CN113072643B

CN113072643B - anti-Glyphacin-3 acid-resistant fully human antibody, immunotoxin thereof, chimeric antigen receptor cell thereof and application

Info

Publication number: CN113072643B
Application number: CN202110303641.4A
Authority: CN
Inventors: 高威; 苟黎明; 陆明; 丁童
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2021-10-15
Anticipated expiration: 2041-03-22
Also published as: CN113072643A

Abstract

The invention relates to an anti-Glypican-3 acid-resistant fully human antibody, immunotoxin thereof, chimeric antigen receptor cell thereof and application thereof. The antibody has at least one of a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR 3. The antibody can be used for preparing immunotoxin or cell, diagnosis kit, medicine or medicine composition; the use of the immunotoxin or cell for the preparation of a medicament or pharmaceutical composition; the medicine or the pharmaceutical composition has the anti-tumor effect on GPC3 positive tumor cells. According to the invention, through a phage display technology and differential screening under different pH conditions, the acid-resistant fully human antibody targeting human GPC3 is obtained, the antibody has strong specificity and high affinity, has relatively stable antigen recognition capability under neutral and acidic conditions, and immunotoxin and cells with the antibody can effectively kill GPC3 positive tumor cells.

Description

anti-Glyphacin-3 acid-resistant fully human antibody, immunotoxin thereof, chimeric antigen receptor cell thereof and application

Technical Field

The invention relates to an anti-Glypican-3 acid-resistant fully human antibody, immunotoxin thereof, chimeric antigen receptor cell thereof and application thereof, belonging to the technical field of biology.

Background

To the best of the inventor's knowledge, liver cancer is the sixth most serious malignant tumor with fatality rate all over the world, and seriously harms human health^[1]. The treatment methods of liver cancer include radical surgical excision, local ablation, chemical arterial embolism, radiotherapy and the like. However, these treatments have generally received little success^[2]. Hepatocellular carcinoma is the most common type of liver cancer, accounts for 83% of all liver cancer cases, and the late-stage survival rate of patients is only 3%^[3]. Glyphican-3 (GPC3) is a member of the heparan sulfate proteoglycan family. A great deal of research finds that GPC3 is specifically and highly expressed in hepatocellular carcinoma, has high sensitivity, is relevant to clinical indexes, and is a hepatocellular carcinoma diagnostic marker and a therapeutic target which are more concerned at present^[4,5]。

The meta-acidic tumor microenvironment is one of the important characteristics of solid tumors^[6]Is also a key factor causing the immune tolerance of solid tumors^[7]. Therefore, the development of acid-resistant antibodies for antibody-based drugs and CAR-T cell therapy is of great interest.

The inventors of the present invention studied anti-tumor-associated fully human antibodies for a long time, and applied 2018.8.27 to an invention patent "anti-GPC 3 fully humanized antibody, chimeric antigen receptor cell and use thereof", and applied 2019.03.22 to an invention patent "anti-Galectin-3 fully humanized single domain antibody and use thereof". The inventors applied this patent with the latest research results.

Disclosure of Invention

The main purposes of the invention are: the problems in the prior art are overcome, the anti-Glyphain-3 acid-resistant fully-humanized antibody, the immunotoxin thereof and the chimeric antigen receptor cell thereof are provided, and the anti-tumor application prospect is achieved.

The technical scheme for solving the technical problems of the invention is as follows:

an anti-Glypican-3 acid-resistant fully human antibody, which comprises a heavy chain and a light chain, and is characterized in that the antibody has at least one of the following technical characteristics:

i. the heavy chain includes a heavy chain CDR1 having the amino acid sequence: GFTFSSYA;

ii. The heavy chain includes a heavy chain CDR2 having the amino acid sequence: ISASGYTT;

iii, the heavy chain comprises heavy chain CDR3 with the amino acid sequence: AKTSSSFDY, respectively;

iv, the light chain comprises a light chain CDR1 having the amino acid sequence: QSISSY;

v, the light chain includes a light chain CDR2 having the amino acid sequence: DAST;

vi, the light chain comprises a light chain CDR3 having the amino acid sequence: QQSTSYPTT are provided.

Preferably, the antibody has at least one of the following technical features:

i. the heavy chain includes a heavy chain CDR1 having the amino acid sequence: GFTFSSYA; the heavy chain includes a heavy chain CDR2 having the amino acid sequence: ISASGYTT; the heavy chain further includes a heavy chain CDR3 having the amino acid sequence: AKTSSSFDY, respectively;

ii. The light chain includes a light chain CDR1 having the amino acid sequence: QSISSY; the light chain includes a light chain CDR2 having the amino acid sequence: DAST; the light chain also includes a light chain CDR3 having the amino acid sequence: QQSTSYPTT are provided.

Preferably, the amino acid sequence of the antibody is shown as SEQ ID NO. 2.

Preferably, the antibody is a Fab fragment, a Fab 'fragment, F (ab)'₂A fragment, a single chain variable fragment (scFv), a disulfide stabilized variable region fragment (dsFv), an IgG molecule, or a bispecific antibody.

Preferably, the antibody has a label including a fluorescent label, an enzymatic label, and a radioactive label.

The present invention also provides:

nucleic acid encoding the anti-Glypican-3 acid-resistant fully human antibody described previously.

Preferably, the nucleic acid sequence is shown as SEQ ID NO. 1.

The present invention also provides:

immunotoxins or cells bearing the anti-Glypican-3 acid-resistant fully human antibody described previously.

Preferably, the immunotoxin comprises anti-Glypican-3 acid-resistant pan-humanized antibody-bacterial toxin, anti-Glypican-3 acid-resistant pan-humanized antibody-bacterial toxin variants; the cells include chimeric antigen receptor T cells, chimeric antigen receptor NK cells, and artificially edited cells.

The present invention also provides:

use of the anti-Glypican-3 acid-resistant fully human antibody described hereinbefore for the preparation of immunotoxins or cells, diagnostic kits, medicaments or pharmaceutical compositions.

Use of the nucleic acid as hereinbefore described for the preparation of an anti-Glypican-3 acid resistant fully human antibody, medicament or pharmaceutical composition.

Use of an immunotoxin or cell as hereinbefore described for the preparation of a medicament or pharmaceutical composition.

Wherein the medicine or the medicine composition has the anti-tumor effect on GPC3 positive tumor cells.

The invention obtains the acid-resistant fully human antibody targeting human GPC3 by differential screening under different pH conditions through a phage display technology, and the antibody has strong specificity and high affinity and has more stable antigen recognition capability under neutral and acidic conditions. The antibody can be used for ELISA, FACS and co-immunoprecipitation detection, and immunotoxins and cells such as chimeric antigen receptor T cells with the antibody can effectively kill GPC3 positive tumor cells. Therefore, the antibody has potential application values in basic research on liver cancer, clinical pathological detection and clinical treatment development.

Drawings

FIG. 1 is a graph showing the binding of enriched phages to the antigen protein by ELISA in example 1 of the present invention.

FIG. 2 is a graph showing the binding of ELISA phage clones to antigen at different pH's in example 1 of the present invention.

FIG. 3 is a graph showing the results of SDS-PAGE detection of purified 42A1IgG and scFv proteins in example 2 of the present invention.

FIG. 4 is a graph showing the results of specific binding detection (ELISA) of the 42A1 antibody to GPC3 protein in example 3 of the present invention.

FIG. 5 is a graph showing the results of specific binding detection (FACS) of the 42A1 antibody on GPC 3-positive cells in example 3 of the present invention.

FIG. 6 is a graph showing the results of detection of specific binding (IP) of the 42A1 antibody to intracellular GPC3 protein in example 3 of the present invention.

FIG. 7 is a graph showing the results of affinity analysis of the 42A1 antibody in example 4 of the present invention.

FIG. 8 is a graph showing the results of analysis of acid resistance of the 42A1 antibody in example 5 of the present invention.

FIG. 9 is a schematic representation of the 42A1 CAR molecule of example 6 of the present invention.

FIG. 10 is a graph showing the results of 42A1 CAR-T cells obtained by lentivirus infection in example 6 of the present invention.

FIG. 11 is a graph showing the results of 42A1 CAR-T cell specific killing of A431-GPC3 cells in example 6 of the present invention.

FIG. 12 is a graph showing the results of SDS-PAGE examining the purified 42A1-PE24 in example 7 of the present invention.

FIG. 13 is a graph showing the results of the WST method for detecting cytotoxicity of 42A1-PE24 in example 7 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the reagents and materials used are commercially available products unless otherwise specified.

Example 1 screening of anti-Glypican-3 acid-resistant fully human antibodies.

Screening was performed using phage display technology at different pH values with human GPC3(Q25-S550) protein as the GPC3 antigen.

The preparation process of the GPC3 antigen is as follows: constructing a pFUSE-hGPC3(Q25-S550) -Fc eukaryotic cell expression vector; the expression vector replaces a signal peptide sequence of GPC3 with a signal peptide sequence of IL-2, and leads GPC3-Fc fusion protein to be secreted into a culture medium; using Lipofectamine^TM2000(Invitrogen, Carlsbad, CA) the expression vector was transfected into HEK293T cells, the supernatant was collected, and GPC3-Fc fusion protein was separated and purified by protein a Argrose (GE Healthcare, Piscataway, NJ) affinity column to obtain GPC3 antigen.

The specific process of phage display is: the plates were coated overnight at 4 ℃ with 10. mu.g/ml purified GPC3-Fc protein; blocking the immune plate with PBS (pH6.0) solution containing 5% skimmed milk powder and 0.1% Tween-20 at room temperature for 1 hour; tomlinson I&J library (Genservice Ltd., Cambridge, UK) at 10¹²Mixing pfu with 5% skimmed milk powder PBS solution 1:1, incubating at room temperature for 2 hours, adding into a sealed immune plate (100 μ l/well), and incubating at room temperature for 1 hour; the immune plate was washed 20 times with 0.1% Tween-20 in PBS (pH6.0); 100 μ l of 100mM Triethylamine elution buffer for 30 min at room temperature; after elution the phage infected TG1 cells in log phase growth, expanded and recovered for the next round of panning. Positive phage enrichment was analyzed by polyclonal phage ELISA after each round of panning.

Polyclonal phage ELISA detection: the plates were coated overnight at 4 ℃ with 5. mu.g/ml purified GPC3-Fc protein and the negative control proteins Frizzled-Fc, BSA and human-IgG; blocking the immune plate with PBS (pH7.4/pH6.0) solution containing 5% skimmed milk powder, 0.1% Tween-20 for 1 hr at room temperature; amplifying and recovering the enriched phage in each round, incubating with 10% skimmed milk powder PBS at room temperature for 2 hours in a ratio of 1:1, adding into a sealed immune plate (100 μ l/well), and incubating at room temperature for 1 hour; the immune plate was washed 5 times with 0.1% Tween-20 in PBS; HRP/Anti-M13 Monoclonal conjugate was treated at a rate of 1: mixing with PBS solution containing 3% skimmed milk powder and 0.05% Tween-20 at ratio of 4000, adding into washed immunoplates (50 μ l/well), and incubating at room temperature for 1 hr; the immune plate was washed 5 times with 0.05% Tween-20 in PBS; adding TMB color developing solution into an immune plate (100 μ l/well), developing at room temperature for 3 min, and adding 0.5M sulfuric acid to stop developing (100 μ l/well); detecting the light absorption value by an enzyme linked immunosorbent assay detector at the wavelength of 450nm and analyzing the affinity of the phage after each round of amplification.

As shown in FIG. 1, the affinity of the enriched phage population for GPC3 antigen was significantly increased, and Frizzled8-hFc and BSA were antigen negative controls.

And (3) performing antigen acid resistance binding analysis on the phage monoclonal enriched in the last round, wherein the specific process is as follows:

monoclonal phage ELISA assay: TG1 cells were infected from the phage pool enriched in the last round and 200 single clones were randomly picked from them, amplified and phage recovered. The plates were coated overnight at 4 ℃ with 5. mu.g/ml purified GPC3-Fc protein and negative control BSA protein; blocking the immune plate with PBS (pH7.4/pH6.0) solution containing 3% skimmed milk powder, 0.05% Tween-20 for 1 hour at room temperature; incubating 200 amplified monoclonal phages with 6% skim milk powder PBS at a ratio of 1:1 for 1 hour at room temperature, adding the phages into an immune plate (50. mu.l/well) coated with GPC3-Fc protein and negative control BSA protein and sealed, and incubating for 1 hour at room temperature; the immune plate was washed 5 times with 0.05% Tween-20 in PBS; HRP/Anti-M13 Monoclonal conjugate was treated at a rate of 1: mixing with PBS solution containing 3% skimmed milk powder and 0.05% Tween-20 at ratio of 4000, adding into washed immunoplates (50 μ l/well), and incubating at room temperature for 1 hr; the immune plate was washed 5 times with 0.05% Tween-20 in PBS; adding TMB color developing solution into an immune plate (100 μ l/well), developing at room temperature for 3 min, and adding 0.5M sulfuric acid to stop developing (100 μ l/well); the absorbance was measured at 450nm using an enzyme linked immunosorbent assay and the binding capacity of the monoclonal phage to the GPC3 protein was analyzed.

The detection result is shown in fig. 2, 4 phage monoclonals which still maintain higher antigen affinity under acidic condition are obtained, the phage monoclonals with positive antigen binding are analyzed, the enriched clone sequences are all the same monoclonal sequence and are named as 42a1, and the DNA sequence in scFv form is SEQ ID NO:1, protein sequence of SEQ ID NO: 2.

SEQ ID NO：1

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTGCTTCTGGTTATACTACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTTCTTCTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTACTTCTTATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA。

SEQ ID NO：2

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISASGYTTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTSSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSTSYPTTFGQGTKVEIK。

in the amino acid sequence, 1-116 is the antibody heavy chain sequence, 132-239 is the antibody light chain sequence, and 117-131 is the connecting peptide sequence. The CDR regions contained therein are as follows: amino acid residues 26-33 (i.e., GFTFSSYA) are heavy chain CDR1, amino acid residues 51-58 (i.e., ISASGYTT) are heavy chain CDR2, and amino acid residues 97-105 (i.e., AKTSSSFDY) are heavy chain CDR 3; amino acid residues 159-164 (i.e., QSISSY) are light chain CDR1, amino acid residues 182-185 (i.e., DAST) are light chain CDR2, and amino acid residues 221-229 (i.e., QQSTSYPTT) are light chain CDR 3.

Furthermore, the antibody format may be selected from Fab fragment, Fab 'fragment, F (ab)'₂A fragment, a single chain variable fragment (scFv), a disulfide stabilized variable region fragment (dsFv), an IgG molecule, or a bispecific antibody. The antibody may optionally have a label including fluorescent, enzymatic, and radioactive labels.

Example 2, 42a1 antibody expression and purification.

In this example, heavy chain and light chain expression vectors of 42a1 antibody were constructed, and then the supernatant was collected after co-transfection in 293T cells, and purified by protein a agarose column separation, and purity of 42a1IgG was determined by SDS-PAGE, as follows.

Molecular cloning: designing a forward primer-VH-F and a reverse primer-VH-R to amplify the heavy chain DNA fragment in the 42A1scFv antibody by means of PCR, and designing a forward primer-VL-F and a reverse primer-VL-R to amplify the light chain DNA fragment in the 42A1scFv by means of PCR; treating the ends of the DNA fragments with restriction enzymes to make the ends of the DNA fragments present sticky ends; the pFUSE backbone and DNA fragment were ligated overnight at 16 ℃ via T4 ligase; the ligation products were transformed into DH 5. alpha. competence, cultured and single clones picked, shaken overnight at 37 ℃; and extracting the plasmid and sequencing to detect whether the plasmid is correctly available.

Expression and purification: 5 million HEK293T cells were seeded in cell culture dishes in DMEM medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, 0.1mg/ml streptomycin and placed in 5% CO₂And culturing in an incubator at 37 ℃. When the cell density reached 60-80%, 5. mu.g of pFUSE-42A1 VH plasmid and 5. mu.g of pFUSE-42A1 VL plasmid were added to 1ml of opti-MEM medium and left to stand for 5 minutes; adding 30 mu g of PEI into opti-MEM culture medium mixed with plasmids, standing for 20 minutes, and replacing HEK293T cells with fresh DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 0.1mg/ml streptomycin during standing; standing for 20 minutes, and adding the mixed opti-MEM culture medium into a HEK293T cell culture dish; the supernatant was recovered every 24 hours, and the 293T cells were replaced with DMEM medium freshly supplemented with 10% fetal bovine serum, 100U/ml penicillin, 0.1mg/ml streptomycin to continue expressing the protein.

Centrifuging the recovered supernatant at 3500rpm and 4 deg.C for 20 min, and vacuum filtering with 0.45 μm microporous filter membrane to further remove debris; the supernatant was purified and purified of 42a1IgG recombinant protein by passing it through a ProteinA Argrose (GE Healthcare, Piscataway, NJ) affinity column.

SDS-PAGE: the concentration of 42A1IgG recombinant protein was measured by BCA method, and 5. mu.g of 42A1IgG protein was subjected to polyacrylamide gel electrophoresis to obtain bands of 42A1IgG recombinant protein in denatured and non-denatured environments, as shown in FIG. 3.

Example 3, 42a1 antibody specificity analysis.

The binding specificity of the 42a1 antibody was analyzed by ELISA assay, flow cytometry (FACS) and IP assay, respectively.

(1) ELISA detection of 42A1 binding specificity to GPC3 antigen protein

The specific process is as follows: immunoplates were coated overnight at 4 ℃ with 5 μ g/ml purified GPC3-his protein and the control protein GPC 5-his; blocking the immune plate for 1 hour at room temperature by using PBS (phosphate buffer solution) containing 3 percent of skimmed milk powder and 0.05 percent of Tween-20; the 42A1IgG recombinant protein was diluted to 5. mu.g/ml with PBS containing 3% skimmed milk powder, 0.05% Tween-20, added to a closed immune plate (50. mu.l/well) and incubated at room temperature for 1 hour; wash the plate 3 times with 0.05% Tween-20 in PBS (340. mu.l/well); the coat anti-human Fcr HRP (Iackson ImmunoResearch) was mixed at a ratio of 1: 2000 ratio, 3% skimmed milk powder, 0.05% Tween-20 in PBS, adding to washed immune plate (50. mu.l/well), and incubating at room temperature for 1 hour; the immune plate was washed 3 times with 0.05% Tween-20 in PBS; adding TMB color developing solution into an immune plate (100 μ l/well), developing at room temperature for 3 min, and adding 0.5M sulfuric acid to stop developing (100 μ l/well); an enzyme linked immunosorbent assay (ELISA) detector is used for detecting an absorbance value at the wavelength of 450nm and analyzing the specificity of the 42A1IgG recombinant protein to GPC3 protein. Note: GPC3-his and GPC5-his fusion proteins were purchased from R & D, infra.

As shown in fig. 4, the results showed that the 42a1 antibody specifically recognized GPC3, but did not recognize the homologous protein GPC5 of GPC 3.

(2) FACS detection of cell binding specificity of 42A1

Culturing and aliquoting A431 and A431-GPC3 cells (50 ten thousand cells/EP tube), centrifuging at 2000rpm for 5 minutes at 4 ℃, discarding the supernatant, resuspending with PBS buffer, centrifuging again, discarding the PBS solution, resuspending the cells with 5. mu.g/ml purified 42A1IgG recombinant protein, 5% BSA in PBS solution, and incubating on ice for 1 hour; centrifugation was carried out at 2000rpm and 4 ℃ for 5 minutes, the supernatant was discarded and resuspended in PBS buffer, and centrifugation was carried out again, the PBS solution was discarded, and Gt F (ab') 2anti-human IgG (γ) R-PE conjugate (life) was added at a ratio of 1: mix 200% in PBS containing 5% BSA and resuspend the cells and incubate on ice for 1 hour; centrifugation at 2000rpm and 4 ℃After 5 minutes, the supernatant was discarded and resuspended in PBS buffer and centrifuged again, the PBS solution was discarded, the cells were resuspended in 0.3ml PBS solution and the PE fluorescence signal was detected by flow cytometry, and the specificity of the 42A1IgG recombinant protein for GPC3 positive cells was analyzed. Note: a431 (human epithelial cancer cell line) was purchased from ATCC (ATCC, Manassas, Va.). In DMEM medium containing 10% fetal calf serum, 100U/ml penicillin and 0.1mg/ml streptomycin, 5% CO was added₂And culturing in an incubator at 37 ℃. GPC3 cDNA was transfected into A431 cells using Lipofectamine TM2000(Invitrogen, Carlsbad, Calif.) and Zeocin selection yielded the GPC3 overexpressing cell line A431-GPC 3.

As shown in fig. 5, the results indicated that the 42a1 antibody specifically recognized a431-GPC3 cells, but not a431 cells.

(3) IP detection of specificity of 42A1 binding to intracellular proteins

Culturing G1 cells and dividing equally (5 million/10 cm dish), digesting and collecting cells and washing the cells once with PBS, centrifuging at 2000rpm for 5 minutes at 4 ℃, discarding supernatant, resuspending with 1ml RIPA buffer (PMSF is added at 1: 100), shaking and mixing the cells, and placing on ice for lysis for 30 minutes; 12000g, 4 degrees C centrifugal 5 minutes, carefully take the supernatant to the new EP tube, utilize Coomassie blue method to determine the protein concentration in the supernatant; each group was prepared by weighing 1mg of total protein, adjusting the total volume to be uniform with RIPA buffer (1: 100 adding PMSF), adding 5. mu.g of 42A1IgG antibody, and incubating overnight on a shaker at 4 ℃; after the incubation is finished, 100 mu l of protein A beads solution is taken from each group, washed once by RIPA buffer (1: 100 adding PMSF), centrifuged for 5 minutes at 2000rpm and 4 ℃, the supernatant is discarded, resuspended by RIPA buffer (1: 100 adding PMSF), 100 mu l of protein A beads are added into each group of protein antibody mixed solution, and incubated for 2 hours in a shaking table at 4 ℃; after the incubation is completed, 1ml of RIPA buffer (1: 100 added PMSF) is added into each group for washing three times, and the mixture is centrifuged at 2000rpm for 5 minutes at 4 ℃; in each group, 40. mu.l of 1X loading buffer was added to precipitated protein A beads (diluted with RIPA buffer containing PMSF at a ratio of 1: 100), and 40. mu.l of the supernatant was subjected to polyacrylamide gel electrophoresis; and simultaneously setting an input group, directly taking 100 mu l of cell lysate, adding 5X loading buffer to carry out polyacrylamide gel electrophoresis, obtaining a band of GPC3 protein and analyzing the specificity of 42A1IgG recombinant protein to GPC3 protein.

As shown in fig. 6, the results indicate that the 42a1 antibody specifically recognizes intracellular GPC3 protein.

Example 4, 42a1 antibody affinity assay.

The affinity of the 42a1 antibody was analyzed by ELISA detection, flow cytometry (FACS), respectively.

(1) ELISA detection 42A1 and GPC3 antigen protein binding affinity

The immunoplates were coated overnight at 4 ℃ using 5. mu.g/ml purified GPC3-Fc protein; blocking the immune plate for 1 hour at room temperature by using PBS (phosphate buffer solution) containing 3 percent of skimmed milk powder and 0.05 percent of Tween-20; diluting 42A1IgG protein to 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125 and 0.009765625 mu g/ml (double dilution) by using PBS solution containing 3% skimmed milk powder and 0.05% Tween-20, adding the diluted solution into a sealed immune plate (50 mu l/hole), and incubating for 1 hour at room temperature; wash the plate 3 times with 0.05% Tween-20 in PBS (340. mu.l/well); coat the coat anti-human Fcr HRP with 1: 2000 ratio, 3% skimmed milk powder, 0.05% Tween-20 in PBS, adding to washed immune plate (50. mu.l/well), and incubating at room temperature for 1 hour; the immune plate was washed 3 times with 0.05% Tween-20 in PBS; adding TMB color developing solution into an immune plate (100 μ l/well), developing at room temperature for 3 min, and adding 0.5M sulfuric acid to stop developing (100 μ l/well); the absorbance was measured at 450nm using an enzyme linked immunosorbent assay and the affinity of the 42A1IgG recombinant protein was analyzed by fitting an affinity curve.

(2) FACS detection of 42A1 cell binding affinity

Culturing and aliquoting A431 and A431-GPC3 cells (50 ten thousand cells/EP tube), centrifuging at 2000rpm for 5 minutes at 4 ℃, discarding the supernatant, resuspending with PBS buffer, centrifuging again, discarding the PBS solution, resuspending the cells with a PBS solution containing 60,40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, 0.009765625. mu.g/ml purified 42A1IgG recombinant protein, 5% BSA, and incubating on ice for 1 hour; centrifugation was carried out at 2000rpm and 4 ℃ for 5 minutes, the supernatant was discarded and resuspended in PBS buffer and centrifuged again, the PBS solution was discarded, and Gt F (ab') 2anti-human IgG (γ) R-PE conj μ gate (life) was added at 1: mix 200% in PBS containing 5% BSA and resuspend the cells and incubate on ice for 1 hour; the cells were centrifuged at 2000rpm at 4 ℃ for 5 minutes, the supernatant was discarded and resuspended in PBS buffer and centrifuged again, the PBS solution was discarded, the cells were resuspended in 0.3ml of PBS solution and the PE fluorescence labeling signal was detected by flow cytometry, and the 42A1IgG recombinant protein affinity curve was fitted and the affinity was calculated.

As shown in FIG. 7, the results revealed that the 42A1 antibody had an affinity of 1.16nM for GPC3-his protein and 21.1nM for A431-GPC3 cells.

Acid resistance analysis of the antibodies of examples 5 and 42A 1.

The affinity of the 42a1 antibody to GPC3-Fc protein was determined by ELISA at different pH. The specific process is as follows:

the immunoplates were coated overnight at 4 ℃ using 5. mu.g/ml purified GPC3-Fc protein; blocking the immune plate for 1 hour at room temperature by using PBS (phosphate buffer solution) containing 3 percent of skimmed milk powder and 0.05 percent of Tween-20; 42A1IgG recombinant protein was diluted to 5. mu.g/ml with PBS solutions (pH7.4/pH6.5/pH5.0/pH4.5) containing 3% skimmed milk powder, 0.05% Tween-20 at different pH values, added to a blocked immune plate (50. mu.l/well) and incubated at room temperature for 1 hour; wash the plate 3 times with 0.05% Tween-20 in PBS (340. mu.l/well); coat the coat anti-human Fcr HRP with 1: 2000 ratio, 3% skimmed milk powder, 0.05% Tween-20 in PBS, adding to washed immune plate (50. mu.l/well), and incubating at room temperature for 1 hour; the immune plate was washed 3 times with 0.05% Tween-20 in PBS; adding TMB color developing solution into an immune plate (100 μ l/well), developing at room temperature for 3 min, and adding 0.5M sulfuric acid to stop developing (100 μ l/well); the absorbance was measured at 450nm using an enzyme linked immunosorbent assay and the affinity of the 42A1IgG recombinant protein was analyzed by fitting an affinity curve.

As shown in FIG. 8, the 42A1 antibody showed 1.17nM affinity for GPC3-Fc protein at pH7.4, 1.18nM affinity for GPC3-Fc protein at pH6.5, 1.35nM affinity for GPC3-Fc protein at pH5.0, and 1.51nM affinity for GPC3-Fc protein at pH 4.5.

Example 6 CAR-T cell construction based on the 42a1 antibody and its anti-tumor effect was evaluated.

1. Construction of 42A1 CAR-T cells.

Second generation 4-1BB type CAR molecules were constructed based on the 42a1 antibody sequence (fig. 9), Human PBMC cells were isolated, T cell activation was induced with Dynabeads Human T-Activator CD3/CD28 and IL-2 targeting, and 42a1 CAR-T cells were constructed using the lentivirus system (fig. 10).

The specific process is as follows:

(1) molecular cloning: design forward primer plvx-CAR-42A1-F and reverse primer plvx-CAR-42A1-R the DNA fragment of 42A1scFv was amplified by PCR method and the ends of the DNA fragment were treated with restriction endonuclease to render them sticky. Then plvx backbone and DNA fragment were ligated by T4 ligase overnight at 16 ℃; the ligation products were transformed into stabl 3 competence, cultured and single clones picked, shaken overnight at 37 ℃; and extracting the plasmid and sequencing to detect whether the plasmid is correctly available.

(2) Virus preparation

5 million HEK293T cells were seeded in 5% CO in a cell culture dish using DMEM medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, 0.1mg/ml streptomycin₂And culturing in an incubator at 37 ℃. When the cell density reached 60-80%, 10. mu.g of plvx-42A1 CAR plasmid, 7.5. mu.g of pSPAX packaging plasmid and 5. mu.g of pMD2G envelope plasmid were mixed into 1ml of opti-MEM medium and allowed to stand at room temperature for 5 minutes; adding 67.5 mu g of PEI into opti-MEM culture medium mixed with plasmid, standing for 20 minutes at room temperature, and replacing 293T cells with fresh DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 0.1mg/ml streptomycin during standing; standing, and adding the mixed opti-MEM culture medium into a 293T cell culture dish; after 10 hours, the 293T cells were supplemented with DMEM medium containing 10% fetal calf serum, 100U/ml penicillin, 0.1mg/ml streptomycin; collecting the first supernatant after the culture medium is replaced for 36 hours, and replacing 293T cells with fresh DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 0.1mg/ml streptomycin; collecting the second supernatant after 36 hours of medium exchange, and ultracentrifuging the collected supernatant for 2 hours (20000rpm, 4 ℃); the supernatant was discarded, and 100. mu.l of 10% fetal bovine serum, 100U/ml penicillin and 0.1mg/ml strand were addedRPMI medium of mycin resuspended viral plaques and virus was lysed overnight at 4 ℃; viral particles were packed in preparation for virus titer and infection of PMBC cells.

HEK293T cells were seeded into 24-well cell culture plates (5 ten thousand cells/well); discarding the culture medium in the pore plate after 24 hours, mixing the subpackaged virus particles with fresh DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 0.1mg/ml streptomycin according to different dilution ratios, and adding the mixture into the pore plate; replacing a fresh DMEM culture medium added with 10% fetal calf serum, 100U/ml penicillin and 0.1mg/ml streptomycin 24 hours after the addition of the virus; HEK293T cells were harvested 48 hours after medium change in the well plates, centrifuged at 2000rpm for 5 minutes at 4 ℃, the supernatant was discarded and resuspended in PBS buffer and centrifuged again, the PBS solution was discarded, the cells were resuspended in 0.3ml PBS solution, the GFP fluorescence signal was detected by flow cytometry and the virus titer was calculated.

(3) T cell activation and infection

PBMC cells were thawed in 24-well plates using RPMI medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, 0.1mg/ml streptomycin, 50U/ml IL-2. Dynabeads Human T-Activator CD3/CD28 was centrifuged at 2000rpm at 4 ℃ for 5 minutes, the supernatant was discarded and resuspended in 1ml PBS buffer and centrifuged again, the PBS solution was discarded, Dynabeads Human T-Activator CD3/CD28 was resuspended in 50. mu.l RPMI medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, 0.1mg/ml streptomycin and added to PBMC cell suspension and mixed well; after 24 hours of activation of PBMC cells by Dynabeads Human T-Activator CD3/CD28, 42A1 CAR lentil virus was added to the cell suspension mixed with polybrene 10. mu.g/ml at an MOI of 15, centrifuged at 1000g and 20 ℃ for 1 hour, and placed in a cell incubator at 37 ℃ for overnight culture;

the cell suspension was centrifuged at 1000rpm for 5 minutes at room temperature, the supernatant was discarded and replaced with RPMI medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, 0.1mg/ml streptomycin, 100U/ml IL-2. Checking the cell state every day after changing the culture medium, and changing the RPMI culture medium which is fresh and added with 10% fetal calf serum, 100U/ml penicillin, 0.1mg/ml streptomycin and 100U/ml IL-2 when the cell density reaches 2million/ml or the culture medium turns yellow; the GFP fluorescence signal was detected by flow cytometry the ninth day after infection with the virus, and after calculating the virus infection efficiency, the activation efficiency and killing efficiency of 42A1 CAR-T cells were measured.

2. The antitumor effect of 42a1 CAR-T cells was evaluated.

To examine the activation of 42a1 CAR-T cells, 42a1 CAR-T cells were co-incubated with a431-GPC3 cells at different effective target ratios, and killing of CAR-T cell target cells was examined after 18 hours.

The specific process is as follows:

2500A 431 cells and A431-GPC3 cells (target cells) and expanded 42A1 CAR-T cells (effector cells) were incubated in 96-well cell culture plates for 18 hours in an effective-to-target ratio of 1.25:1, 2.5:1, 5:1, respectively, and then the supernatant was collected after RPMI medium supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 0.1mg/ml streptomycin; the killing efficiency of 42A1 CAR-T cells was analyzed by detecting the content of Lactate Dehydrogenase (LDH) released from lysed cells in the supernatant using the CytoTox96 Non-Radioactive cytoxicity Assay (promega) kit.

As shown in fig. 11, the experimental results show that the 42a1 CAR-T cells can significantly kill a431-GPC3 cells without affecting the a431 cells, demonstrating that the 42a1 CAR-T cells can effectively kill GPC3 positive tumor cells.

Example 7 immunotoxins were constructed based on the 42a1 antibody and evaluated for anti-tumor effect.

To explore the possibility of using 42A1 as a suitable antibody for the construction of potent immunotoxins, fusion of 42A1scFv to a truncated and deimmunized pseudomonas exotoxin (mPE24) constructed 42A1-mPE24, a novel anti-GPC 3 immunotoxin; purifying by chromatography, and detecting the purity of 42A1-PE24 by SDS-PAGE; cells were then treated with 42A1-PE24 to determine cytotoxicity in vitro.

The specific process is as follows:

(1) construction of 42A1-PE24 immunotoxin: the sequence of 42a1scFv was fused to a truncated and deimmunized pseudomonas exotoxin (PE24) fragment and then cloned into the pMH212 vector. BL21 competent cells were transfected with the 42A1-PE24 plasmid and induced with 1mM IPTG for 1.5 h. The inclusion bodies were collected, washed and lysed with lysozyme to obtain the original extract of the recombinant protein. Recombinant proteins were denatured and remodeled by overnight dialysis. And (3) obtaining the remolded recombinant protein by using an ion exchange column chromatography.

(2) SDS-PAGE: the concentration of the 42A1-PE24 recombinant protein was measured by BCA method, and 5. mu.g of 42A1-PE24 protein was subjected to polyacrylamide gel electrophoresis to obtain bands of the 42A1-PE24 recombinant protein in denatured and non-denatured environments, as shown in FIG. 12.

(3) Cytotoxicity assay of 42a1-PE24 immunotoxin: will be 10 in total⁴Individual cells were seeded into 96-well plates and cultured overnight at 80-90% density, and different concentrations of immunotoxin were added to the wells after 24 h. After 72 hours, OD450nm values were measured using the CCK-8 kit (Beyotime, Songjiang, Shanghai) to analyze cytotoxicity of immunotoxicity. As shown in FIG. 13, the results indicated that 42A1-PE24 inhibited the growth of GPC 3-positive cancer cells, with an IC50 value of 1.99nm, but had no significant effect on GPC 3-negative cells.

The 42A1 antibody of the invention can be used for preparing chimeric antigen receptor T cells, chimeric antigen receptor NK cells or artificially edited cells; in addition to being useful for preparing 42a1 antibody-bacterial toxins, it can also be used for preparing 42a1 antibody-bacterial toxin variants.

In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Reference to the literature

[1].J.Bruix,K.H.Han,G.Gores,J.M.Llovet,V.Mazzaferro Liver cancer:Approaching a personalized care J.Hepatol.,62(2015),pp.S144-S156

[2].Sung,H,Ferlay,J,Siegel,RL,Laversanne,M,Soerjomataram,I,Jemal,A,Bray,F.Global Cancer Statistics 2020:GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36Cancers in 185Countries.CA Cancer J Clin.2020.https://doi.org/10.3322/caac.21660

[3].Farazi P A,Depinho R A.Hepatocellular carcinoma pathogenesis:from genes to environment.[J].Nature Reviews Cancer,2006,6(9):674-687.

[4].Filmus J,Capurro M.Glypican-3:a marker and a therapeutic target in hepatocellular carcinoma[J].Febs Journal,2013,280(10):2471-2476.

[5].Gao W,Kim H,Feng M,et al.Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3for liver cancer therapy[J].Hepatology,2014,60(2):576–58

[6].Karuri A,Dobrowsky E,Tannock I.Selective cellular acidification and toxicity of weak organic acids in an acidic microenvironment[J].BRITISH JOURNAL OF CANCER,1993

[7].Bohn T,Rapp S,Luther N,et al.Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages[J].Nature Immunology,2018

Sequence listing

<110> Nanjing university of medical science

<120> anti-Glypican-3 acid-resistant fully human antibody, immunotoxin thereof, chimeric antigen receptor cell thereof and application

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<170> SIPOSequenceListing 1.0

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<211> 717

<212> DNA

<213> Artificial Sequence

<400> 1

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcatct atttctgctt ctggttatac tacagcttac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaacttct 300

tcttcttttg actactgggg ccagggaacc ctggtcaccg tctcgagcgg tggaggcggt 360

tcaggcggag gtggcagcgg cggtggcggg tcgacggaca tccagatgac ccagtctcca 420

tcctccctgt ctgcatctgt aggagacaga gtcaccatca cttgccgggc aagtcagagc 480

attagcagct atttaaattg gtatcagcag aaaccaggga aagcccctaa gctcctgatc 540

tatgatgcat ccactttgca aagtggggtc ccatcaaggt tcagtggcag tggatctggg 600

acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac ttactactgt 660

caacagtcta cttcttatcc tactacgttc ggccaaggga ccaaggtgga aatcaaa 717

<210> 2

<211> 239

<212> PRT

<213> Artificial Sequence

<400> 2

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ala Ser Gly Tyr Thr Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Ser Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

145 150 155 160

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Asp Ala Ser Thr Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Thr

210 215 220

Ser Tyr Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

225 230 235

Claims

1. An anti-Glypican-3 acid-resistant fully human antibody, which comprises a heavy chain and a light chain, and is characterized in that the antibody has the following all technical characteristics:

2. The anti-Glypican-3 acid-resistant fully human antibody according to claim 1, which is characterized in that the amino acid sequence of the antibody is represented by SEQ ID NO. 2.

3. The anti-Glypican-3 acid-resistant fully human antibody according to claim 1, which is a Fab fragment, a Fab 'fragment, a F (ab)' 2 fragment, a single chain variable fragment scFv, a disulfide-stabilized variable region fragment dsFv, an IgG molecule or a bispecific antibody.

4. The anti-Glypican-3 acid-resistant fully human antibody according to claim 1 or 2, which is characterized in that the antibody has a label comprising a fluorescent label, an enzyme label, and a radioactive label.

5. Nucleic acid encoding the anti-Glypican-3 acid-resistant fully human antibody according to any one of claims 1 to 4.

6. The nucleic acid of claim 5, wherein the nucleic acid sequence is as shown in SEQ ID NO. 1.

7. An immunotoxin or cell bearing the anti-Glypican-3 acid-resistant fully human antibody of any one of claims 1 to 4.

8. The immunotoxin or cell according to claim 7, wherein the immunotoxin comprises anti-Glypican-3 acid-fast pan-derived antibody-bacterial toxin, anti-Glypican-3 acid-fast pan-derived antibody-bacterial toxin variants; the cells are artificially edited cells and comprise chimeric antigen receptor T cells and chimeric antigen receptor NK cells.

9. Use of the anti-Glypican-3 acid-resistant fully human antibody according to any one of claims 1 to 4 for the preparation of an immunotoxin or cell, a diagnostic kit, a medicament or a pharmaceutical composition; the medicine or the medicine composition has an anti-tumor effect on GPC3 positive tumor cells.

10. Use of the nucleic acid of claim 5 or 6 for the preparation of an anti-Glypican-3 acid-resistant fully human antibody, a medicament or a pharmaceutical composition; the medicine or the medicine composition has an anti-tumor effect on GPC3 positive tumor cells.

11. Use of the immunotoxin or cell of claim 7 or 8 for the preparation of a medicament or pharmaceutical composition; the medicine or the medicine composition has an anti-tumor effect on GPC3 positive tumor cells.