CN101974535B - Preparation and application of novel anti-ErbB2 humanized antibody MIL12 - Google Patents

Abstract

The invention discloses a group of novel gene sequences, the amino acid sequence of the antibody light and heavy chain variable regions encoded by it is the same as that of the antibody Herceptin which has been on the market, but its expression level in mammalian cells is much higher than that of Herceptin. The results of in vitro biological function evaluation indicated that the above-mentioned antibody could specifically recognize the erbB2 antigen and mediate the ADCC effect to kill erbB2-positive tumor cells, and its biological activity was similar to that of Herceptin. The invention also discloses the preparation method of the above antibody.

Description

Preparation and Application of Novel Anti-ErbB2 Humanized Antibody MIL12

technical field

The present invention relates to novel antibodies and uses thereof. By optimizing codons, a novel anti-ErbB2 humanized antibody MIL12 was obtained. The gene was fully synthesized by molecular biology technology, and after eukaryotic expression and purification, the function of the new antibody was evaluated by immunology and cell biology experiments.

Background technique

As a member of the epidermal growth factor receptor family (ErbB family), ErbB2 (HER2, HER2/neu) is a transmembrane glycoprotein with tyrosine kinase activity. The molecular structure of ErbB2 can be divided into three parts: the extracellular domain, the transmembrane domain and the intracellular domain. The extracellular domain can be divided into D1 and D3, two very similar barrel-shaped domains surrounded by β-sheets, and D2 and D4. Two cysteine-rich domains, part of the intracellular domain have tyrosine kinase activity. The formation of ErbB2 homologous or heterologous dimers is accompanied by the phosphorylation of C-terminal tyrosine residues in cells, which causes the activation of proteins containing SH2, SH3 and PTB domains in cells, and recruits and activates downstream related proteins, through Signaling cascades leading to cell proliferation and differentiation. ErbB2 is overexpressed in a variety of tumor tissues, including breast cancer (25-30%), ovarian cancer (18-43%), non-small cell lung cancer (13-55%), prostate cancer (5-46%), gastric cancer (21-64%), head and neck tumors (16-50%) and other malignant tumors derived from epithelial cells, while the expression level is very low or not expressed in adult normal tissues. Clinical studies have also found that tumor patients with high ErbB2 expression are often insensitive to radiotherapy and chemotherapy, and are prone to tumor metastasis, and the prognosis of patients is poor. Therefore, it has become an ideal target molecule for tumor immunotherapy, and it is also a hot spot in current tumor treatment research.

(trastuzumab)上市，应用于ErbB2过表达的乳腺癌患者的临床治疗。目前，Herceptin已成为广泛应用于转移性乳腺癌的一线临床治疗药物。In 1998, the US Food and Drug Administration (FDA) approved Herceptin, a humanized monoclonal antibody targeting ErbB2 developed by Genetech.

(trastuzumab) was launched for the clinical treatment of breast cancer patients with ErbB2 overexpression. At present, Herceptin has become the first-line clinical treatment drug widely used in metastatic breast cancer.

Herceptin may inhibit the growth of breast cancer cells overexpressing ErbB2 through the following pathways: (1) specifically binding to ErbB2, blocking ligand-mediated cell signaling, and affecting epithelial cell growth; (2) accelerating ErbB2 protein receptors (3) Improve the ability of immune cells to attack and kill tumor cells through ADCC, and enhance the cytotoxicity caused by chemotherapy; (4) Down-regulate vascular growth factor and other angiogenesis factors, and inhibit the growth of tumor vascular tissue.

The main expression method of whole antibody molecules is mammalian cell expression, and the production capacity of antibodies in the world is far below the demand. In addition to being limited by factors such as culture scale, production process, and purification process, the production of antibodies largely depends on the expression level of antibody genes in mammalian cells.

In this study, on the basis of Herceptin antibody, a new anti-human ErbB2 humanized antibody variable region coding gene was designed through computer-aided analysis and molecular biology techniques. Same, but expressed at much higher levels than Herceptin in mammalian cells.

Contents of the invention

The present invention discloses a group of novel antibody variable region genes. The gene sequence shown in Sequence 1 in the sequence listing encodes the light chain variable region amino acids of antibody MIL12; the gene sequence shown in Sequence 2 in the sequence listing encodes the heavy chain of antibody MIL12. Variable region amino acids.

The present invention discloses a new anti-human ErbB2 humanized antibody MIL12, the amino acid sequence of the light chain variable region is encoded by sequence 1; the amino acid sequence of the heavy chain variable region is encoded by sequence 2.

A feature of the anti-human ErbB2 humanized antibody MIL12 disclosed in the present invention is that the expression level of the antibody in mammalian cells is significantly better than that of Herceptin.

A characteristic of the antibody MIL12 disclosed in the present invention is that it can specifically recognize and bind the target antigen ErbB2.

One feature of the antibody MIL12 disclosed in the present invention is to effectively inhibit and kill ErbB2-positive tumor cells.

The invention discloses the application prospect that the antibody MIL12 can be used to produce drugs for treating improper proliferation-related diseases.

The present invention also discloses a method for preparing the antibody MIL12, and the specific implementation process is as follows:

1) Using computer-aided analysis to obtain new antibody genes;

2) Synthesize antibody genes using fully synthetic PCR technology;

3) Expression and identification of antibodies;

4) Identification of antibody biological activity.

The design and preparation process of the MIL12 antibody will be described in detail below with reference to the above steps. The methods for designing and preparing the antibodies of the present invention are merely illustrative and not limiting; other known methods or modified methods can also be used.

1) Using computer-aided analysis to obtain new antibody genes

According to the amino acid sequence of the antibody, the codons commonly used in mammalian cells are selected for reverse translation, rationally optimized by bioinformatics technology, the stability of the gene and its expression level in mammalian cells are increased, and the corresponding antibody gene is obtained.

2) Synthesize antibody genes using fully synthetic PCR technology;

According to the obtained antibody gene. Utilize gene synthesis PCR technology, design corresponding primers, obtain full-length gene by overlapping PCR method; clone into cloning vector, and carry out sequence determination.

3) Expression and identification of antibodies

a) Antibody expression: construct the antibody gene into a eukaryotic expression vector, transiently transfect eukaryotic expression cells by liposome transfection, electroporation, etc., and harvest the supernatant after culturing for a long enough time, which contains the expressed target antibody .

b) Double-sandwich enzyme-linked immunosorbent assay (ELISA): After coating with a suitable antibody as a capture antibody, after blocking, add the sample to be tested and a standard sample for capture reaction; then use a suitable enzyme-labeled secondary antibody for color development reaction to determine the content of the target protein in the sample.

c) SDS-PAGE: Prepare a polyacrylamide gel with a suitable concentration according to the size of the target protein, prepare a reduction electrophoresis sample or a non-reduction electrophoresis sample for polyacrylamide gel electrophoresis according to the needs of the experiment, and separate the protein; after Coomassie brilliant blue staining , to determine the molecular weight of the target protein.

4) Biological activity identification of anti-ErbB2 antibody

a) Flow cytometry analysis: collect the target cells; after washing with buffer (PBS+2% fetal bovine serum), add primary antibody, react at 4°C for 30mins; wash with buffer twice, add corresponding secondary antibody, and protect from light at 4°C React for 30mins, wash with buffer twice, resuspend in the sheath fluid for flow cytometric analysis directly or fix with 1% paraformaldehyde, and store in the dark at 4°C.

b) Growth inhibition experiment: ErbB2-positive cells were used as target cells, and antibodies of different concentrations were added to act on the target cells, and negative control, positive control and experimental groups were set up; after 48 hours of action, the growth inhibitory activity of the antibody on the target cells was measured by the MTT method.

c) Antibody-dependent cytotoxicity test (ADCC): mark ErbB2 positive cells as target cells, and use lymphocyte separation medium to separate peripheral blood mononuclear cells from peripheral blood as effector cells; after mixing according to the corresponding ratio, add different amounts Antibody killing experiments were carried out on a round-bottom 96-well culture plate, and experimental wells, target cell spontaneous release wells, maximum release wells and background wells were set up; incubate at 37°C for 2h. Use a multifunctional microplate reader to detect and calculate the killing rate.

Description of drawings

Figure 1 Electrophoresis analysis of antibody MIL12 gene. Among them, lane 1 is the molecular weight standard of nucleic acid DL2000; lane 2 is the gene of the variable region of the light chain; lane 3 is the gene of the variable region of the heavy chain.

Figure 2 Comparison of the average expression levels of antibodies.

Figure 3 SDS-PAGE analysis of the molecular weight of the antibody MIL12. Lane 1 is MIL12; Lane 2 is Herceptin; Lane 3 is human IgG; M indicates the standard protein molecular weight marker.

Figure 4 Antibody MIL12 specifically recognizes target cells such as SKVO3, MCF7, SKBR3 expressing ErbB2 antigen. A. MCF7 cells; B. SKBR3 cells; C. SKOV3 cells.

Figure 5 MIL12 specifically recognizes exogenously expressed ErbB2 antigen. A. R2 represents ErbB2-EGFP transfected positive cells; B. MIL12 recognizes exogenously expressed ErbB2.

Figure 6 Flow cytometric analysis technique to determine the affinity of MIL12 antibody and Hercepin to target cells.

Figure 7 MIL12 inhibits the growth of tumor cell lines. A. The antibody inhibits the growth of MCF7 cells; B. The antibody inhibits the growth of SKOV3 cells.

Figure 8 Antibody-mediated ADCC killing of tumor cell lines. A. Antibody-mediated ADCC kills MCF7 cells; B. Antibody-mediated ADCC kills SKOV3 cells.

Example 1 Rational Design and Gene Synthesis of Anti-ErbB2 Antibody MIL12

1. Materials:

The primer design software was biosun software, and the primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.; Pyrobest DNA polymerase was a product of TAKARA Company; dNTP was a product of TAKARA Company; pGEM-T Easy vector system was a product of Invitrogen Company; T4 DNA ligase was a product of NEB Company Products; gene sequencing was completed by Beijing Nuosai Genome Research Center Co., Ltd.; other related reagents were purchased from the market.

2. Method results

1. Gene acquisition of antibody MIL12

According to the amino acid sequence of the Herceptin antibody, codons with high frequency in mammalian cells were selected, reverse-translated, rationally optimized by bioinformatics technology, and the stability of the gene and its expression level in mammalian cells were increased to obtain The antibody light chain variable region gene sequence (SEQ ID NO: 1) and heavy chain variable region gene sequence (SEQ ID NO: 2). According to the requirements of constructing antibody eukaryotic expression vectors in Example 2, EcoR V and Xho I restriction sites were respectively introduced at the 5' end and 3' end of the antibody light chain variable region gene; Pvu II and BstE II restriction sites were introduced into the 'end and 3' end respectively.

2. Primer design

According to the principle of total gene synthesis, use computer-aided design software to design primers, considering the secondary structure of the primers, GC content and other related parameters. Ten primers were designed for each gene, numbered P1, P2, P3, P4, P5, P6, P7, P8, P9, and P10, for total gene synthesis.

3. Whole gene synthesis

1) Dilute with sterile water after primer synthesis, as follows:

a) Centrifuge the primer tube at 12000RPM for 2mins, dilute the primer at a concentration of 100uM, and store at -20°C;

b) Mix a small amount of primers P2-P9 to a final concentration of 10uM as a working solution;

c) Mix and dilute a small amount of primers P1 and P10 to a final concentration of 10uM as the use solution P1/P10;

2) Total gene synthesis, using Pyrobest DNA polymerase, the method is as follows:

a) Take 1uL of P2~P9 mixed primers as a template, and prepare the following Overlap PCR system:

P2～P9 mixed primers 1uL

dNTP 3uL

10×Buffer 5uL

Pyrobest DNA polymerase 0.3uL

P1/PI0 primers 4uL (primers not added first)

Make up to a total volume of 50uL with sterile water

b) The above PCR system is carried out as follows:

After the reaction, it was lowered to room temperature.

Add primers P1/P10 and perform the following PCR reaction:

After the reaction, it was lowered to room temperature.

c) Separating the PCR product by 1-2% agarose gel electrophoresis, recovering a 350bp fragment for the antibody heavy chain gene, and recovering a 320bp fragment for the light chain gene.

d) Recovery and tailing (Pyrobest DNA polymerase cannot add tail A to the 3' end of the PCR product, so it cannot be directly connected to the T vector), and make the following 10uL system:

Recovery fragment 8.2uL

10×Buffer 1uL

dNTP 0.5uL

Ordinary Taq enzyme 0.3uL

The reaction conditions are as follows: 72°C for 20mins, after the reaction is completed, cool down to room temperature.

e) Take the tailed product and connect it to the pGEM-T Easy vector:

Tailed product 4.2uL

2×Connect Buf 5uL

T carrier 0.5uL

T4 ligase 0.3uL

After ligation at room temperature for 2 hours or overnight at 4°C, the ligation product was transformed into Escherichia coli JM109, and spread on LB agar medium containing 100 μg/mL ampicillin (final concentration). The obtained clones were cultured in LB liquid medium containing 100 μg/mL ampicillin (final concentration), and the plasmids were extracted with a plasmid extraction kit (Broadtech Corporation), and the obtained plasmids were identified by nucleic acid sequencing.

After Overlap PCR amplification, agarose gel electrophoresis analysis was performed to obtain the target band of specific size (Figure 1); the product was successfully cloned into the pGEM-TEasy vector through molecular biology techniques such as recovery, tailing, and cloning; it was identified by sequencing , the synthesized gene is consistent with the target sequence.

According to the gene information of Herceptin antibody, we also synthesized the original gene sequence of Herceptin using a similar method.

Example 2 Expression, identification and biological function identification of anti-ErbB2 antibody MIL12

1. Materials

Antibody eukaryotic expression vector pTGS-FRT-DHFR was constructed by our company and applied for national patent (patent authorization number: ZL200510064335.0); primers were designed by biosun software and synthesized by Shanghai Yingjun Biotechnology Co., Ltd.; liposomes and MTT were Invitrogen The company's products, goat anti-human IgG, horseradish-labeled goat anti-human IgG and human IgG were prepared by our company; endonucleases were products of NEB; SKVO3, MCF7, SKBR3 were purchased from ATCC; DELFIA EuTDA cytotoxicity kit It is a product of PE Company; other reagents are commercial products.

1. Method

1. Antibody expression

1.1 Construction of antibody eukaryotic expression vector

The expression vector pTGS-FRT-DHFR (patent authorization number: ZL200510064335.0) obtained by our company containing the constant region gene of human IgG1 antibody was selected as the expression vector of the antibody of the present invention. The MIL12 antibody and Herceptin antibody light and heavy chain variable region gene cloning vectors obtained as described in Example 1 were digested with corresponding endonucleases (the heavy chain variable region gene was digested with Pvu II and BstE II, the light chain variable region gene was digested with After EcoR V and Xho I digestion), it was ligated with the vector digested by the same endonuclease. Eukaryotic expression vectors were constructed by transformation and other common techniques in molecular biology. Taking the construction of the MIL 12 antibody expression vector as an example, the specific implementation is as follows:

a) As described in Example 1, the obtained antibody MIL12 light and heavy chain variable region genes were constructed into pGEM-TEasy vectors, and the obtained vectors were named pGEM-T-MIL12VL and pGEM-T-MIL12VH respectively;

b) pGEM-T-MIL12VL was digested with EcoR V and Xho I;

c) Take 1 μg of pTGS-FRT-DHFR vector and digest it with EcoR V and Xho I. The pTGS-FRT-DHFR vector digested with EcoR V and Xho I and the antibody MIL12 light chain variable region gene digested with EcoR V and Xho I were connected with DNA ligase T4. The ligation product was transformed into JM109 Escherichia coli and spread on LB agar medium containing 100 μg/mL ampicillin (final concentration). The obtained clones were cultured in LB liquid medium containing 100 μg/mL ampicillin (final concentration), and the plasmids were extracted with a plasmid extraction kit (Broadtech Corporation). After the extracted plasmid was digested by EcoR V and Xho I, it was analyzed by 1% agarose gel electrophoresis, and a clone carrying the antibody MIL12 light chain variable region gene was selected.

As a result of the above procedures, the plasmid pTGS-MIL12VL carrying the light chain variable region gene of the anti-ErbB2 humanized antibody MIL12 was obtained.

d) Digest pGEM-T-MIL12VH with Pvu II and BstE II;

e) Take 1 μg of pTGS-MIL12VL vector and digest it with Pvu II and BstE II. The resulting Pvu II and BstE II digested pTGS-MIL12VL vector and the antibody MIL12 heavy chain variable region gene digested with Pvu II and BstE II were ligated with DNA ligase T4. The ligation product was transformed into JM109 Escherichia coli and spread on LB agar medium containing 100 μg/mL ampicillin (final concentration). The obtained clones were cultured in LB liquid medium containing 100 μg/mL ampicillin (final concentration), and the plasmids were extracted with a plasmid extraction kit (Broadtech Corporation). After the extracted plasmid was digested with Pvu II and BstE II, it was analyzed by 1% agarose gel electrophoresis, and a clone carrying the heavy chain variable region gene of the antibody MIL12 was selected.

As a result of the above procedure, the plasmid pTGS-MIL12 carrying the heavy chain variable region gene of the anti-ErbB2 humanized antibody MIL12 was obtained on the basis of pTGS-MIL12VL.

1.2 Antibody expression

The eukaryotic expression vectors of the obtained antibodies MIL12 and Herceptin were transiently transfected into CHO cells by a liposome-mediated method, and the supernatant was harvested after 48 hours, and the expression of the target antibodies was analyzed by double-sandwich ELISA and SDS-PAGE experiments Condition.

Goat anti-human IgG and horseradish-labeled goat anti-human IgG were used to detect the antibody content in the supernatant by double-sandwich ELISA. The untransfected supernatant was used as a negative control, and pure human IgG was used as a standard. The results of ELISA experiments showed that both MIL12 antibody and Hercepitn antibody were expressed in the supernatant. Among them, the expression level of MIL12 antibody was about 9.3±2.13 μg/mL, much higher than the expression level of Herceptin antibody at 4.3±2.43 μg/mL (Figure 2). The SDS-PAGE experiment was carried out in a non-reduced form, and the results showed that the expressed MIL12 antibody had the same molecular weight as the Hercepin antibody, both of which were 155KDa (Figure 3).

2. Functional identification of antibodies

2.1 Identification of MIL12 monoclonal antibody

The recognition function of the obtained antibody MIL12 was identified by using SKVO3, MCF7, SKBR3 and other tumor cell lines expressing ErbB2 antigen. The results of flow cytometry analysis ( FIG. 4 ) showed that the MIL12 antibody could specifically recognize tumor cell lines such as SKVO3, MCF7, and SKBR3. ErbB2 extracellular region and transmembrane region genes were constructed on the pEGFP-N1 vector, and EGFP was fused and expressed. The results showed ( FIG. 5 ) that MIL12 could specifically recognize the exogenously expressed ErbB2 antigen; suggesting that the antibody MIL12 obtained in the present invention could specifically bind ErbB2 antigen.

Further using flow cytometric analysis technology, the binding of different concentrations of antibodies to target cells was determined, and the results showed that the affinity of MIL12 antibody was similar to that of Hercepin ( FIG. 6 ).

2.2 Function of inhibiting and killing tumor cells

SKVO3, MCF7 and other tumor cell lines were used to verify the functions of the obtained antibodies in inhibiting tumor cell growth and killing tumor cells.

SKVO3 and MCF7 were used for growth inhibition experiments (Figure 7). Compared with the human IgG control group, MIL12 had obvious inhibitory activity on the growth of SKVO3 and MCF7 cells, and the inhibitory activity was similar to the positive control Hercetpin.

In the ADCC experiment, labeled SKVO3 and MCF7 cells were used as target cells, and human peripheral blood mononuclear cells were used as effector cells. When the effect-to-target ratio was 50:1, the antibody could effectively kill the target cells (Figure 8), and the results were consistent with positive results. Control Hercetpin was similar.

Claims (3)

1. An antibody light chain variable region gene, the sequence of which is shown in Sequence 1; 2. An antibody heavy chain variable region gene, the sequence of which is shown in SEQ ID NO: 2. 3. The application of the antibody light chain variable region gene of claim 1 and the antibody heavy chain variable region gene of claim 2 in the preparation of antibodies.

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