CN113527500B

CN113527500B - Fully human monoclonal antibody of glypican 3, chimeric antigen receptor and application thereof

Info

Publication number: CN113527500B
Application number: CN202110807826.9A
Authority: CN
Inventors: 蒋小滔; 刘璇; 吴砂
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2022-09-20
Anticipated expiration: 2041-07-16
Also published as: CN113527500A

Abstract

The invention relates to a fully human monoclonal antibody to glypican 3 comprising heavy chain CDRs 1-CDR3 and light chain CDRs 1-CDR3, wherein the heavy chain CDRs 1-CDR3 comprise amino acid sequences 1-3; the light chain CDRs 1-CDR3 include amino acid sequences 4-6. The invention also relates to a preparation method of the fully human monoclonal antibody of the glypican 3; and a fully human chimeric antigen receptor based on the fully human monoclonal antibody to glypican 3. The invention also relates to the application of the fully human monoclonal antibody of glypican 3 as a medicament in the fields of tumor resistance and liver cancer resistance.

Description

Fully human monoclonal antibody of glypican 3, chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the field of biological genes, and particularly relates to a fully human monoclonal antibody of glypican 3, a chimeric antigen receptor and application thereof.

Background

China is a big liver cancer country, and in 2020, 39 thousands of people die of liver cancer in China, rank the second among all cancers and are second to lung cancer, thus bringing serious threat to the health of people.

Surgery, radiotherapy and chemotherapy are three traditional treatment methods for treating tumors clinically at present. Surgery can resect macroscopic or imagewise visible tumors, but is ineffective for macroscopic or imagewise invisible tumor cells that metastasize through the blood and lymphatic circulation. Moreover, the recurrence rate after the liver cancer operation is very high, and the 5-year survival rate is only about 10 percent. Radiotherapy and chemotherapy both belong to nonspecific therapies, and except killing tumor cells, the traditional Chinese medicine composition has obvious inhibition on normal tissues and cells, especially hematopoietic stem cells with vigorous division, thereby bringing obvious side effects. In 2008, Sorafenib (Sorafenib) was the first approved molecular targeted drug for clinical treatment of liver cancer; in 12 months in 2017, regorafenib (Stivarga) is approved to be marketed in china as a target drug for second-line treatment of liver cancer. However, their actual efficacy is still far from satisfactory and the overall survival time of the patients can only be extended by 2-3 months. Therefore, there is a need to develop new and effective therapeutic methods for liver cancer.

Chimeric antigen receptor T cells (CAR-T) are immunotherapy developed in recent years and have shown a strong antitumor effect in the treatment of hematological tumors. Figure 1 shows a schematic diagram of the process of Chimeric Antigen Receptor (CAR) binding to T cells to form CAR-T. It can be seen that CAR-T combines the advantages of antibodies and T cells, respectively, with the advantages of (1) its extracellular domain is the variable domain of antibodies, and can specifically recognize tumor antigens; (2) the intracellular domains are the T cell activation signaling molecule CD3 ζ and the costimulatory molecule CD28 or 4-1 BB. The us FDA approved first and second CAR-T products in the world, in 8 and 10 months of 2017, one after the other, for the treatment of acute lymphocytic leukemia and diffuse large B-cell lymphoma in children and young adults, respectively. Clinical trials with CAR-T as a therapeutic have seen explosive growth in recent years worldwide. By 6 months 2021, the worldwide registration of CAR-T clinical trials (https:// clinicalterals. gov /) was as many as 1220, of which 448 were developed in China, accounting for 36.72%. 22/6/2021, the official approval by the national drug administration (NMPA) of the double star katter CAR-T product aliskiren injection (i.e., CAR-T cell injection against human CD 19) is on the market, which means that china has come to the cellular immunotherapy for the first year.

Glypican 3(glypican-3, abbreviated as GPC3) is a heparan sulfate glycoprotein and is mainly expressed on the surface of cell membranes. Figure 2 shows a schematic of the structure of glypican 3. The GPC3 core protein comprises 580 amino acids, has a molecular weight of 70kD, and has a Furin protease cleavage site in the middle. The Furin protease cleaves Arg358 and Cys359 to form a 40kD amino-terminal subunit and a 30kD carboxy-terminal subunit, both subunits being linked by a disulfide bond; the carboxyl terminus is anchored to the cell membrane by covalent binding to Glycosylphosphatidylinositol (GPI), with two heparan sulfate side chains located at Cys495 and Cys508 respectively.

GPC3 has high specificity, and can be highly expressed in adult liver cancer, and various children malignant solid tumors such as yolk sac tumor, hepatoblastoma, undifferentiated sarcoma, Wilms' sarcoma, rhabdomyosarcoma, and immature teratoma; it is hardly expressed in normal human tissues, so that it is expected to become one of the ideal targets for immunotherapy of liver cancer and malignant solid tumors in children.

In the existing report, CN109021108A discloses an anti-GPC 3 fully humanized antibody, CAR thereof and application thereof. However, it has drawbacks in that: in the process of screening for antibodies, full-length GPC3 was used, whereas natural full-length GPC3, which has its carboxy terminus close to the cell membrane and is covered with a large amount of polysaccharide (as shown in fig. 2), was difficult to recognize by antibodies, thereby affecting the anti-tumor effect.

Therefore, it is necessary to provide a more optimized technical solution based on the existing reports, so as to overcome the above-mentioned drawbacks.

Disclosure of Invention

Based on the above, the invention discloses a fully human monoclonal antibody of glypican 3, and a glypican 3 fully human CAR prepared by the fully human monoclonal antibody of glypican 3 through molecular biology, synthetic biology and immunology methods. Compared with the prior art, the method has the following advantages: (1) the fully human monoclonal antibody of glypican 3 related by the invention is obtained by using 40KD amino terminal subunit as screening antigen, and the recognition epitope of the monoclonal antibody is the amino terminal subunit of GPC 3. And the amino-terminal subunit is positioned at the outer side of the carboxyl-terminal subunit, so that the amino-terminal subunit is more easily recognized by immune cells, and the tumor cells are better mediated to be killed. (2) The fully human monoclonal antibody has the affinity of 0.667nM for glypican 3, which is higher than that of the antibodies disclosed in the prior art, and has stronger antigen recognition capability.

The term "monoclonal antibody" as referred to herein refers to a highly homogeneous antibody produced by a single B cell clone and directed against only a particular epitope of an antigen.

The term "chimeric antigen receptor" as referred to herein, refers to an artificial receptor that has been genetically engineered to confer a novel ability to target a particular antigen to immune cells, such as T lymphocytes. These receptors are chimeric in that they incorporate into one receptor a domain in the antibody that specifically recognizes the antigen and a T cell activation domain.

The term "heavy chain CDR", i.e., complementarity determining region of heavy chain, as referred to herein, refers to the core conserved structure of the heavy chain variable region of an antibody, and is the key region determining antigen binding of an antibody, including CDR1, CDR2 and CDR 3.

The term "light chain CDR", i.e., complementarity determining region of the light chain, as referred to herein, refers to the core conserved structure of the antibody light chain variable region, and is the key region determining antigen binding of the antibody, including CDR1, CDR2 and CDR 3.

It is an object of the present invention to provide a fully human monoclonal antibody to glypican 3.

A fully human monoclonal antibody to glypican 3 comprising heavy chain CDRs 1-CDR3 and light chain CDRs 1-CDR3,

wherein,

the heavy chain CDRs 1-CDR3 comprise amino acid sequences 1-3;

the light chain CDRs 1-CDR3 include amino acid sequences 4-6;

the amino acid sequence 1-6 is shown as SEQ ID No.1-SEQ ID No. 6.

Further, the amino acid sequence of the heavy chain is shown as SEQ ID No. 7.

Further, the amino acid sequence of the light chain is shown as SEQ ID No. 8.

Further, the fully human monoclonal antibody to glypican 3 is selected from the group consisting of a single domain antibody, a Fab fragment antibody, and F (ab') ₂ A fragment antibody, a single chain variable fragment antibody, or a bispecific antibody.

Another object of the present invention is to provide a method for preparing the fully human monoclonal antibody against glypican 3, which comprises the steps of:

s1, connecting the amino terminal of glypican 3 with an amino acid sequence to obtain a modified glypican 3 antigen,

s2, carrying out phage display technology screening experiment on the modified glypican 3 antigen to obtain the fully human monoclonal antibody of glypican 3.

Further, the amino acid sequence of the modified glypican 3 antigen is shown as SEQ ID No. 14.

Further, the screening times are 1-5 times.

Another object of the present invention is to provide a fully human CAR prepared from the fully human monoclonal antibody against glypican 3.

Further, the fully human CAR comprises an amino acid sequence of a CD8 hinge region, namely an amino acid sequence shown in SEQ ID No. 9.

Further, the fully human CAR comprises the amino acid sequence of the transmembrane region of human CD28, namely the amino acid sequence shown in SEQ ID No. 10.

Further, the fully human CAR comprises the amino acid sequence of the intracellular domain of human 4-1BB, i.e., the amino acid sequence shown in SEQ ID No. 11.

Further, the fully human CAR comprises the amino acid sequence of the tyrosine activation motif of the intracellular domain immunoreceptor of human CD3 ζ, namely the amino acid sequence shown in SEQ ID No. 12;

preferably, the fully human CAR has an amino acid sequence shown as SEQ ID No. 13.

Another object of the present invention is to provide the use of the fully human monoclonal antibody against glypican 3 as an antitumor agent.

The invention also aims to provide the application of the fully human monoclonal antibody of glypican 3 as a medicament for resisting liver cancer.

The invention has the following beneficial effects:

(1) the amino terminal of the glypican 3 is taken as a screening antigen, and the obtained fully human monoclonal antibody of the glypican 3 can better identify tumor cells by screening; the CAR-T cell of the fully human monoclonal antibody based on the glypican 3 has better effect of killing tumor cells;

(2) the fully human monoclonal antibody of glypican 3 related by the invention has the affinity of 0.667nM for glypican 3, is higher than the antibodies disclosed in the prior art, and has stronger antigen recognition capability.

Drawings

Figure 1 shows a schematic diagram of the process of CAR binding to T cells to form CAR-T.

Figure 2 shows a schematic of the structure of glypican 3.

FIG. 3 shows the schematic diagram of construction of glypican 3 amino-terminal subunit prokaryotic expression vectors.

FIG. 4 shows SDS-PAGE detection of glypican 3 amino-terminal subunit expression.

Wherein: first column M1: protein labeling; second column NC: bacterial lysate not induced with IPTG; third column 1: after IPTG was added, the bacterial lysate was induced for 16h at 15 ℃; fourth column 2: after IPTG was added, the bacterial lysate was induced at 37 ℃ for 4 h; fifth column NC 1: the supernatant after centrifugation of bacterial lysate not induced by IPTG addition; sixth column 3: after IPTG is added, the bacterial lysate is induced for 16h at 15 ℃ to obtain a supernatant after centrifugation; seventh column 4: after IPTG is added, the supernatant fluid after centrifugation of bacterial lysate is induced for 4h at 37 ℃; eighth column NC 2: a precipitate from centrifugation of a bacterial lysate not induced with IPTG; ninth column 5: adding IPTG, and inducing the bacterial lysate for 16h at 15 ℃ to obtain a centrifuged precipitate; tenth column 6: after IPTG was added, the centrifuged pellet of bacterial lysate was induced at 37 ℃ for 4 h.

FIG. 5 shows SDS-PAGE detection of glypican 3 amino-terminal subunit expression.

Wherein, the first column M: protein labeling; second column 1: adding IPTG, and inducing the bacterial lysate for 16h at 15 ℃ to obtain a centrifuged precipitate; third column 2: adding IPTG, and inducing the bacterial lysate for 4h at 37 ℃ to obtain a centrifuged precipitate; the primary antibody is a mouse anti-His monoclonal antibody, and the secondary antibody is an HRP-labeled anti-mouse antibody.

FIG. 6 shows ELISA detection of phage binding to the amino-terminal subunit of glypican 3 after three rounds of screening.

FIG. 7 shows the results of the phage positive clone ELISA assay.

FIG. 8 shows jxt-mAb1 (subclass IgG1) detected by SDS-PAGE.

Wherein the first column M1: protein labeling; second column R: under reducing conditions; third column NR: under non-reducing conditions.

FIG. 9 shows the ELISA detection results for recombinantly expressed jxt-mAb 1.

Figure 10 shows a schematic diagram of a glypican 3 fully human CAR structure.

Figure 11 shows the expression of CAR molecules in T cells.

FIG. 12 shows the binding of the immunofluorescent tested jxt-mAb1 to the human liver cancer cell line HepG 2.

FIG. 13 shows the results of the jxt-mAb1 immunoblot detection.

FIG. 14 shows that jxt-mAb1 was used for immunohistochemical detection of normal tissues in liver cancer and paracancer.

Figure 15 shows a schematic of cytokine secretion after phosphatidylinositosan 3-based fully human CAR-T co-culture with tumor cells.

Figure 16 shows a schematic of glypican 3-based fully human CAR-T killing of tumor cells.

FIG. 17 shows a schematic representation of the killing of the hepatoma cell line hepG2 by fully human CAR-T based on glypican 3.

Detailed Description

In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. The starting materials, reactions and work-up procedures which are given in the examples are, unless otherwise stated, those which are customary on the market and are known to the person skilled in the art.

Example 1

Preparation of modified glypican 3 antigen-glypican 3 amino-terminal subunit

The preparation of the amino-terminal subunit of glypican 3 comprises the following steps:

s1, referring to an amino acid sequence (https:// www.uniprot.org/uniprot/P51654) of uniprot website human phosphatidylinositol proteoglycan 3, wherein the amino terminal sequence is Met1-Arg 358; to facilitate purification of the protein, 6 His amino acids were added at the carboxy terminus to form amino acid sequence 14. The amino acid sequence 14 is shown as SEQ ID No. 14;

s2, optimizing DNA codon of the amino acid sequence 14 according to prokaryotic expression, and adding Shine-Dalgarno sequence (namely amino acid sequence 15, SEQ ID No. 15: AGGAGGACAGCT) before an initiation codon to improve translation of the transcribed gene. FIG. 3 shows a schematic diagram of construction of a prokaryotic expression vector of amino-terminal subunit of glypican 3, wherein the prokaryotic expression vector is pET-30a (+) (Sigma-Aldrich, # 69909); the positive clone is identified by double enzyme digestion or Polymerase Chain Reaction (PCR) of restriction enzyme NdeI (NEB, # R0111S) and HindIII ((NEB, # R3104), prokaryotic expression plasmid and phosphatidylinositolglycan 3 amino-terminal subunit cloning vector synthesized by the company, after gel cutting and recovery, T4 ligase 16 ℃ is connected overnight, BL21 competent cells (Takara, #9126) are transformed, and double enzyme digestion or Polymerase Chain Reaction (PCR) is carried out;

s3, selecting BL21 clone identified as positive by double enzyme digestion, and inoculating the clone to an LB culture medium containing 50 mu g/ml kanamycin; a bacterial shaker, culturing at 37 ℃ and 200 rpm; detecting OD600, adding IPTG (Shanghai Biotech, # B541007) with final concentration of 0.5mM when OD600 reaches 0.6-0.8, and culturing at 37 deg.C for 4h (or 15 deg.C for 16 h); after completion of the culture, the bacteria were collected by centrifugation, added with lysis buffer (50mM Tris (hydroxymethyl) aminomethane (Tris),150mM NaCl, 5% glycerol, pH 8.0), and subjected to ultrasonic lysis for 1 min;

SDS-PAGE detection of protein expression: mixing the bacterial lysate with the sample buffer solution, boiling at 100 ℃ for 10min, centrifuging at 10000rpm for 5min, and absorbing the supernatant for electrophoresis; after completion of the electrophoresis, coomassie brilliant blue staining was performed.

FIG. 4 shows SDS-PAGE detection of glypican 3 amino-terminal subunit expression, showing that BL21 transformed with glypican 3 amino-terminal subunit plasmid produced a protein of about 45kD under IPTG induction, consistent with the molecular weight of the protein of interest;

s4, confirming the expression of the protein by western blot (Westernblot):

proteins from SDS-PAGE were electroporated onto PVDF membrane, and then detected by adding anti-His protein antibody (GenScript, # A00186).

FIG. 5 shows SDS-PAGE detection of glypican 3 amino-terminal subunit expression, showing that a distinct band is detectable at 45kD, consistent with the molecular weight of the target protein, and a small amount of dimer is detectable near 90 kD;

s5, purifying the amino terminal subunit of glypican 3:

after the bacteria are subjected to ultrasonic lysis, centrifuging, and washing the precipitate twice by using 4M urea; then dissolving the inclusion body by 8M urea; protein dialysis renaturation, renaturation buffer solution is: 50mM Tris,150mM NaCl,0.5M L-arginine, 10% glycerol, pH 8.0;

after renaturation of the protein, the protein is purified by a nickel column, and the purity of the purified protein is measured by SDS-PAGE; by Pierce ^TM High capacity endotoxin removal spin columns (cat # 88276) remove endotoxin; filtering with 0.22 μm filter membrane for sterilization, and subpackaging at-80 deg.C for freezing.

Example 2

Screening of fully human monoclonal antibodies against the amino-terminal subunit of glypican 3

The modified glypican 3 antigen (glypican 3 amino-terminal subunit) prepared in the previous step is screened by adopting a phage display technology, and three rounds of screening are carried out.

Human Single chain antibody (scFv) phage display library from Creative-Biolabs (HuScL-2: human Single chain antibody library, library size 1.42X 10 ⁹ )

First round screening: the antigen coating concentration is 20 mug/ml;

and (3) second screening: the antigen coating concentration is 5 mug/ml;

and (3) third screening: the antigen coating concentration was 1. mu.g/ml.

The specific processes of the three rounds of screening are as follows: diluting glypican 3 amino-terminal subunit antigen with PBS (phosphate buffer solution) with the pH value of 7.4, adding a 96-hole enzyme label plate, 100 mu l/hole, and standing overnight at 4 ℃; blocking the ELISA plate for 1h at 37 ℃ by using a PBS solution containing 5% of skimmed milk powder and 0.1% of Tween-20; 0.1% PBST diluted phage pool (final concentration 1X 10) was added ¹² pfu/ml) was incubated at room temperature for 1 h; wash plate 10 times with 0.1% PBST; mu.l of 0.2M Gly-HCl50, pH 2.0, was then eluted at room temperature for 10min and then neutralized with 15. mu.l of 1M Tris-HCl pH 8.0. The neutralized phage were infected with TG1 cells in the logarithmic growth phase, expanded and recovered for the next round of screening. After screening, positive phage enrichment was analyzed by ELISA.

Binding assay of phage to glypican 3 amino-terminal subunit:

antigen coated at 5 μ g/ml, and BSA negative control; carrying out temperature blocking on a 0.25% casein blocking solution for 1 h; amplifying and recovering the enriched phage in each round, diluting the amplified phage by one time with 0.1% PBST, adding the diluted phage into a sealed immune plate (100 mu.l/hole), and incubating the phage for 1h at room temperature; 0.1% PBST wash 6 times; adding HRP-anti-M13 monoclonal antibody (1:5000), incubating at 37 deg.C for 30min, washing the plate 6 times with 0.2% PBST, developing color of TMB, and washing with 2M H ₂ SO ₄ The OD450nm was terminated, tested, and the affinity of the phage was analyzed after each round of amplification.

Figure 6 shows ELISA detection of phage binding to the amino-terminal subunit of glypican 3 after three rounds of screening, demonstrating that after three rounds of enrichment, the affinity of the phage population for the amino-terminal subunit of glypican 3 was significantly increased, without binding to the negative control BSA.

Third round of binding assay of enriched phage monoclonals to glypican 3 amino-terminal subunit:

after the third round of selection, 5. mu.l of the eluate was mixed with 100. mu.l of TG1 cells in the middle stage of growth, added to the top agar and inverted, from which 50 single clones were randomly picked, amplified and recoveredA bacteriophage. Antigen coated at 5 μ g/ml, and BSA negative control; blocking with 0.25% casein blocking solution at room temperature for 1 h; the 50 phage single clones preserved were diluted one time with 0.1% PBST, added to a closed immunoplate (100. mu.l/well), and incubated at room temperature for 1 h; wash 6 times with 0.1% PBST; adding HRP-anti-M13 monoclonal antibody (1:5000), incubating at 37 deg.C for 30min, washing the plate with 0.2% PBST for 6 times, developing color with TMB, adding 2M H ₂ SO ₄ The reaction was stopped, tested for OD450nm, and monoclonal phages were analyzed for their ability to bind to the amino-terminal subunit of glypican 3.

FIG. 7 shows the result of ELISA detection of phage positive clones, and 16 antigens were found to bind to the positive clones, with a positive rate of 32%.

Amplifying 16 positive phage clones in the last step, extracting phage DNA, identifying by agarose gel electrophoresis, and sequencing by a company; the gene sequences were analyzed, and 9 out of 16 clones (1, 3, 13, 19, 31, 32, 39, 47 and 49, respectively) were found to have identical sequences, and were designated jxt-mAb1, which is a fully human monoclonal antibody to glypican 3. The DNA sequence of the scFv form is SEQ ID No.16, and the amino acid sequence is SEQ ID No. 17. In the amino acid sequence of jxt-mAb1, 1-118 are the antibody heavy chain sequence SEQ ID No. 7; 134-241 is the antibody light chain sequence SEQ ID No. 8; 119-133 is a linker peptide sequence; 26-35 are SEQ ID No. 1; 50-66 are SEQ ID No. 2; 97-107 is SEQ ID No. 3; 157-167 is SEQ ID No. 4; 183-189 is SEQ ID No. 5; 222-230 is SEQ ID No. 6.

Example 3

Expression and identification of fully human monoclonal antibody jxt-mAb1 against the amino-terminal subunit of glypican 3

S1. expression of jxt-mAb 1: the amino acid sequence of the heavy chain (SEQ ID No.7) and the amino acid sequence of the light chain (SEQ ID No.8) based on jxt-mAb1 were tested to express and purify the full-length human IgG1 antibody. FIG. 8 shows the results of the SDS-PAGE detection of jxt-mAb1 (subclass IgG1), indicating successful expression of jxt-mAb1 of purified IgG1 class, with a purity of 95% or more.

S2. binding assay for jxt-mAb1 to anti-glypican 3: commercial human full-length glypican 3(Acrobiosystems, cat # GP3-H52H4) was coated at a concentration of 5. mu.g/ml, control 5. mu.g/ml BSA; 0.1% PBST containing 5% skimmed milk powder, and sealing at room temperature for 1 h; diluting jxt-mAb1 with 5% skimmed milk powder in 0.1% PBST, adding into sealed immunoplate, and incubating at room temperature for 1 hr; washing with PBST, adding HRP-labeled goat anti-human IgG, and incubating at room temperature for 0.5 h; washing with PBST for 5 times, adding TMB, developing at room temperature in dark place for 15min, and adding 2M sulfuric acid to stop developing; and finally, detecting the light absorption value by using an enzyme-linked immunosorbent assay detector under the wavelength of 450 nm.

FIG. 9 shows the results of the ELISA assay of jxt-mAb1, indicating that jxt-mAb1 specifically recognizes GPC3, but does not recognize the unrelated protein BSA. The kd value was calculated to be 0.667nM after curve fitting by the Graphpadprism software.

Example 4

Construction of fully human CAR-T based on glypican 3

S1, constructing a second generation 4-1BB type CAR molecule comprising the amino acid sequence of human CD8 hinge region (SEQ ID No.9), the amino acid sequence of human CD28 transmembrane region (SEQ ID No.10), the amino acid sequence of human 4-1BB intracellular region (SEQ ID No.11), and the amino acid sequence of human CD3 zeta intracellular region immunoreceptor tyrosine activation motif (SEQ ID No.12), based on the sequence of glypican 3 fully human monoclonal antibody jxt-mAb1 in example 3; the complete structure is shown in FIG. 10, and the amino acid sequence is shown in SEQ ID No. 13;

s2, construction of a lentiviral vector: the amino acid sequence of 4-1BB type CAR molecule is subjected to codon optimization aiming at human source, and the titer of the finally prepared lentivirus is 2.01 multiplied by 10 ⁸ IFU/ml；

S3, separating PBMC of healthy adults by a density gradient centrifugation method, separating total T cells by a magnetic bead (stemcell) negative selection method:

culturing in RPMI1640 medium containing 300U/ml recombinant human IL-2 and 10% fetal calf serum; anti-CD 3/CD28(stemcell, ImmunoCult) ^TM HumanCD3/CD28TCellactivator, cat # 10971) activates T cells for 48 h; then lentivirus (MOI 20) was added followed by polybrene to a final concentration of 8. mu.g/ml, 1800rpm, and centrifugation at 32 ℃The heart is kept for 2 hours, and the mixture is put into a cell culture box at 37 ℃ for overnight culture; adding lentivirus (MOI 15), centrifuging at 1800rpm at 32 deg.C for 2h, performing second transfection, culturing in 37 deg.C cell culture box, and culturing; alternate day fluid change (RPMI 1640 medium containing 300U/ml recombinant human IL-2, 10% fetal bovine serum); finally, on day 5 post-virus infection, T cells were harvested and CAR expression was detected by flow cytometry (detection antibody was FITC-labeled goat anti-mouse IgGF (ab') 2).

FIG. 11 shows expression of 4-1BB type CAR molecules in T cells, indicating successful expression of 4-1BB type CAR molecules in T cells at day 5 after lentivirus transfection with a transfection efficiency of 23.79%.

Test example 1

Glypican 3 fully human monoclonal antibody jxt-mAb1 specifically binds to human hepatoma cells

Binding to human hepatoma cell line HepG 2: HepG2 cells were purchased from Guangzhou Securio organisms (catalog No.: CC 0101). DMEM complete medium containing 10% fetal bovine serum was placed in 5% CO ₂ Culturing overnight in an incubator at 37 ℃; the next day, the medium was decanted, jxt-mAb1 (diluted with PBS containing 1% BSA) was added to a final concentration of 2. mu.g/ml, and the reaction was carried out at room temperature for 15 min; washing with PBS (containing 1% BSA) for 3 times, adding FITC-goat anti-human IgG, and reacting at room temperature for 15 min; after washing 3 times with PBS (containing 1% BSA), the results were observed by inverted fluorescence microscopy.

FIG. 12 shows immunofluorescence detection of jxt-mAb1 binding to human liver cancer cell line HepG2, indicating that jxt-mAb1 binds to human liver cancer cell line HepG 2.

Immunoblot detection jxt-mAb1 recognition of native glypican 3

Collecting hepG2 cells (cell density 70-80%) in a T75 culture bottle, centrifuging at 4000rpm for 5min, removing supernatant, washing with PBS for three times, and freezing the cell precipitate at-80 deg.C; after 24h, the cell pellet was removed, the cell pellet was shaken for 1min and then dispersed, 30. mu.l of RIPA cell lysis buffer (thermolisher, cat # 89900) containing PMSF was added, and shaking was carried out at 4 ℃ for 0.5 h; shaking for 5min, centrifuging at 13000rpm for 10min, and taking supernatant to another ep tube; the Bradford assay measures the concentration of extracted whole cell protein (bioradprotienkit); SDS-PAGE was performed to give 50ug protein per well; after electrophoresis, transferring the membrane, and sealing with 0.2% PBST containing 5% skimmed milk powder for 1 h; add 5. mu.g/ml jxt-mAb1 (0.2% diluted in PBST) and react for 1h on a shaker at room temperature; washing for 6 times, and adding HRP (horse radish peroxidase) -labeled goat anti-human IgG; after 6 washes, a chemiluminescent chromogenic solution (ThermoFisher scientific, #32209) was added for 1min at room temperature and observed on a 6200ECL chemiluminescent imaging system.

FIG. 13 shows the results of the jxt-mAb1 immunoblot assay, indicating that jxt-mAb1 can bind to the protein of human hepatoma cell line HepG2, with two binding bands: one at 70kD and one at 40kD, corresponding to the full length and amino terminal subunit size of GPC 3; moreover, this binding is specific, since it does not bind to the extracted proteins of 293T cells.

Immunohistochemical detection of jxt-mAb1 binding to patient-derived liver cancer tissue

The method comprises the following steps: placing the paraffin section of the liver cancer tissue in a thermostat at 60 ℃ and baking for 120 min; dewaxed and hydrated sequentially with the following solvents (hydration time indicated in parentheses): xylene (10min) → absolute ethanol (5min × 2 times) → 95% ethanol (5min × 2) → 90% (5min) → 85% ethanol (5min) → 80% ethanol (5min) → 75% ethanol (5 min); washing with distilled water for 2 times, adding 3% H ₂ O ₂ Incubating at room temperature for 30 min; washing with distilled water for 2 times; antigen retrieval: high-pressure repair or microwave repair; washing with PBS for 3 times, each for 5min, and sealing for 30 min; 5. mu.g/ml of jxt-mAb1 (diluted in 1% BSA in PBS) was added and incubated at 37 ℃ for 2 h; washing with PBS for 5min for 3 times; adding a goat anti-mouse secondary antibody marked by HRP, and incubating for 45min at 37 ℃; after washing with PBS, DAB color development, hematoxylin counterstaining, dehydration. The resulting transparent, neutral resin mounting sheet was observed under a microscope.

FIG. 14 shows jxt-mAb1 immunohistochemical detection of liver cancer and paracancerous normal tissues, indicating that jxt-mAb1 binds to patient-derived liver cancer cells, but not paracancerous normal tissues, with strong specificity.

Test example 2

Antitumor effect of fully human CAR-T based on glypican 3

S1, adding the fully human CAR-T (effector cell) based on glypican 3 and the human hepatoma cell line HepG2 cell (target cell) in different ratios (1:1, 3:1 and 9:1) into a 96-well culture plate for co-culture;

s2, after 20h of culture, collecting supernatant, and detecting the concentrations of IL-2 and IFN-gamma in the supernatant by ELISA.

Figure 15 shows a schematic of cytokine secretion after co-culture of glypican 3-based fully human CAR-T with tumor cells, demonstrating that glypican 3-based CAR-T cells were co-cultured with tumors to produce large amounts of IL-2 and IFN- γ; and neither IFN-. gamma.nor IL-2 was produced after co-culture with 293T cells.

S3, detecting the killing rate of effector cells to target cells:

detection was performed by LDH method (Promega, CytoTox96 nonradioactive cytotoxicity assay, cat # G1780).

FIG. 16 shows a schematic of glypican 3-based fully human CAR-T killing of tumor cells, demonstrating that glypican 3-based fully human CAR-T can specifically kill GPC3 positive human hepatoma cell line HepG2 cells, but not GPC3 negative 293T cells; moreover, the killing is dose-dependent, i.e., the higher the effective target ratio, the greater the killing rate.

Adding propidium iodide (Thermofisiher, ReadyFlow) to the cells ^TM Reagent, cargo number: r37169) under an inverted fluorescent microscope.

FIG. 17 shows a schematic of glypican 3-based fully human CAR-T killing of the liver cancer cell line hepG2, indicating that glypican 3-based CAR-T can specifically kill GPC3 positive human liver cancer cell line HepG2 cells; whereas Mock-T cells that were not transfected with CAR were unable to kill the target cells.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> Small Tao

<120> fully human monoclonal antibody of glypican 3, chimeric antigen receptor and application thereof

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Artificial sequence ()

<400> 1

Gly Tyr Ser Phe Thr Ser Tyr Trp Met His

1 5 10

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence ()

<400> 2

Asn Ile Tyr Pro Gly Gly Ala Asn Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 3

<211> 11

<212> PRT

<213> Artificial sequence ()

<400> 3

Ala Arg Gly Ser Tyr Leu Tyr Gly Met Asp Tyr

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence ()

<400> 4

Arg Ala Ser Gln Gly Ile Ser Val Trp Leu Ser

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence ()

<400> 5

Lys Ala Ser Asn Leu His Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence ()

<400> 6

Gln Gln Gly Asn Ser Tyr Pro Trp Thr

1 5

<210> 7

<211> 118

<212> PRT

<213> Artificial sequence ()

<400> 7

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Gly Ala Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Ser Tyr Leu Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 8

<211> 108

<212> PRT

<213> Artificial sequence ()

<400> 8

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Lys Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Gly Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys Arg

100 105

<210> 9

<211> 45

<212> PRT

<213> Artificial sequence ()

<400> 9

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 10

<211> 27

<212> PRT

<213> Artificial sequence ()

<400> 10

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 11

<211> 42

<212> PRT

<213> Artificial sequence ()

<400> 11

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 12

<211> 48

<212> PRT

<213> Artificial sequence ()

<400> 12

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

<210> 13

<211> 424

<212> PRT

<213> Artificial sequence ()

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Val

20 25 30

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

35 40 45

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

50 55 60

Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Gln Ser Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Gly

100 105 110

Asn Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys

115 120 125

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

145 150 155 160

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

165 170 175

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

180 185 190

Gly Asn Ile Tyr Pro Gly Gly Ala Asn Thr Asn Tyr Ala Gln Lys Phe

195 200 205

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

210 215 220

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

225 230 235 240

Ala Arg Gly Ser Tyr Leu Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

245 250 255

Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys

305 310 315 320

Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys

420

<210> 14

<211> 364

<212> PRT

<213> Artificial sequence ()

<400> 14

Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu

1 5 10 15

Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp

20 25 30

Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly

35 40 45

Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val

50 55 60

Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys

65 70 75 80

Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala

85 90 95

Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln

100 105 110

Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala

115 120 125

Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe

130 135 140

Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp

145 150 155 160

Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro

165 170 175

Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu

180 185 190

Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe

195 200 205

Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln

210 215 220

Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile

225 230 235 240

Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu

245 250 255

Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys

260 265 270

Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly

275 280 285

Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu

290 295 300

Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu

305 310 315 320

Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys

325 330 335

Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser

340 345 350

Gln Gln Arg Gln Tyr Arg His His His His His His

355 360

<210> 15

<211> 12

<212> DNA

<213> Artificial sequence ()

<400> 15

aggaggacag ct 12

<210> 16

<211> 723

<212> DNA

<213> Artificial sequence ()

<400> 16

caagttcaac tggttcagtc aggtgccgaa gttaaaaaac ccggtgccag cgttaaagta 60

tcttgcaagg cctctggtta ttcttttacg tcatactgga tgcattgggt acgacaggcc 120

ccaggtcaag gcctggagtg gatggggaat atctacccag gcggcgcaaa cacgaattat 180

gctcagaagt tccaaggcag agtcaccatg acaagagaca cgtccacttc caccgtgtat 240

atggaactta gctcattgcg ctctgaggac acggcggtgt attattgcgc gcgaggcagt 300

tacctttatg gtatggacta ttggggtcag ggaacccttg taaccgtttc cagcggagga 360

gggggtagtg gcgggggagg ttcaggcggt gggggaagcg agattgtgtt gacgcaatca 420

ccatcatctg taagtgcgag cgtcggggac agagtaacta ttacatgccg agcgagccag 480

ggcatctcag tatggcttag ttggtaccaa caaaagcctg ggaaagcgcc caagctgctt 540

atctacaaag ccagtaatct tcacagtggc gtgccagcgc ggttttccgg ttcaggcagt 600

ggtacagact ttaccttgac aataagtggg ctccaatcag aggattttgc ttcatattat 660

tgccaacaag gtaattccta tccgtggact tttggaggtg gaaccaaact ggaggtaaag 720

cgg 723

<210> 17

<211> 241

<212> PRT

<213> Artificial sequence ()

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Gly Ala Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Ser Tyr Leu Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Val

130 135 140

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

145 150 155 160

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

165 170 175

Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Ser Gly Val Pro

180 185 190

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

195 200 205

Ser Gly Leu Gln Ser Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Gly

210 215 220

Asn Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Lys

225 230 235 240

Arg

Claims

1. A fully human monoclonal antibody to glypican 3, characterized in that the fully human monoclonal antibody to glypican 3 comprises heavy chain CDRs 1-CDR3 and light chain CDRs 1-CDR3,

wherein,

the heavy chain CDR1-CDR3 are respectively shown as amino acid sequences 1-3;

the light chain CDRs 1-CDR3 are shown in amino acid sequences 4-6, respectively;

the amino acid sequences 1-6 are shown as SEQ ID number 1-SEQ ID number 6.

2. The fully human monoclonal antibody to glypican 3 according to claim 1, characterized in that the amino acid sequence of the heavy chain is shown as SEQ ID number 7.

3. The fully human monoclonal antibody to glypican 3 according to claim 1, characterized in that the amino acid sequence of the light chain is represented by SEQ ID number 8.

4. The fully human monoclonal antibody to glypican 3 according to claim 1, characterized in that the fully human monoclonal antibody to glypican 3 is selected from the group consisting of an Fab fragment antibody, an F (ab') ₂ A fragment antibody, a single chain variable fragment antibody, or a bispecific antibody.

5. A fully human chimeric antigen receptor prepared from a fully human monoclonal antibody to glypican 3 according to any one of claims 1 to 4.

6. Use of a fully human monoclonal antibody to glypican 3 according to any one of claims 1 to 4 as a medicament for the preparation of an anti-liver cancer medicament.