CN105085680A - Humanized anti-PD-1 and c-MET bispecific antibody, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of biological technology, and specifically relates to a recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody and its preparation method and application. The present invention designs recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody molecules by comparing human antibody libraries, computer molecular structure simulation, molecular docking simulation and sequence analysis, including affinity maturation, structure optimization, human Humanization and codon optimization, and on the basis of optimizing the signal peptide sequence and plasmid expression vector replicon, the expression plasmid of recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody was constructed. The bispecific antibody also has the characteristics of low immunogenicity, simple and safe preparation process, and high product yield, purity, and activity. A new specific drug for treating non-small cell lung cancer can be further prepared.

Description

Humanized anti-PD-1 and c-MET bispecific antibody and its preparation method and application

technical field

The invention belongs to the field of biological technology, and in particular relates to a humanized anti-PD-1 and c-MET bispecific antibody and its preparation method and application.

Background technique

The prior art discloses that monoclonal antibodies have been widely used in tumor therapy. Studies have shown that traditional monoclonal antibodies kill tumor cells mainly by recruiting immune effector cells with Fcγ receptors, activating complement, inducing apoptosis, and inhibiting growth factors; however, because T cells do not have Fcγ receptors on the surface, traditional monoclonal antibodies Monoclonal antibodies cannot recruit T cells for treatment, thus limiting the therapeutic effect of monoclonal antibodies.

Studies have shown that T cells play an important role in tumor therapy, and inducing T cell responses in animal models can cause tumor regression and inhibit tumor recurrence. Some studies have reported that infusing tumor-induced specific T cells back into melanoma patients can effectively prevent tumor metastasis. Cytotoxic T lymphocytes (CTLs) kill target cells mainly through two stages: effector-target cell binding stage and cell lysis stage, therefore, can bridge T cells and tumor-associated antigens (TAA) and mediate targeted cytotoxicity Bispecific antibodies against tumor cells by T lymphocytes (rCTL) have broad application prospects.

Activation of T cells requires a dual signal. The first signal (recognition signal) comes from the combination of TCR and antigenic peptide; the second signal (activation signal) comes from the combination of costimulatory receptor and its ligand. Among them, co-stimulatory receptors include activating receptors (mainly CD28) and inhibitory receptors (including CTLA-4, PD-1 and BTLA, etc.). PD-1, Programmed cell death protein 1, is a member of the immunoglobulin superfamily, expressed in activated T cells, B cells, and macrophages, and is mainly involved in suppressing the immune response of activated T cells.

Antineoplastic drugs are usually expressed in normal tissues because their targets are usually expressed in normal tissues, which can easily cause toxic reactions in normal tissues, resulting in serious side effects. c-MET, the hepatocyte growth factor receptor (HGFR), encoded by the proto-oncogene c-Met, is a receptor tyrosine kinase (RTK), which has almost no activity in normal tissues, but in many tumors Usually abnormally active.

With the rapid development of computer science, computer-aided drug design has become a brand-new technology for research and development of new drugs in recent years, which has significantly accelerated the speed and efficiency of new drug design. The continuous analysis of protein structure and the continuous growth of PDB data make it possible for computer-aided design of protein drugs. Research on protein drugs based on protein spatial structure has attracted much attention.

Contents of the invention

The purpose of the present invention is to provide screening, modification, design, expression and purification of bispecific humanized antibodies targeting PD-1 and c-MET and their preparation methods through rational selection of targets.

Another object of the present invention is to provide the application of the bispecific antibody in the preparation of drugs for the treatment of non-small cell lung cancer.

The present invention designs recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody molecules by comparing human antibody libraries, computer molecular structure simulation, molecular docking simulation and sequence analysis, including affinity maturation, structure optimization, human Humanization and codon optimization, and on the basis of optimizing the signal peptide sequence and plasmid expression vector replicon, the expression plasmid of recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody was constructed.

More specifically, the present invention transforms the antibody sequence obtained by screening the phage library by comparing the human antibody library with the computer molecular simulation software DiscoveryStudio, and performs affinity maturation and humanization while maintaining its specificity. Two different antibody formats (FullAntibody, SMIPAntibody) are designed by deleting certain domains in the complete antibody (such as CL, CH1, etc.), and the light chain and heavy chain of the same specificity are connected by connecting short peptide fragments Gly4Ser of different lengths. At the same time, combined with the results of computer molecular simulation, the site-directed mutagenesis technology was used to modify its Fc region, and the "KnobsintoHoles" technology was applied to enable two antibody chains with different targeting specificities to interact to form heterodimers.

Technical scheme of the present invention carries out by following method and step:

1) Computer-aided design of humanized anti-PD-1 and c-MET bispecific antibodies;

2) Selection of connecting peptide between the light chain and the heavy chain of the bispecific antibody and the modification of the Fc region,

The light chain and heavy chain of the same specificity are connected by connecting short peptide fragments Gly4Ser of different lengths; combined with the results of computer molecular simulation, the Fc region is modified by site-directed mutagenesis technology, and the "KnobsintoHoles" technology is used to make it have different target specificities The two antibody chains are capable of interacting to form a heterodimer;

3) Mammalian cell expression of humanized anti-PD-1 and c-MET bispecific antibody,

The humanized anti-PD-1 and c-MET bispecific antibody described in the present invention is not limited to a specific expression system, including eukaryotic expression system and prokaryotic expression system;

4) Purification of humanized anti-PD-1 and c-MET bispecific antibody

Cell culture supernatant was applied to rProteinA affinity chromatography column, equilibrated with 0.02MPB (pH7.0) buffer, then eluted with 0.1MGlycine-HCl (pH3.0) buffer, and the eluted peaks were collected. Freeze-dry the rProteinA affinity chromatography elution peak collection solution, and store the freeze-dried powder at low temperature for later use;

5) Activity determination of humanized anti-PD-1 and c-MET bispecific antibody.

In the present invention, the determination experiments of humanized anti-PD-1 and c-MET bispecific antibodies were carried out, including: 1) specificity and affinity analysis and determination; 2) stimulation of T cell proliferation and cytokine secretion; 3) in vitro Inhibition of tumor cell proliferation experiment; 4) in vivo tumor inhibition experiment; and 5) pharmacodynamics experiment; the experimental results show that the humanized anti-PD-1 and c-MET bispecific antibody of the present invention also has low immunogenicity , and the preparation process is simple and safe, and the product has the characteristics of high yield, purity and activity. A new specific drug for treating non-small cell lung cancer can be further prepared.

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis identification of the transient expression of FullAntibody.

Figure 2 is the SDS-PAGE electrophoresis identification of the purification of FullAntibody.

Figure 3 is the SDS-PAGE electrophoresis identification of transient expression of SMIPAntibody.

Figure 4 is the SDS-PAGE electrophoresis identification of SMIPAntibody purification.

Figure 5 is the flow cytometric detection of FullAntibody specificity.

Figure 6 is the flow cytometric detection of SMIPAntibody specificity.

Figure 7 is a Western Blot analysis of the inhibition of c-MET downstream signal transduction pathway activation in tumor cells by FullAntibody and SMIPAntibody.

Detailed ways

Example 1

1) Sequence design of humanized anti-PD-1 and anti-c-MET bispecific antibody and construction of expression plasmid

By comparing human antibody libraries, computer molecular structure simulation, molecular docking simulation and sequence analysis, design recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody molecules (including affinity maturation, structure optimization, humanization) and codon optimization), and on the basis of optimizing the signal peptide sequence and plasmid expression vector replicon, construct the expression plasmid of recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody;

2) Expression of humanized anti-PD-1 and c-MET bispecific antibody

Acclimate 293E and CHO cells to adapt to serum-free suspension culture;

Recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody expression plasmids were transiently transfected into 293E cells, and protein expression and purification conditions were optimized, mainly including transfection conditions (cell density during transfection, transfection plasmid The amount used and its ratio to the transfection reagent PEI, etc.), the optimization of culture conditions (optimization of medium, selection of rotation speed, etc.) and the selection of parameters in the purification process, while constructing CHO cell stable transfection strains, screening to obtain high expression cloning;

3) Purification of humanized anti-PD-1 and c-MET bispecific antibody

Cell culture supernatant was applied to rProteinA affinity chromatography column, equilibrated with 0.02MPB (pH7.0) buffer, then eluted with 0.1MGlycine-HCl (pH3.0) buffer, and the eluted peaks were collected. Freeze-dry the rProteinA affinity chromatography elution peak collection solution, and store the freeze-dried powder at low temperature until use.

Example 2

Determination of humanized anti-PD-1 and c-MET bispecific antibody

1) Specificity and affinity analysis and determination

Use biomacromolecule interaction analyzers (Biacore, ITC, etc.) to track the interaction between recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies and the extracellular domain of target receptors in real time, and analyze their binding specificity Sex, affinity and kinetic properties;

The binding affinity and specificity of recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies to target receptors (PD-1, c-MET) on the surface of living cells were analyzed by flow cytometry;

2) Stimulate T cell proliferation and cytokine secretion

Human peripheral blood was cultured from whole blood, pretreated with recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies, and then stimulated with leucoagglutinin (PHA-L) to evaluate recombinant humanized anti-PD-1 1 and the ability of anti-c-MET bispecific antibody to enhance the proliferation of T cells stimulated by PHA-L and the secretion of cytokines (IL-2, IFN-γ);

3) In vitro inhibition of tumor cell proliferation experiment

In vitro expansion and culture of tumor cell lines expressing c-MET on the cell surface (such as MKN45, MDA-MB-231, A549, etc.), adding recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies for pretreatment, and then Stimulate with hepatocyte growth factor (HGF), and evaluate the functions of recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies to antagonize HGF-stimulated tumor cell growth, invasion and other related signaling pathways, such as AKT and ERK1 /2. Changes in STAT3, etc. and changes in proliferation and invasion capabilities;

4) In vivo tumor inhibition experiment

SCID mice transplanted with human PBMC were subcutaneously implanted with tumor cells, injected with recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies, and evaluated recombinant humanized anti-PD-1 and anti-c-MET bispecific antibodies ability to inhibit tumorigenesis;

5) Pharmacodynamic experiment

The recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody was used to carry out the non-small cell lung cancer model experiment in experimental animals, and the effective dose, dose-effect and time-effect relationship and toxic and side effects were evaluated; the results showed that the The recombinant humanized anti-PD-1 and anti-c-MET bispecific antibody can significantly inhibit the growth of non-small cell lung cancer cells, and has a more obvious dose-effect and time-effect relationship, with little side effects; it can be further prepared to treat non-small cell lung cancer Drugs for non-small cell lung cancer.

Claims (5)

1. anti-PD-1 and the c-MET bi-specific antibody of humanization, it is characterized in that, by the antibody formation of the structural domain in two kinds of disappearance complete antibodies, the connection short peptide stretch Gly4Ser of different lengths is utilized to connect same specific light chain and heavy chain, through the transformation of Fc district, make the heterodimer of two antibody chains formation with different targeting specific.

2. the preparation method of anti-PD-1 and the c-MET bi-specific antibody of humanization according to claim 1, is characterized in that, comprise step:

1) anti-PD-1 and the c-MET bi-specific antibody of computer aided design (CAD) humanization;

2) selection of connection peptides and the transformation in Fc district between bi-specific antibody light chain and heavy chain,

The connection short peptide stretch Gly4Ser of different lengths is utilized to connect same specific light chain and heavy chain; Simultaneously in conjunction with computer molecular simulation result, utilize site-directed mutagenesis technique Dui Qi Fc district to transform, by " KnobsintoHoles " technology, two antibody chains with different targeting specific are interacted and form heterodimer;

3) mammalian cell expression of anti-PD-1 and the c-MET bi-specific antibody of humanization is expressed,

4) purifying of anti-PD-1 and the c-MET bi-specific antibody of humanization

5) humanization anti-PD-1 and c-MET bi-specific antibody determination of activity.

3., by the preparation method of claim 2, it is characterized in that, step 3) in: expression system comprises eukaryotic expression system and prokaryotic expression system.

4., by the preparation method of claim 2, it is characterized in that, step 4) in, cells and supernatant, upper rProteinA affinity column, balances through 0.02MPB (pH7.0) damping fluid, then use 0.1MGlycine-HCl (pH3.0) buffer solution elution, collect elution peak; Lyophilize rProteinA affinity chromatography elution peak collects liquid, and lyophilized powder is stand-by in cryopreservation.

5. the purposes of anti-PD-1 and the c-MET bi-specific antibody of the humanization of claim 1 in the medicine of preparation treatment nonsmall-cell lung cancer.