 antigen binding protein described in the first aspect in the preparation of a tumor detection product.

[0057] The CA15-3 antigen binding protein of the present invention can be applied to the diagnosis of CA15-3 lymphocyte-related diseases, such as early diagnosis of breast cancer.

[0058] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The CA15-3 antigen binding protein obtained by the present invention can specifically bind to the CA15-3 target, and the CA15-3 antigen binding protein can be used for the diagnosis of diseases related to changes in CA15-3 concentration, such as early diagnosis of breast cancer; it has great potential in immunological research and the development of new treatment strategies. The CA15-3 antigen binding protein obtained by the present invention can be used directly or by various labeling and packaging into a kit for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is the result of the potency test.

[0062] Figure 2 This is the result of SDS-PAGE electrophoresis.

[0063] Figure 3 Affinity determination results.

[0064] Figure 4 It is the result of antibody pair matching test. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0066] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0067] Example 1 Animal Immunization and Serum Titer Test

[0068] In this example, each polypeptide (CA15-3 polypeptide) was used to immunize 2 rabbits (purchased from Guangdong Medical Experimental Animal Center, name: ordinary New Zealand rabbit, specification: about 1.8 kg), and the polypeptide was outsourced and synthesized by GenScript.

[0069] 1. Take 1 mL of blood from the ears of two rabbits respectively, and collect serum for use as a control. Immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen into 1.5 mL of PBS, mix 1.5 mL of antigen + 1.5 mL of complete adjuvant, and oscillate on an oscillator for more than 15 minutes until the oil-in-water state; immunize each rabbit with 1.5 mL on the back.

[0070] 2. Two weeks later, the second immunization was carried out. Each rabbit was immunized with 0.5 mg of antigen. It was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 mL of PBS. The mixture was 1 mL of antigen + 1 mL of incomplete adjuvant and oscillated on an oscillator for more than 15 minutes until it reached the oil-in-water state. Each rabbit was immunized with 1 mL on the back.

[0071] 3. One week later, each rabbit was immunized with 0.5 mg of antigen, which was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 mL of PBS, and mixed as 1 mL of antigen + 1 mL of incomplete adjuvant. The mixture was shaken on an oscillator for more than 15 minutes until the mixture reached the oil-in-water state. Each rabbit was immunized with 1 mL on its back.

[0072] 4. One week later, each rabbit was immunized with 0.5 mg of antigen, which was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 mL of PBS, and mixed as 1 mL of antigen + 1 mL of incomplete adjuvant. The mixture was shaken on an oscillator for more than 15 minutes until it reached the oil-in-water state. Each rabbit was immunized with 1 mL on its back.

[0073] 5. One week later, collect 1 mL of blood from the ear and test the titer by ELISA. If it reaches 1:100,000 or above, perform shock immunization.

[0074] 6. For shock immunization, 1 mg of antigen was immunized per rabbit. Emulsified with complete adjuvant, 2 mg of antigen was diluted into 1.5 mL of PBS, and mixed as 1.5 mL of antigen + 1.5 mL of complete adjuvant. Oscillate on an oscillator for more than 15 minutes until the mixture reaches the oil-in-water state. 1.5 mL was applied to the back of each rabbit for immunization.

[0075] 7. Three days later, blood was collected from the ears. 5 mL of blood was collected from each rabbit for titer measurement. If the titer was in line with expectations, whole blood was collected in anticoagulant tubes and serum was separated.

[0076] The materials involved in the above operations in this embodiment are all commonly used reagents and consumables.

[0077] Example 2 Antiserum titer ELISA test

[0078] 1. Dilute CA15-3 natural antigen (purchased from XEMA, R229C) to 1 μg / mL with PBS, add 100 μL to each well of the immunoplate, and incubate overnight in a 4°C refrigerator.

[0079] 2. Wash the ELISA plate 3 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel the last time.

[0080] 3. Add 300 μL of 3% BSA blocking solution to each well and incubate at 37°C for 1 hour.

[0081] 4. Wash the ELISA plate 3 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel for the last wash.

[0082] 5. Dilute the antiserum with PBS, add 100 μL to each well of the immunoplate, and incubate at 37°C for 1 hour.

[0083] 6. Wash the ELISA plate 6 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel for the last wash.

[0084] 5. Add 100 μL of PBS-diluted Anti-rabbit Antibody (HRP) (anti-rabbit secondary antibody, Beijing Sino Biological, Cat. No. SSA003) to each well and incubate at room temperature for 1 hour.

[0085] 6. Wash the ELISA plate 6 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel for the last wash.

[0086] 7. Take out Solution A and Solution B of TMB substrate colorimetric solution from 4°C, return to room temperature, mix in equal proportions to prepare an appropriate amount of colorimetric solution, add 100 μL to each well of the ELISA plate, and react for 10 minutes in the dark.

[0087] 8. Add 50 μL 1M HCl to each well to terminate the color development reaction and read the OD450 value with a microplate reader.

[0088] The titer test results are as follows Figure 1 As shown, the titer is greater than 1:102400, indicating successful immunization.

[0089] Example 3 Screening of immune rabbit antibody scFv display library

[0090] Rabbit spleen cells were ground and RNA was extracted to establish a cDNA library. The antibody variable region sequences in the library were amplified by literature methods and a phage display library was constructed for antibody screening. After three rounds of panning, 192 monoclones were picked and 34 positive clones were obtained, of which CF24 and CF128 were the preferred two.

[0091] The amino acid sequence of the heavy chain variable region of CF24 is shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of CF24 is shown in SEQ ID NO. 2. The amino acid sequence of the heavy chain variable region of CF128 is shown in SEQ ID NO. 3; the amino acid sequence of the light chain variable region of CF128 is shown in SEQ ID NO. 4.

[0092] Example 4 Expression and purification of positive cloned antibodies

[0093] Recombinant antibody expression: The light and heavy chain sequences of CF24 and CF128 were constructed into pCDNA3.1(+) vectors after the genes were synthesized and the codon sequences were optimized by Guangzhou Liuhe. After obtaining the vector, the plasmid was extracted, and then the light and heavy chain vectors were mixed at a molar ratio of 1:1. Take 300μg of plasmid, add 12.5mL OPM 293CD05 culture medium and 800μL 1mg / mL PEI (polyethyleneimine, POLYSCIENCE) solution and mix well, let stand at room temperature for half an hour, add to 250mL of HEK293F cells being cultured, put in a shaker, 37℃, 8% CO2, 110rpm culture, add OPM 293 cell supplement every other day, culture for 7 days, and collect cell supernatant by centrifugation.

[0094] Purification of recombinant antibodies: Add 3 mL of Protein A filler (Sino Biological) to the supernatant, incubate at room temperature for half an hour, remove the filler and load it into the empty purification column, add 20 mL of PBS solution to rinse, then add 10 mL of 100 mM glycine solution at pH 3.0 for elution, and add 1 M Tris solution to neutralize the eluate to pH 7.0. Concentrate the neutralized eluate to 1 mL, determine the protein concentration, and run the representative antibody on SDS-PAGE gel for purity identification. The electrophoresis results are as follows: Figure 2 As shown, Figure 2 This is the result of positive antibody purification. Figure 2 In the figure, lane M is the molecular weight standard, lane 1 is the recombinant antibody CF24, and lane 2 is the recombinant antibody CF128. Figure 2 It can be seen that the antibody reaches the electrophoresis purity standard, and no obvious other bands are observed except the main band.

[0095] Example 5 Affinity ELISA determination of positive clones

[0096] 1. CA15-3 natural antigen (purchased from XEMA, R229C) was coated on the enzyme strip overnight. Wash the ELISA plate 3 times with PBST, 300μL per well, and pat off the residual liquid on a paper towel the last time. 2. Add 200μL of 3% BSA-PBS and block at 37℃ for 2 hours. Wash the ELISA plate 3 times with PBST, 300μL per well, and pat off the residual liquid on a paper towel the last time. 3. Dilute the antibody at different concentrations, add 100μL, and incubate at 37℃ for 1 hour. Wash the ELISA plate 3 times with PBST, 300μL per well, and pat off the residual liquid on a paper towel the last time. 4. Add 100μL of diluted commercial horseradish peroxidase-conjugated goat anti-rabbit antibody (HRP) and incubate at 37℃ for 1 hour. Wash the ELISA plate 6 times with PBST, 300μL per well, and pat off the residual liquid on a paper towel the last time. 5. Use tetramethylbenzidine microporous peroxidase substrate (TMB) as substrate for color development for 10 minutes. 6. Add 50 μL of 0.1 mol / L sulfuric acid to terminate the reaction and measure the absorbance at 450 nm.

[0097] The affinity test results are shown in Figure 3 According to the ELISA results, the affinity of the two antibodies was calculated by the origin software 10 -11 about.

[0098] Example 6 Antibody Pairing Range Test

[0099] 1. Coat the immunoplate with CA15-3 antibody overnight. Wash the plate with PBST 3 times, 300 μL per well, and tap the remaining liquid on a paper towel for the last wash.

[0100] 2. Add 200 μL of 3% BSA-PBS and block at 37°C for 2 hours. Wash the ELISA plate 3 times with PBST, 300 μL per well, and tap off any remaining liquid on a paper towel for the last wash.

[0101] 3. Dilute the CA15-3 natural antigen at different concentrations, add 100 μL, and incubate at 37°C for 2 hours. Wash the ELISA plate 6 times with PBST, 300 μL per well, and tap off the residual liquid on a paper towel the last time. 4. Add 100 μL of diluted anti-CA15-3-HRP antibody and incubate at 37°C for 1 hour. Wash the ELISA plate 6 times with PBST, 300 μL per well, and tap off the residual liquid on a paper towel the last time. 5. Use tetramethylbenzidine microporous peroxidase substrate (TMB) as a substrate for color development for 10 minutes. 6. Add 50 μL of 0.1 mol / L sulfuric acid to terminate the reaction, and measure its absorbance at 450 nm.

[0102] The results of the antibody pair test are shown in Figure 4 The linear range is 6.25-100 U / mL (R 2 >0.99).

[0103] In summary, the anti-CA15-3 protein monoclonal antibody (CA15-3 antigen binding protein) provided by the present invention is a rabbit monoclonal antibody. Compared with mouse antibodies and polyclonal antibodies, rabbit antibodies naturally have higher affinity and stronger specificity. The affinity of the two antibodies is 10 -11 The results of the antibody pair matching test showed that its linear range was 6.25-100U / mL, and the combined antibody pair had strong specificity and high accuracy.

[0104] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A CA15-3 antigen binding protein, characterized in that The CA15-3 antigen binding protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR3 having a sequence as shown in SEQ ID NO.7 or SEQ ID NO.13; The light chain variable region includes CDR3 having a sequence as shown in SEQ ID NO.10 or SEQ ID NO.

16.

2. The CA15-3 antigen binding protein according to claim 1, characterized in that The heavy chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.5 or SEQ ID NO.11; Preferably, the heavy chain variable region comprises a CDR2 having a sequence as shown in SEQ ID NO.6 or SEQ ID NO.12; Preferably, the light chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.8 or SEQ ID NO.14; Preferably, the light chain variable region comprises CDR2 having a sequence as shown in SEQ ID NO.9 or SEQ ID NO.

15.

3. The CA15-3 antigen binding protein according to claim 1 or 2, characterized in that The heavy chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.5, a CDR2 having a sequence as shown in SEQ ID NO.6, and a CDR3 having a sequence as shown in SEQ ID NO.7; and the light chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.8, a CDR2 having a sequence as shown in SEQ ID NO.9, and a CDR3 having a sequence as shown in SEQ ID NO.10; or, The heavy chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.11, a CDR2 having a sequence as shown in SEQ ID NO.12, and a CDR3 having a sequence as shown in SEQ ID NO.13; and the light chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.14, a CDR2 having a sequence as shown in SEQ ID NO.15, and a CDR3 having a sequence as shown in SEQ ID NO.

16.

4. The CA15-3 antigen binding protein according to any one of claims 1 to 3, characterized in that The heavy chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.1 or SEQ ID NO.3; and / or, The light chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.2 or SEQ ID NO.

4.

5. The CA15-3 antigen binding protein according to any one of claims 1 to 4, characterized in that The sequence of the heavy chain variable region includes SEQ ID NO.1 or SEQ ID NO.3; Preferably, the sequence of the light chain variable region includes SEQ ID NO.2 or SEQ ID NO.

4.

6. The CA15-3 antigen binding protein according to claim 5, characterized in that The sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO.1, and the sequence of the light chain variable region includes the sequence shown in SEQ ID NO.2; or, The sequence of the heavy chain variable region includes that shown in SEQ ID NO.3, and the sequence of the light chain variable region includes that shown in SEQ ID NO.

4.

7. The CA15-3 antigen binding protein according to any one of claims 1 to 6, characterized in that The antigen binding protein is stronger than or equal to 10 -11 It specifically binds to human CA15-3 with an affinity of nmol / L.

8. The CA15-3 antigen binding protein according to any one of claims 1 to 7, characterized in that The antigen binding protein is an antibody; Preferably, the antibody is a monoclonal antibody; Preferably, the constant region of the monoclonal antibody is a rabbit-derived constant region.

9. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CA15-3 antigen binding protein according to any one of claims 1 to 8.

10. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 9.

11. A host cell, characterized in that The host cell contains at least one copy of the expression vector according to claim 10; or the nucleic acid molecule according to claim 9 is integrated into the genome of the host cell.

12. A reagent for detecting CA15-3 protein in a sample, characterized in that: The reagent comprises the CA15-3 antigen binding protein according to any one of claims 1 to 8.

13. A kit, characterized in that: The kit comprises the CA15-3 antigen binding protein according to any one of claims 1 to 8; or the kit comprises the reagent for detecting CA15-3 protein in a sample according to claim 12.

14. Use of the CA15-3 antigen binding protein according to any one of claims 1 to 8 in the preparation of a tumor detection product.

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