CN111378038A

CN111378038A - Human monoclonal antibody aiming at human cell programmed death receptor-1 and application thereof

Info

Publication number: CN111378038A
Application number: CN201811628902.4A
Authority: CN
Inventors: 应天雷; 吴艳玲
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2020-07-07

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 aiming at human cell programmed death receptor-1 and application thereof

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a fully human monoclonal antibody aiming at human PD1(Programmed cell death 1, PD-1), an amino acid sequence of an antigen binding fragment thereof and a nucleotide sequence for coding the proteins.

Background

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

Studies show that the T cells in a resting state do not express PD-1, when the T cells are activated, the PD-1 is combined with ligands PDL1 and PDL2 thereof, the activity of effector T cells is inhibited through mTOR and PI3K/AKT pathways, the function of Treg is enhanced, and the apoptosis of anergic and antigen-specific T lymphocytes is induced, so that the generation of IL-2, TNF- α, IFN-gamma and the like is inhibited, the tumor cells are just the immune check points utilizing the inhibition, and the recognition, attack and killing of the T cells are avoided through the expression of excessive PDL1, so that the tumor cells are normally proliferated and survived, and the monoclonal antibody prevents the binding of the PD-1 and the ligands thereof, so that the T cells kill the tumor cells again.

Based on the foundation and the current situation of the prior art, the inventor of the application intends to provide a novel anti-PD1 fully human antibody, which has high affinity and can be used for immunotherapy of various solid tumors.

Disclosure of Invention

The invention aims to provide a novel anti-PD1 fully human antibody based on the basis and the current situation of the prior art, in particular to a fully human monoclonal antibody specifically binding to PD1 and an amino acid sequence thereof, and discloses a nucleotide sequence for encoding the monoclonal antibody, a vector for expressing the monoclonal antibody and a host cell.

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

The purpose of the invention is realized by the following technical scheme:

1. enrichment screening of specific single-domain antibodies aiming at human PD-1 protein;

2. detecting by polyclonal phage-enzyme-linked immunoassay;

3. soluble expression and purification of antibodies:

4. detecting the binding capacity of the PD-1 antibody and the PD-1(ECD) -hFc protein:

5. the cell binding capacity of monoclonal antibody P2-G12 was determined by Hematology.

More specifically, the present invention provides the CDRs of the heavy and light chain complementarity determining regions of a fully human monoclonal antibody or antigen-binding fragment against human PD1 antigen, said antibody CDR regions consisting 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;

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

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;

the fully human monoclonal antibody or antigen binding fragment of the invention directed to human PD1 antigen is an antibody directed to the epitope of human PD-1 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 CDR regions, FR regions, and/or said anti-PD-1 monoclonal antibodies as described previously; the polynucleotide has a nucleotide sequence shown as 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, which is a virus plasmid.

The invention discloses a host cell, which contains the expression vector or integrates the nucleic acid molecule in the genome.

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

In the present invention, the preferred monoclonal antibody is IgG 1.

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

The invention discloses a bispecific antibody which contains the monoclonal antibody or the antigen binding fragment.

The invention discloses an immunoconjugate, which comprises the monoclonal antibody or the antigen binding fragment or the bispecific antibody, and an effector molecule; the conjugated molecule is a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.

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

Further, the invention provides an application of the anti-PD-1 fully human monoclonal antibody or the antigen binding fragment in preparation of a medicament for detecting PD-1 in a biological sample or 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 Complementarity Determining Region (CDR) of the light chain and/or heavy chain variable region of P2-G12; the CDR region or partial or whole gene of the antibody can be used for transforming and producing different forms of genetic engineering antibodies in prokaryotic and eukaryotic cells and any expression system, and further used for clinically diagnosing or treating tumors.

The invention provides a fully human monoclonal antibody aiming at human PD1, an amino acid sequence of an antigen binding fragment thereof, nucleotide sequences for coding the proteins, a corresponding expression vector, a host cell capable of expressing the antibody, and a production method of antibody Fab and IgG1 forms, wherein the antibody is an antibody which is effectively expressed in prokaryotic and eukaryotic cells and specifically binds to PD1, and by utilizing a CDR region or a part or a whole gene of the antibody, different forms of genetically engineered antibodies can be modified and produced in the prokaryotic and eukaryotic cells and any expression system, and the genetically engineered antibodies can be further used for preparing medicines for preventing or treating various types of malignant tumors.

Drawings

FIG. 1 screening and enrichment of antibodies specific to human PD-1 protein,

and (3) performing 4 rounds of phage screening on the antibody library by using the PD-1(ECD) -hFc marked by biotin, detecting the enrichment of specific antibodies by using polyclonal phage ELISA, and gradually enriching the antibodies from the second round of screening to the most remarkable enrichment of fourth round of antibodies, thereby indicating that the antibodies against PD-1 are enriched.

FIG. 2 shows the Fab form of PD-1 antibody expressed from E.coli, which was purified by nickel column affinity chromatography and then checked for purity by SDS-PAGE.

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

FIG. 4 FACS tests the binding capacity of P2-G12 to HEK293T cells overexpressing the full-length human PD-1 protein.

Detailed Description

Example 1 enrichment screening for Single Domain antibodies specific for human PD-1 protein

Screening methods the previously constructed library size of 1.5 × 10 was used as described in the reported literature¹¹The ultra-large phage-displayed fully human Fab antibody library of (4) was screened against biotin-labeled PD-1(ECD) -hFc fusion proteins. Immobilization of Biotin-labeled PD-1(ECD) -hFc fusion proteins on streptavidin-coated magnetic beads, 10¹²The antibody displayed by each phage is incubated with 5,4,2,1 microgram of antigen for two hours in 1,2,3,4 rounds at normal temperature, and the phage used in each round of screening is 10¹²An

EXAMPLE 2 polyclonal phage-ELISA assay

After multiple rounds of screening, the phage antibody obtained after each round of screening is subjected toThe library was tested by polyclonal phage ELISA to determine antibody enrichment. Mu.g/ml antigen was coated 50. mu.l per well on a 96-well ELISA plate overnight at 4 ℃. Blocking with 3% (m/v) skimmed milk powder in PBS (MPBS) for 1h, adding 10¹¹The phage antibody library supernatants obtained after 1,2,3, and 4 rounds of screening were incubated with the coated antigen at 37 ℃ for 1.5 h. Unbound phage were washed away and bound phage were detected with HRP-anti-M13 antibody. ABTS was added for color development, absorbance values at 405nm were read in a microplate reader, and antibodies showing specificity were enriched in round 3 according to the polyclonal phage ELISA results (as shown in FIG. 1).

EXAMPLE 3 soluble expression and purification of antibodies

For the expression and purification of the antibody Fab fragment, the selected phage plasmid of the positive clone is transferred into an E.coli HB2151 competent cell, a single colony is selected from an overnight-grown 2YT-AG plate, the single colony is inoculated into an SB culture medium, IPTG induced expression is carried out at the temperature of 30 ℃, bacteria are harvested and purified by Ni-NTA, the purity is detected by SDS-PAGE electrophoresis, and the result shows that the purity reaches more than 85 percent, so that the antibody Fab fragment can be used for subsequent experiments;

for construction of an IgG1 expression vector, firstly, Fab of a P2-G12 antibody is used as a template, a heavy chain and a light chain on a Fab phage plasmid are amplified by PCR, a PCR heavy chain product and a eukaryotic expression vector pTT-IgG1 are subjected to BsmBI enzyme digestion, a PCR light chain product and a pTT-IgG1 vector are subjected to SfiI enzyme digestion, the heavy chain and the light chain are cloned into pTT-IgG1 respectively under the action of T4 ligase, competent cells are transformed, clones are selected for sequencing, correct clones are identified as IgG1-Hc and IgG1-Lc through sequencing, then the IgG1-Hc and IgG1-Lc recombinant plasmids are co-transfected into Expi293 cells for transient expression, and affinity chromatography purification is carried out by Protein G resin.

Example 4 detection of the binding Capacity of the PD-1 antibody to the PD-1(ECD) -hFc protein

PD-1(ECD) -hFc protein is coated on an ELISA plate, an antibody diluted by gradient concentration is added for incubation, the HRP-anti-Fab antibody detects the binding capacity of the antibody and the PD-1(ECD) -hFc, and the ELISA result shown in figure 3 shows that P2-G12 can well bind the PD-1 protein and the binding capacity is enhanced by 100 times after the protein is converted into IgG 1.

EXAMPLE 5 cellular binding Capacity of monoclonal antibodies P2-G12 determined by flow cytometry

Cells in the logarithmic growth phase were collected and, after incubation with antibodies at different concentrations, detected by adding anti-human IgG1-FITC labeled antibody, it was shown that P2-G12 could bind to HEK293T cells overexpressing PD-1, but not to HEK293T cells themselves.

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Claims

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.