CN111378038A - Human monoclonal antibody against human programmed death receptor-1 and its application - Google Patents

Abstract

The invention belongs to the technical field of biological pharmacy, and relates to fully human monoclonal antibodies aiming at human PD1, amino acid sequences of antigen binding fragments thereof, nucleotide sequences for coding the proteins, corresponding expression vectors, host cells capable of expressing the antibodies, and a production method of antibody Fab and IgG1 forms, wherein the antibodies are determined by CDR specific gene sequences of complementarity determining regions existing in variable regions of genes of a light chain and a heavy chain of the antibodies, and effectively expressed antibodies specifically binding to PD1 are obtained in prokaryotic and eukaryotic cells.

Description

Human monoclonal antibody against human programmed death receptor-1 and its application

technical field

The invention belongs to the technical field of biopharmaceuticals, and particularly relates to a fully human monoclonal antibody against human PD1 (Programmed cell death 1, PD-1), the amino acid sequence of its antigen-binding fragment and the nucleotide sequences encoding these proteins.

Background technique

Programmed cell death-1 (PD-1), also known as CD279, is a protein encoded by the PDCD1 gene in humans. PD-1 protein is a type I transmembrane protein containing 268 amino acids. Its main structure consists of an extracellular immunoglobulin variable region (IgV)-like domain (amino acids 2-170), a hydrophobic transmembrane region (171 -191 amino acids) and the intracellular region (192-288 amino acids). As a member of the immunoglobulin superfamily, PD-1 is an important immunosuppressive molecule, which is mainly on the surface of activated T cells, B cells, NK cells, monocytes and dendritic cells.

Studies have shown that T cells in a resting state do not express PD-1. When T cells are activated, PD-1 binds to its ligands PDL1 and PDL2, and inhibits the activity of effector T cells through the mTOR and PI3K/AKT pathways. The function of Treg, induction of anergy and apoptosis of antigen-specific T lymphocytes, thereby inhibiting the production of IL-2, TNF-α and IFN-γ, etc.; tumor cells use this inhibitory immune checkpoint , by expressing too much PDL1 to escape the recognition, attack and killing of T cells, so as to normal proliferation and survival. Monoclonal antibodies block the binding of PD-1 to its ligands, thereby allowing T cells to re-kill tumor cells.

Based on the basis and status of the prior art, the inventors of the present application intend to provide a novel anti-PD1 fully human antibody with high affinity, which can be used for immunotherapy of various solid tumors.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel anti-PD1 fully human antibody based on the basis and current situation of the prior art, specifically a fully human monoclonal antibody that specifically binds to PD1 and its amino acid sequence, disclosed in the present invention The nucleotide sequences encoding the monoclonal antibodies of the present invention, vectors and host cells for expressing the monoclonal antibodies of the present invention are described.

The invention also discloses the antigen-binding fragment of the monoclonal antibody, the bispecific antibody, and the conjugate with the effector molecule.

The object of the present invention is achieved through the following technical solutions:

1. Enrichment screening for human PD-1 protein-specific single-domain antibodies;

2. Polyclonal phage-ELISA detection;

3. Soluble expression and purification of antibodies:

4. Detect the binding ability of PD-1 antibody to PD-1(ECD)-hFc protein:

5. The cell-binding ability of the monoclonal antibody P2-G12 was determined by flow cytometry.

More specifically, the present invention provides a fully human monoclonal antibody against human PD1 antigen or the complementarity determining region CDR of the heavy chain and light chain of the antigen-binding fragment, the CDR region of the antibody mainly comprises:

The light chain variable region of the antibody contains CDR1 of SEQ ID NO:3, CDR2 of SEQ ID NO:4, and CDR3 of SEQ ID NO:5; the heavy chain variable region of the antibody contains CDR1 of SEQ ID NO:6, SEQ ID NO:6 CDR2 of ID NO:7, and CDR3 of SEQ ID NO:8;

The described fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen includes framework region FR and above-mentioned complementarity determining region CDR, and described FR region mainly includes:

The light chain framework region of the antibody consists of amino acids 1-23, 30-46, 50-85, and/or 96-105 of SEQ ID NO:1, and the heavy chain framework region of the antibody consists of SEQ ID NO:2 1-25, 34-50, 59-96, and/or 115-125 amino acids;

The fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen of the present invention is an antibody against human PD-1 epitope, and has the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 .

The present invention discloses a polynucleotide encoding a protein selected from the group consisting of the aforementioned CDR region, FR region, and/or the aforementioned anti-PD-1 monoclonal antibody; the aforementioned polynuclear The nucleotide has the nucleotide sequence shown in SEQ ID NO: 9 or 15; the nucleic acid molecule is operably linked to a promoter;

The invention discloses an expression vector, which contains the nucleic acid molecule;

The invention discloses a plasmid, and the plasmid is a virus plasmid.

The present invention discloses a host cell, which contains the above-mentioned expression vector, or integrates the above-mentioned nucleic acid molecule into its genome.

In the present invention, the anti-PD-1 monoclonal antibody, CDR region and FR region are fully human.

In the present invention, the preferred monoclonal antibody is IgG1.

In the present invention, preferred antigen-binding fragments are scFv, Fv, Fab or F(ab) ₂ .

The present invention discloses a bispecific antibody, which contains the above-mentioned monoclonal antibody or antigen-binding fragment.

The present invention discloses an immunoconjugate, the immunoconjugate contains the above-mentioned monoclonal antibody or antigen-binding fragment, or the bispecific antibody, coupled with an effector molecule; the conjugated molecule Is a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.

The present invention discloses a CAR-T cell, the cell surface displays the above-mentioned monoclonal antibody or antigen-binding fragment.

Further, the present invention provides the use of the anti-PD-1 fully human monoclonal antibody or antigen-binding fragment for preparing and detecting PD-1 in biological samples or for preparing medicine for treating tumors.

In some embodiments of the invention, the amino acid sequence of the light chain variable region and/or the amino acid sequence of the heavy chain variable region of the monoclonal antibody, or antigen-binding fragment, comprises at least one P2-G12 light chain and/or The complementarity determining region (CDR) of the variable region of the heavy chain; using this antibody CDR region or part or the whole gene, different forms of genetically engineered antibodies can be engineered and produced in prokaryotic and eukaryotic cells and any expression system, and further used for clinical use to diagnose or treat tumors.

The present invention provides a fully human monoclonal antibody against human PD1, the amino acid sequence of its antigen-binding fragment and the nucleotide sequence encoding these proteins, a corresponding expression vector and host cells capable of expressing the antibody, as well as antibody Fab and IgG1 A production method of the form, the antibody is an antibody that specifically binds to PD1 that is efficiently expressed in prokaryotic and eukaryotic cells, and can be expressed in prokaryotic and eukaryotic cells and any expression system using this antibody CDR region or part or whole gene The genetically engineered antibodies in different forms are modified and produced, and further used in the preparation of medicines for preventing or treating various types of malignant tumors.

Description of drawings

Figure 1. Screening enrichment of antibodies specific for human PD-1 protein, where,

The antibody library was screened with biotin-labeled PD-1(ECD)-hFc for 4 rounds, and polyclonal phage ELISA was used to detect the enrichment of specific antibodies. After the second round of screening, the antibodies were gradually enriched until the fourth round. Antibody enrichment was most significant, indicating that anti-PD-1 antibodies were enriched.

Figure 2. The PD-1 antibody in Fab form expressed from E. coli was expressed and purified by nickel column affinity chromatography, and its purity was checked by SDS-PAGE.

Figure 3. ELISA detects the binding affinity of the Fab or IgG1 forms of the specific antibody P2-G12 to PD-1.

Figure 4. FACS detection of the binding ability of P2-G12 to HEK293T cells overexpressing human full-length PD-1 protein.

Detailed ways

Example 1 Enrichment screening of human PD-1 protein-specific single-domain antibodies

The screening method was described in the reported literature, using the previously constructed super-large phage-displayed fully human Fab antibody library with a capacity of 1.5 × 10 ¹¹ to target the biotin-labeled PD-1(ECD)-hFc fusion protein. Screen for antibodies. Biotin-labeled PD-1(ECD)-hFc fusion protein was immobilized on streptavidin-coated magnetic beads, and 10 ¹² phage-displayed antibodies were mixed with 5 at room temperature for 1, 2, 3, and 4 rounds, respectively. ,4,2,1 μg antigen was incubated for two hours, and the phages used in each round of screening were 10 ¹²

Example 2 Detection of polyclonal phage-enzyme-linked immunosorbent assay

After multiple rounds of screening, polyclonal phage ELISA was performed on the phage antibody library obtained after each round of screening to determine antibody enrichment. 2 μg/ml antigen was coated in 50 μl per well on a 96-well ELISA plate overnight at 4°C. After blocking with PBS (MPBS) containing 3% (m/v) nonfat dry milk for 1 h, 10 ¹¹ phage antibody library supernatants and coated antigens obtained after the first, second, third and fourth rounds of screening were added at 37°C. Incubate for 1.5h. Unbound phage is washed away, and bound phage can be detected with HRP-anti-M13 antibody. ABTS was added to develop color, and the absorbance value at 405 nm was read on a microplate reader. According to the results of polyclonal phage ELISA (as shown in Figure 1), specific antibodies were enriched in the third round.

Example 3 Soluble Expression and Purification of Antibodies

For the expression and purification of antibody Fab fragments, as previously described, the phage plasmids of the selected positive clones were transferred into E. coli HB2151 competent cells, and a single colony was picked from the 2YT-AG plate grown overnight and inoculated into SB In the medium, IPTG was induced to express at 30°C, and the bacteria were harvested for purification with Ni-NTA. The purity was detected by SDS-PAGE electrophoresis. The result showed that the purity was over 85%, which could be used for subsequent experiments;

For the construction of the IgG1 expression vector, the Fab of the P2-G12 antibody was used as the template, and the heavy and light chains on the Fab phage plasmid were amplified by PCR, and the PCR heavy chain product and the eukaryotic expression vector pTT-IgG1 were digested with BsmBI. , PCR light chain product and pTT-IgG1 vector were digested with SfiI, the heavy chain and light chain were cloned into pTT-IgG1 under the action of T4 ligase, transformed into competent cells, and the clones were selected for sequencing. The clones were IgG1-Hc and IgG1-Lc, and then the IgG1-Hc and IgG1-Lc recombinant plasmids were co-transfected into Expi293 cells for transient expression and purified by affinity chromatography with Protein G resin.

Example 4 Detection of the binding ability of PD-1 antibody to PD-1(ECD)-hFc protein

The PD-1(ECD)-hFc protein was coated on the ELISA plate, and incubated with the antibody diluted in gradient concentration. The HRP-anti-Fab antibody detected the binding ability of the antibody to PD-1(ECD)-hFc, as shown in Figure 3 The ELISA results of P2-G12 showed that P2-G12 could bind PD-1 protein well, and its binding ability was enhanced 100 times after being converted into IgG1.

Example 5 The cell-binding ability of the monoclonal antibody P2-G12 was determined by flow cytometry

The cells in logarithmic growth phase were collected, incubated with different concentrations of antibodies, and then detected by adding anti-human IgG1-FITC-labeled antibodies. The results showed that P2-G12 could bind to HEK293T cells overexpressing PD-1, but not to HEK293T cells. There is no non-specific binding per se.

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Claims (17)

1. A fully human monoclonal antibody or antigen-binding fragment directed against human PD1 antigen having CDR regions of the heavy and light chain complementarity determining regions, wherein the CDR regions of said antibody consist essentially of:

the variable region of the light chain of the antibody contains CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 4, and CDR3 of SEQ ID NO. 5; the heavy chain variable region of the antibody contains CDR1 of SEQ ID NO. 6, CDR2 of SEQ ID NO. 7, and CDR3 of SEQ ID NO. 8.

2. The Complementarity Determining Regions (CDRs) of the heavy chain and the light chain of the fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen according to claim 1, wherein the antibody or antigen-binding fragment comprises the framework region FR and the complementarity determining region CDRs as claimed in claim 1, and wherein the FR regions consist essentially of:

the light chain framework region of the antibody consists of amino acids 1-23, 30-46, 50-85, and/or 96-105 of SEQ ID NO:1, and the heavy chain framework region of the antibody consists of amino acids 1-25, 34-50, 59-96, and/or 115-125 of SEQ ID NO: 2.

3. The CDR of complementarity determining regions of the heavy chain and the light chain of the fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen according to claim 1, wherein the fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen is an antibody against the epitope of human PD-1 and has the amino acid sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2.

4. A polynucleotide encoding a protein selected from the group consisting of: the CDR region of claim 1, the FR region of claim 2, and/or the anti-PD-1 monoclonal antibody of claim 3.

5. The polynucleotide of claim 4, having a nucleotide sequence as set forth in SEQ ID NO 9 or 15.

6. The nucleic acid molecule of claim 4, operably linked to a promoter.

7. An expression vector comprising the nucleic acid molecule of claim 4.

8. The expression vector of claim 7, wherein the expression vector is a viral plasmid.

9. A host cell comprising the expression vector of claim 7, or having integrated into its genome the nucleic acid molecule of claim 4.

10. The CDRs of Complementarity Determining Regions (CDRs) of the heavy chain and the light chain of the fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen according to claims 1-3, wherein the anti-PD-1 monoclonal antibody, the CDR regions and the FR regions are fully human.

11. The CDRs of Complementarity Determining Regions (CDRs) of the heavy chain and the light chain of a fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen of claims 1-3, wherein the monoclonal antibody is IgG 1.

12. The CDR of the heavy and light chain complementarity determining regions of a fully human monoclonal antibody or antigen-binding fragment thereof against human PD1 antigen of claims 1-3, wherein the antigen-binding fragment is scFv, Fv, Fab or F (ab)₂。

13. A bispecific antibody comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-3.

14. An immunoconjugate comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-3, or the bispecific antibody of claim 13, conjugated to an effector molecule.

15. The conjugate of claim 14, wherein the conjugate molecule is a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

16. A CAR-T cell characterized in that the cell surface displays the monoclonal antibody or antigen-binding fragment of any one of claims 1-3.

17. Use of the anti-PD-1 fully human monoclonal antibody or antigen-binding fragment of claim 3 in the preparation of a medicament for detecting PD-1 in a biological sample or for treating a tumor.

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