CN116041523A

CN116041523A - Preparation method of recombinant antibody applied to tumor marker CA724

Info

Publication number: CN116041523A
Application number: CN202211629298.3A
Authority: CN
Inventors: 李永刚; 李娜
Original assignee: Nanjing Genstars Biotech Co ltd
Current assignee: Nanjing Genstars Biotech Co ltd
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2023-05-02

Abstract

The invention discloses a preparation method of a recombinant antibody applied to a tumor marker CA724, wherein the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2. The amino acid sequence of the variable region of the heavy chain of the antibody is shown as SEQ ID NO.3, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 4. The variable region sequences of the CDR1, the CDR2 and the CDR3 of the antibody heavy chain are shown in SEQ ID NO. 5-7, and the variable region sequences of the CDR1, the CDR2 and the CDR3 of the antibody light chain are shown in SEQ ID NO. 8-10. A nucleic acid molecule encoding the anti-CA 724 antibody. The antibody provided by the invention has high activity and is stable among antibody batches. The invention has important significance for further realizing stable expression and large-scale production of the anti-CA 724 antibody.

Description

Preparation method of recombinant antibody applied to tumor marker CA724

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a preparation method of a recombinant antibody applied to tumor CA 724.

Background

Carbohydrate antigen CA724 (carbohydrate antigen, CA 724) is a non-specific tumor marker, and is one of assay markers for detecting gastric cancer and various digestive tract cancers. CA724 has a molecular weight of 220-400KD and comprises two high molecular weight glycoproteins recognizable by anti-CA 724 antibodies: CC49 and B72.3.CA724 is one of the indexes with highest correlation with gastric cancer, the serum level of the CA724 is far higher than that of gastric benign lesion patients and healthy people, the content of serum of normal people is less than 6U/mL, and the serum detection level is rapidly increased when cancers such as digestive tract tumors, gastric cancer and ovarian cancer occur, so that the detection of CA724 by using the anti-CA 724 antibody has great clinical reference value in the gastric cancer diagnosis process. At present, the monoclonal antibody for detecting CA724 is basically purchased from foreign countries, and has defects in sensitivity and specificity, so that the monoclonal antibody for resisting CA724 with better sensitivity and specificity needs to be provided.

Disclosure of Invention

The invention aims to provide a novel anti-CA 724 antibody and application thereof. The antibody provided by the invention has high activity and is stable among antibody batches. The invention has important significance for further realizing stable expression and large-scale production of the anti-CA 724 antibody.

The technical scheme adopted by the invention is as follows:

an anti-CA 724 antibody or antigen-binding fragment thereof having at least one heavy chain CDR domain selected from SEQ ID NOs 5, 6, 7 and/or at least one heavy chain CDR domain selected from SEQ ID NOs: 8. 9, 10.

The anti-C724 antibody, or antigen-binding fragment thereof, of the invention, the heavy chain variable region of which comprises the following three complementarity determining region CDRs: SEQ ID NO:5, CDR1, SEQ ID NO:6 and CDR2 as set forth in SEQ ID NO: CDR3 shown in fig. 7; the light chain variable region of the antibody comprises the following three complementarity determining region CDRs: SEQ ID NO:8, CDR1, SEQ ID NO:9 and CDR2 as set forth in SEQ ID NO:10, CDR3.

The heavy chain amino acid sequence of the anti-CA 724 antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2.

The amino acid sequence of the variable region of the heavy chain of the anti-CA 724 antibody is shown as SEQ ID NO.3, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 4.

A nucleic acid molecule encoding the anti-CA 724 antibody.

The nucleotide sequence of the heavy chain of the antibody is shown as SEQ ID NO. 11; the nucleotide sequence of the antibody light chain is shown as SEQ ID NO. 12.

A vector comprising the nucleic acid molecule described above.

A host cell comprising a nucleic acid molecule or a vector as described above.

An antibody conjugate obtained by coupling said anti-CA 724 antibody with a carrier or a drug, or by coupling said antibody with a chemical or biological label.

A kit for detecting a CA724 protein, comprising said anti-CA 724 antibody.

The application of the antibody or the nucleic acid molecule in preparing a diagnostic agent for diagnosing cancer or detecting a CA724 product.

Advantageous effects

The invention provides a novel anti-CA 724 antibody which has higher activity and good stability, accelerates the stability within 20% after seven days, can well identify CA724, has high affinity and specificity, and provides a novel choice for C724 detection.

Drawings

FIG. 1 SDS-PAGE gel of the supernatant of CA724 cell line, wherein lane 1 is the reducing band and lane 2 is the non-reducing band.

FIG. 2 is a microscopic representation of the status of a monoclonal cell.

FIG. 3 is a chromatogram of anti-CA 724 antibody purity.

Detailed Description

Example 1 antibody acquisition

1.1 antigen immunization Process

Antigen preparation process: taking saccharide antigen CA724, taking CA724 protein as immunogen, diluting the antigen to 1mg/mL, taking 50 mu L of antigen solution for the first immunization, and 50 mu L of cytokine adjuvant interleukin-2; subsequent immunization was performed with 50. Mu.L of antigen solution and 50. Mu.L of cytokine adjuvant. The antigen solution and the adjuvant are respectively sucked by two 2mL syringes, the air in the syringes is discharged as much as possible, the three-way connection is used, the antigen is pushed into the adjuvant, and the connected syringes are repeatedly pushed for about 20min-40min until the emulsified antigen is not diffused in water.

Immunization of mice procedure: healthy laboratory mice are selected, and the healthy laboratory mice have no obvious external injury, are actively fed and are active, and are suitable for mice with the age of 6 to 8 weeks. The intraperitoneal injection method is adopted, the immunization is carried out once every 21 days, after the immunization is carried out twice, the tail is broken and the blood is taken after 7 days of each immunization, the titer is detected, and the immunization can be stopped when the titer meets the requirement, and the fusion is carried out.

1.2 cell fusion Process

Material preparation:

preparing HAT culture medium, and subpackaging DMEM and PEG; surgical instruments, 50mL plastic centrifuge tubes, 50mL glass centrifuge tubes.

SP2/0 cells were resuscitated one week in advance, cultured and allowed to grow well.

Feeder cells were harvested and plated one day in advance with HAT medium, and 5 feeder cell plates were required to fuse one mouse.

Fusion:

process SP2/0: blowing off, transferring to a 50mL plastic centrifuge tube, centrifuging for 5min at 1000r, discarding the supernatant, adding DMEM, blowing off, centrifuging for 5min at 1000r, adding DMEM to about 10mL, blowing off, and counting for later use.

Extraction of spleen cells:

mice were bled from the orbital sockets, sacrificed by cervical removal and the blood samples were left as positive controls.

The spleens of mice were isolated, the spleens were rinsed outside with DMEM, the spleens were rinsed inside with DMEM, and the rinse was collected and transferred to a 10mL glass centrifuge tube for counting.

Centrifuging at 1000r for 5min, discarding supernatant, adding DMEM, blowing off, transferring to a 50mL glass centrifuge tube, transferring the SP2/0 processed previously to the 50mL glass centrifuge tube at the same time, centrifuging at 1000r for 5min, discarding supernatant, and grinding on the back of hand. The mixture was subjected to a warm water bath at 37℃and 1mL of PEG was added thereto over 1min, followed by shaking and standing for 90s. 1mL of DMEM is added within 1min, 1mL of DMEM is added within 30s, the mixture is gradually accelerated, the DMEM is added to 40-50 mL, the mixture is kept stand for 10min at 37 ℃, centrifugation is carried out for 6min at 800r, the supernatant is discarded, and HAT is added for plating.

Half liquid exchange is carried out after 3 days, full liquid exchange is carried out after one week, and HAT culture medium is exchanged for culture.

1.3 subclone selection procedure

Observing the cell state, detecting the whole plate after the cell clusters which can be observed by naked eyes exist in 96 holes, and selecting the hole with higher positive to subclone, namely a positive Kong Yikuai plate.

After the cell clusters observed by naked eyes exist in the 6 holes, subcloning and picking are carried out, each plate selects 12 holes of single cell clusters for subcloning and picking, the cells with high positive and good cell states are selected for next subcloning, at least three subcloning is carried out, and finally, the monoclonal cell clusters with high positive and good cell states are selected for strain fixing.

Transferring the selected cell mass to 24-hole culture, and transferring to a small square bottle for culture after the 24-hole cells grow well. After the cells in the vial grow well, freezing and preserving are carried out to prepare ascites.

1.4 ascites preparation Process

Mice were injected with paraffin one week in advance, 0.5mL per mouse.

After the cells in the vial grew well, the cells were blown off, transferred to a 10mL glass centrifuge tube, centrifuged at 1000r for 5min, the supernatant was discarded, 1mL of LDMEM was added, counted, and 0.8X10 of each mouse was injected ⁶ Cells were diluted by injecting 0.5mL per mouse, and then cells were injected intraperitoneally into mice.

Observing the state of the mice, and collecting the ascites after the abdomen of the mice is obviously enlarged.

1.5 antibody purification procedure

Reagent preparation: equilibration/dialysis buffer: 10mM PB (pH 7.5) +100mM NaCl

100% saturated ammonium sulfate

Column cleaning liquid: 20mM Tris (pH 8.0) +1.5M NaCl

Preparing equipment: q column, low temperature high speed centrifuge, balance, peristaltic pump, nucleic acid protein detector.

33% ammonium sulfate precipitation

Sample preparation: the frozen ascites is melted in a constant temperature water bath at 25 ℃, and the oil is removed by filtration through filter paper.

120mL of ascites is measured, 10mM PB (pH 7.5) +100mM NaCl which is 1 time of the volume of the ascites is added and evenly mixed; adding saturated ammonium sulfate solution with volume of 1 time of ascites volume, stirring and precipitating at 20-25deg.C for 10min, wherein the saturation degree of ammonium sulfate is 33%; centrifuging at 9000rpm and 5 ℃ for 10min, collecting precipitate, and discarding supernatant; the precipitate was suspended and mixed uniformly with 10mM PB (pH 7.5) +100mM NaCl in 2/3 times the volume of ascites;

dialysis for desalting: dialysis was performed twice with 10mM PB (pH 7.5) +100mM NaCl dialysate at a dialysis ratio of 1:50, each dialysis time is not less than 5 hours and not more than 25 hours. After dialysis, the antibody was recovered and filtered with filter paper to remove impurities for further use. Column purification: filling 50mL of Q column, cleaning with 2 times of 0.1M NaOH, and then washing with 5 times of ultrapure water for later use; balancing the column with a balancing buffer solution with the volume of 5 times of the column, and adjusting the display value of the protein ultraviolet detector to 0 after the column is balanced to serve as a base line; sampling the dialyzed sample, wherein the sampling speed is 5-6mL/min, collecting Q column flow-through liquid in the whole process, and starting to collect when the detection value of A280 is more than 0.2 until the detection value is reduced to 0.2, stopping collecting, and collecting by a branch pipe, wherein the sample is the target protein; after the completion of the protein collection, the column was washed with 3 column volumes of 20mM Tris (pH 8.0) +1.5MNaCl until no protein was eluted; the packing was rinsed with 5 volumes of ultrapure water and a second Q column was prepared with 5 volumes of 10mM PB (pH 7.5) +100mM NaCl equilibrated column;

sampling the Q column sample for the first time, and collecting the target protein and synchronizing the steps;

after the collection of the proteins, the column was washed with 3 volumes of 20mM Tris (pH 8.0) +1.5M NaCl until no protein flowed out, the packing was washed with 5 volumes of ultrapure water, and the column was kept at 5-8deg.C with 20% ethanol filled for a long period of time; antibody treatment: measuring the volume of the collected antibody, and immediately adjusting the NaCl concentration of the preservation solution to 300mM by using 5M NaCl; diluting with 10mM PB (pH 7.5) +300mM NaCl buffer, adjusting antibody concentration to 3mg/mL, adding 100% Proclin300 to final concentration of 0.1%, filtering with 0.22 μm filter membrane, sterilizing, and packaging, inspecting and warehousing as required.

1.6 antibody sequencing Process

Carrying out reverse transcription on the sample to obtain cDNA; amplifying and obtaining heavy and light chain variable regions; cloning the variable region genes into pMD18-T vectors respectively, and sequencing; the IMGT/V-QUEST is used for sequencing result comparison, and then the complete sequence is obtained, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2. The amino acid sequence of the variable region of the heavy chain of the antibody is shown as SEQ ID NO.3, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 4.

EXAMPLE 2 recombinant expression and Large Scale culture expression

2.1CHO cell culture

Recovering CHO cells by using a CD CHO culture medium added with 1 mM-10 mM glutamine, placing the recovered CHO cells in a shaking table with the rotating speed of 120-150rpm, the temperature of 37 ℃ and the carbon dioxide of 5% -8%, and carrying out timed counting and subculture to ensure that the cells are in a logarithmic growth phase.

2.2 electric transfer of recombinant plasmid into CHO cells

Regulating CHO cell density to 1-2X 10 ⁷ Placing cells in a centrifuge tube, blowing and mixing 30-50 μg endotoxinfree recombinant CA724 plasmid with 700 μl cells, transferring into an electric rotating cup, electric rotating, counting the next day, and counting according to the number of living cells 1.0X10) ⁴ cells/well are plated in 96-well plates.

2.3 pressure screening of stably transformed cell lines

The cells were cultured under pressure using a screening pressure of 20-50. Mu. Mol/L MSX for about 14-25 days, the cells were screened under a microscope for the formation of cell mass wells, and the confluency was recorded, and after 3-5 days, they were transferred into 24-well plates for 3-5 days, and then into 6-well plates for 3-5 days, and after 3-5 days, the cell supernatants were collected and SDS-PAGE was performed to detect the expression of the objective antibodies, and the results are shown in FIG. 1. Cell holes with higher expression level of the screening antibody are amplified to 30ml of culture medium, and the cell density reaches 1.5-3.0x10 ⁶ cells were frozen at cells/ml as positive cell lines.

2.4 subclone cell line selection

Resuscitating positive cell strain, culturing in a carbon dioxide shaker at 37deg.C and carbon dioxide of 5% -8% at rotation speed of 120-150rpm/min to obtain cell density of 1.5-3.0X10 ⁶ Passage at cells/ml, ensuring that the passage inoculation density is 2.0-5.0X10 ⁵ cells/ml. After passaging twice, the cells were diluted with medium and inoculated into 96-well plates at 0.5 cells/well for stationary culture. Single cell wells were selected under the microscope and labeled (fig. 2). Use culture after 10-20 daysThe subcloned cell strains are gradually expanded to 24-pore plates, 6-pore plates and culture flasks by the culture medium, and IgG high-expression cell strains are screened by ELISA method. The cell density reaches 1.5-3.0X10 ⁶ The subcloned cell lines were frozen at cells/ml.

2.5 Mass production and purification of antibodies

Resuscitate subcloned cells, passaged twice until the cell state is stable, and expand culture to the desired system using shake flask or fermenter, during which time supplements such as glucose are added. After 14 days, the cell supernatant was collected and purified using an affinity column using staphylococcal protein a (protein a) to give anti-CA 724 antibody with a purity of greater than 90% (figure 3).

Example 3 application of antibody in detection of carbohydrate antigen CA724

The Roche CA724 monoclonal antibody is used as a control antibody (the Roche CA724 monoclonal antibody is used as a standard rod of the CA724 antibody industry and is used as the control antibody). The control sample and the antibody are used as coating matched with the Roche CA724 antibody marked sample, the activity result of the CA724 antibody of the invention is detected, and the following test results are obtained by checking the calibration disk and 2 blood samples with Roche's constant value: and 3.1, taking out the plate coated in advance, the enzyme working solution prepared in advance and the standard pole fixed value sample, recovering the room temperature, and marking the experimental layout.

3.2 sample addition: a25. Mu.L aliquot of blood and 100. Mu.L/well of CA 724-labeled sample, respectively, were added and the enzyme-working solution was an enzyme-labeled sample diluted with an enzyme diluent and incubated at 37℃for 60min. Control and blank wells were left.

3.3 washing the plate: the plates were washed 5 times with PBST and dried.

3.4 luminescence reading: luminescent substrates A and B (A and B are semi-finished products of outsourcing kit manufacturers, the approximate component of A is 0.1% luminol (Tris buffer solution), the approximate component of B is 0.01% hydrogen peroxide (LM buffer solution)), and 50 uL/hole is added after uniform mixing (in-situ preparation); and measuring the luminescence value by using a luminometer within 1-5 min.

3.5, judging the result: according to the data, the difference of the linear activity of the reagent and the marker post is analyzed.

As shown by the detection result, compared with the control antibody, the activity of the CA724 antibody is relatively higher, so that the activity of the CA724 antibody produced by the invention is detected to be qualified.

The heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.1:

INRSSAETRDRKWKGTWQAPRGAQLVGKWHTFQGILSHSKNDSLLSEHISVPLSQYE

CLRGLPKVWDYSCEPPLLIVLLIRYLDRKVSIQLTPHPAIYFNLMLAPFISCLRNICLQQ

GHKKFLFLINRSSKFFSSIYNFFLIMPNFVQLDVDPADLTPVIATIISTSSSHPGFPYVIFH

VPSDPRALLFLFTLVSPNHLEFLCVYGGKWLKFVFLQRLFNIALITILSTQLCSVTFVII

QVS

the light chain amino acid sequence is shown as SEQ ID NO.2:

HPKAMQYTSDSVYFEGFDIPVLCMLCFCFISDGPIRIVPSDLNFSLPASHPGIIYQCFITL

NILTIIRSYSENHRMIKIVFPLLYYCYKSDTIGRSHRSSLDPLANTSEILLARIISCYAFASF

QMGPSEFHKPCMLPPSHNEVDLIGDFWSTTRIGSTFRARYKVVGPQNITLVGPQNITS

DSRTAVTRFTSSC

variable region amino acid sequence of antibody heavy chain SEQ ID No.3:

INRSSAETRDRKWKGTWQAPRGAQLVGKWHTFQGILSHSKNDSLLSEHISVPLSQYE

CLRGLPKVWDYSCEPPLLIVLLIRYLDRKVSIQLTPHPAIYFNL

variable region amino acid sequence of antibody light chain SEQ ID No.4:

HPKAMQYTSDSVYFEGFDIPVLCMLCFCFISDGPIRIVPSDLNFSLPASHPGIIYQCFITL

NILTIIRSYSENHRMIKIVFPLLYYCYKSDTIGRS

CDR1, CDR2, CDR3 of antibody heavy chains

SEQ ID NO.5: CDR1 variable region sequence of antibody heavy chain: GTWQAPRG

SEQ ID NO.6: CDR2 variable region sequences of antibody heavy chain: SLLSEHIS

SEQ ID NO.7: CDR3 variable region sequences of antibody heavy chain: VSIQLTP

CDR1, CDR2, CDR3 of a light chain

SEQ ID NO.8: CDR1 variable region sequence of antibody light chain: AMQYTS

SEQ ID NO.9: CDR2 variable region sequences of antibody light chain: LCFCFISD

SEQ ID NO.10: CDR3 variable region sequences of antibody heavy chain: CYKSDT

Nucleotide sequence of antibody heavy chain SEQ ID No.11:

ATTAATAGGTCATCAGCTGAAACCAGAGACAGAAAATGGAAAGGAACATGGCAGG

CACCCAGAGGTGCCCAGTTGTGAGTGGGTAAGTGGCACACATTTCAGGGCATCCT

ATCCCATTCTAAAAATGATAGTCTCCTCTCAGAGCACATATCTGTACCTCTTTCACA

GTATGAGTGTTTGTAGAGATAGGGCCTTCCCAAAGTGTGGGATTACAGCTGTGAGC

CACCACTATTAATAGTATTGTTATAAATCAGATACCTAGATAGGAAGGTCTCATAGAT

CCAGCTGACTTGACCCCACCCAGCCATATATTTTAATTTGATGTTGGCACCTTTCATA

TCCTGTTTAAGAAATATTTGCCTACAGCAAGGTCACAAAAAATTTTTGTTTTTGATA

AATAGAAGTTCTAAATTCTAATTTAGTTCTATTTATAATTTTTTTCTTTAGTAGATAAT

GCCAAATTTTGTTCAACTCGATGTAGATCCAGCTGACTTGACCCCGGTGATTGCAA

CAATTTAAATTTCCACAAGCAGTTCCCACCCAGGCTTTCCTTATGTTATTTTCCATGT

CCCTTCTGATCCACGAGCTTTATTGTTTTAACTTTTCACATTAGTATCTCCAAATCAC

TTGGAATTCCTGTGTGTTTATGGTGGAAAGTAGTGGTTAAAATTTGTTTTTCTCCAA

CGGCTGTTCAATATAGCACTTATTTAATAAACTATACTTTCCACACAACTCTGCAGT

GTGACCTTTGTTATAATTCAAGTGTCTGT

antibody light chain nucleotide sequence SEQ ID No.12:

CACCCAAAGGCTATGCAATATTAGACAAGTGACTCTGTTTATTTCGAGGGTTTTGAC

ATTCCGGTTTTGTGCATGCTATGCTTTTGCTTCATTTCAGATGGGCCCATCAGAATT

GTCCCTTCTGATCTTAATTTTTCTCTTTAGCCAGCATCCCATCCAGGTATAATTTATC

AATGTTTCATAACTCTCAATATTCTTACTATTATAAGATCTTATTCTGAGAATCATAGG

ATGATCAAAATTGTGTTTCCATTATAGCTCTATTAATAGTATTGTTATAAATCAGATAC

CTAGATAGGAAGGTCTCATAGATCCAGCTGACTTGACCCCCTAGCCAACACCAGCG

AGATCTGACTTTTGGCAAGAATAATCTCATGCTATGCTTTTGCTTCATTTCAGATGG

GCCCATCAGAATTCCATTAGAAACCATGCATGCTCCCACCTTCTCATAATGAAGTTG

ACCTGATAGGAGATTTTTGGTCAACCACAAGGATAGGGTCAACCTTCAGAGCACG

CTATAAAGTGGTTGGTCCTCAAAATATCACTTTGGTTGGTCCTCAAAATATCACTTG

ATCTGACTCTAGGACCGCAGTGACTAGGTTTACATCATAATCATGC

Claims

1. an anti-CA 724 antibody is characterized in that the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2.

2. The anti-CA 724 antibody of claim 1, wherein the variable region of the heavy chain of the antibody has the amino acid sequence shown in SEQ ID No.3 and the variable region of the light chain has the amino acid sequence shown in SEQ ID No. 4.

3. The anti-CA 724 antibody of claim 1, wherein the variable region sequences of CDR1, CDR2 and CDR3 of the heavy chain are shown in SEQ ID nos. 5-7 and the variable region sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID nos. 8-10.

4. A nucleic acid molecule encoding the anti-CA 724 antibody of claim 1.

5. The nucleic acid molecule of claim 5, wherein the nucleotide sequence of the heavy chain of the antibody is set forth in SEQ id No. 11; the nucleotide sequence of the antibody light chain is shown as SEQ ID NO. 12.

6. A vector comprising the nucleic acid molecule of claim 4.

7. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 6.

8. An antibody conjugate, which is obtained by coupling the anti-CA 724 antibody of claim 1 with a carrier or a drug, or by coupling the antibody of claim 1 with a chemical or biological label.

9. A kit for detecting C724 protein, comprising the anti-CA 724 antibody of any one of claims 1-2.

10. Use of the antibody of claim 1 or the nucleic acid molecule of claim 4 for the preparation of a diagnostic agent for diagnosing cancer or a product for detecting CA 724.