CN108203464B - High-affinity, high-specificity and high-functionality anti-human PD-1 antibody with multiple antigen recognition epitopes - Google Patents

Abstract

The invention discloses an anti-human PD-1 antibody with high affinity, high specificity and multiple antigen recognition epitopes and higher functionality. The PD-1 monoclonal antibody can be specifically combined with PD-L1, and can effectively block the combination of PD-L1 and PD-1 protein, specifically relieve the immune negative regulation of PD-1 and activate T cells to secrete cytokines. The functions of the antibody approach or reach the level of the current PD-1 targeting drug Keytruda (Pembrolizumab), and the antibody has different epitopes from Keytruda and has larger diversity.

Description

High-affinity, high-specificity and high-functionality anti-human PD-1 antibody with multiple antigen recognition epitopes

Technical Field

The invention belongs to the fields of tumor immunotherapy and molecular immunology, and particularly relates to a high-affinity, high-specificity and multi-antigen recognition epitope anti-human PD-1 antibody with higher functionality.

Background

Tumor immunotherapy has become a most important means for tumor therapy, and only after cancer cells are identified, an immune system can effectively attack the cancer cells, canceration is the result that normal somatic cells lose normal cell regulation function, and gene mutation is accumulated to a certain degree (Tian et al, 2011), but the cancer cells can disguise themselves as normal somatic cells, so that the attack of the immune system is ingeniously avoided. The tumor immunotherapy enhances the immune function of a human body, so that the T cells can better identify cancer cells, thereby playing a role in killing the cancer cells.

The immune system is the most effective mechanism for defending the body from invading exogenous objects, and a plurality of immune organs, tissues, cells and molecules cooperate with each other to achieve the aims of protecting the body from being infected from the outside and maintaining the homeostasis, and the mutual balance requires the coordination of a plurality of immune checkpoint proteins, the stimulating immune checkpoint protein enhances the defense reaction of the immune system, the inhibiting immune checkpoint protein controls the over-strong immune system, and prevents the generation of autoimmune reaction (Chen et al, 2013). The tumor immunotherapy is to improve the immunity of the anti-tumor, and amplify and strengthen the function of T cells in a proper way, so that the enhancement of the immunity can not generate autoimmune diseases. T cells play a dominant role in cellular immune response, mature T cells are distributed to various immune organs, and under the induction of antigens, the T cells are activated, proliferated and differentiated to form different effector T cells and memory T cells, and the activation of the T cells requires the synergistic action of double signals and cytokines. The primary signal of T cell activation is achieved by specific binding of the TCR on its surface to the antigen presented by the pMHC, however, T cell activation is not achieved solely by the action of the primary signal, and a secondary signal is required, which is antigen independent and different from one species of T cell, from the signal of interaction with its cell surface receptor or ligand and the corresponding co-stimulatory factor expressed on the APC. Stimulation of these two signals involves different immune checkpoint proteins, which can be divided into stimulatory and inhibitory immune checkpoint proteins, depending on the effect produced. In the development of therapeutic drugs for immune checkpoint proteins, agonists or stimulatory antibodies are developed for stimulatory immune checkpoint proteins, and inhibitors or inhibitory antibodies are developed for inhibitory immune checkpoint proteins.

PD-1 (Programmedcell death protein 1 or CD279), a membrane receptor expressed on T cells, progenitor B cells, activated B cells, and bone marrow cells (Bennett et al 2003), is a member of the B cell immunoglobulin supergene family, and is a member of the CD28 membrane receptor family with CD28, CTLA-4, ICOS and BTLA. PD-1 has two known ligands, PD-L1 and PD-L2, PD-1 plays a role in inhibiting immune regulation, when PD-1 is combined with its ligand (PD-L1 or PD-L2), it inhibits the proliferation of T cells, the secretion and function of cytokines through information conduction, and the current research shows that mRNA of PD-L1 is expressed in all tissues, but in tumor cells, PD-L1 is highly expressed, and through combining with PD-1 on T cells, the tumor cells inhibit the activation and proliferation of T cells, so that the tumor cells can escape immunologically. The relation between PD-1 and PD-L1 or PD-L2 has become an important link in tumor immunotherapy, the signal path between PD-1/PD-L1 or PD-1/PD-L2 has a direct relation with the occurrence and treatment of tumors, monoclonal antibody drugs capable of specifically blocking the binding of PD-1 and PD-L1 or PD-1 and PD-L2 are developed, the monoclonal antibody drugs are used for blocking the binding of PD-1 and PD-L1 or the binding of PD-1 and PD-L2 to generate negative regulation effect on T cells and enhancing the immune response of T cells to various antigens to carry out tumor immunotherapy, and the monoclonal antibody is a very useful treatment method.

Disclosure of Invention

The invention aims to provide a human PD-1 monoclonal antibody and application thereof.

Another object of the present invention is to provide a gene encoding the above human PD-1 monoclonal antibody.

Still another object of the present invention is to provide a method for preparing the above human PD-1 monoclonal antibody.

It is still another object of the present invention to provide an anti-tumor agent.

The purpose of the invention can be realized by the following technical scheme:

a human PD-1 monoclonal antibody, the protein sequence of which contains a heavy chain variable region and a light chain variable region, the monoclonal antibody is selected from any one of the following (1) - (3):

(1) the heavy chain variable region has the sequence shown in SEQ ID NO: 1, and the light chain variable region has an amino acid sequence shown as SEQ ID NO: 3;

(2) the heavy chain variable region has the sequence shown in SEQ ID NO: 5, and the light chain variable region has an amino acid sequence shown as SEQ ID NO: 7;

(3) the heavy chain variable region has the sequence shown in SEQ ID NO: 9, and the light chain variable region has an amino acid sequence shown as SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

The coding gene of the human PD-1 monoclonal antibody. The coding gene is selected from any one of the following (4) to (6):

(4) comprises the nucleotide sequence shown in SEQ ID NO: 2, and the nucleotide sequence shown as SEQ ID NO: 4, and a nucleotide sequence encoding the variable region of the monoclonal antibody light chain;

(5) comprises the nucleotide sequence shown in SEQ ID NO: 6, and the nucleotide sequence shown as SEQ ID NO: 8, and a nucleotide sequence encoding the variable region of the monoclonal antibody light chain;

(6) comprises the nucleotide sequence shown in SEQ ID NO: 10, and the nucleotide sequence shown as SEQ ID NO: 12, and a nucleotide sequence encoding the variable region of the monoclonal antibody light chain.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the coding gene.

The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain are applied to the preparation of the human PD-1 monoclonal antibody.

A method for preparing human PD-1 monoclonal antibody is to transfect competent cells with recombinant expression vector containing the coding gene and culture to obtain human PD-1 monoclonal antibody.

The unique V-region nucleotide/protein sequences of clones 1H10C4D4, 2E5E4D11, 154G5A5B7 were obtained by the skilled person as follows:

1) immunizing a mouse by using the recombinant expressed human PD-1 extracellular region to obtain an immune response aiming at the human PD-1;

2) fusing spleen cells of the mouse in the step 1), and screening obtained hybridoma cells to obtain a positive parent clone which specifically recognizes human PD-1 and can block the combination of PD-1 and PD-L1 protein;

3) subcloning the positive parent clone obtained in the step 2) to obtain a stable hybridoma cell strain;

4) sequencing the hybridoma cell strain obtained in the step 3) to obtain variable region coding sequences of the light chain and the heavy chain of the antibody.

Producing functional human PD-1 monoclonal antibody by recombinant antibody with the variable region coding sequence obtained in step 4).

The monoclonal antibody can be specifically bound with human PD-1, can block the binding of PD-1 and PD-L1 protein, and can relieve the immune negative regulation of PD-1 and activate T cells to secrete cytokines.

The functional human PD-1 monoclonal antibody is applied to the preparation of antitumor drugs.

An anti-tumor preparation comprising the functional human PD-1 monoclonal antibody.

The invention has the advantages of

The PD-1 monoclonal antibody can be specifically combined with PD-1, and can effectively block the combination of PD-L1 and PD-1 protein, specifically relieve the immune negative regulation of PD-1 and activate T cells to secrete cytokines. The functions of the antibody approach or reach the level of the current PD-1 targeting drug Keytruda (Pembrolizumab), and the antibody has different epitopes from Keytruda and has larger diversity.

Drawings

FIG. 1: detection of serum titers in mice after immunization

FIG. 2: the purified monoclonal antibody can specifically bind to human PD-1 recombinant protein

PD-1 antibody cloning and PD-1-Fc recombinant protein binding ELISA assay, Keytruda, EC₅₀=9.15 ng/ml; 1H10C4D4， EC₅₀=6.81 ng/ml; 2E5E4E11， EC₅₀=4.65 ng/ml; 154G5A5B7， EC₅₀=8.72 ng/ml。

FIG. 3: purified monoclonal antibodies are capable of specifically binding to cell lines expressing human PD-1

The PD-1 antibody clone bound to stable cells expressing PD-1 CHO-K1/GLP 1/G.alpha.15 (peak 2) but not to maternal CHO (peak 1).

FIG. 4: the purified monoclonal antibody can block the binding of PD-1 and PD-L1 protein

The binding of the recombinant protein PD-1 and the recombinant protein PD-L1 was inhibited by the PD-1 antibody clone. Keytruda, IC₅₀=0.2241 μg/ml; 1H10C4D4， IC₅₀=0.4934 μg/ml; 2E5E4E11， IC₅₀=0.4289 μg/ml; 154G5A5B7， IC₅₀=0.3662 μg/ml。

FIG. 5: the purified monoclonal antibody can relieve the immune negative regulation of PD-1 and stimulate T cells to secreteinterleukin 2

A. CD4⁺Co-culturing T cells and allogeneic dendritic cells, adding PD-1 antibody clones with different concentrations into a co-culture system, wherein the PD-1 antibody clone blocks the combination of PD-1 expressed on CD4+ T cells and PD-Ll expressed on dendritic cells, so that the activation of the T cells and the secretion of IL-2 are increased, and Keyruda: EC₅₀=0.1444 μg/ml; 1H10C4D4: EC₅₀=0.0640 μg/ml; 2E5E4E11: EC₅₀=0.9291 μg/ml; 154G5A5B7: EC₅₀=1.123 μg/ml。

B. PD-1 expressing utility cells were co-cultured with PD-L1 expressing target cells and Relative Light Units (RLU) were used as a measure of utility cell activation. Adding different concentrations of PD-1 antibody clones to the co-culture system, the PD-1 antibody clones blocking the binding of PD-1 expressed on utility cells and PD-Ll expressed on target cells, resulting in activation of utility cells, increased RLU reading, Keytruda: EC₅₀=0.3273 μg/ml; 1H10C4D4: EC₅₀=1.131 μg/ml; 2E5E4E11: EC₅₀=1.008 μg/ml; 154G5A5B7: EC₅₀= 1.422 μg/ml。

Detailed Description

The present invention relates to a human PD-1 antibody having functionality, and embodiments of the present invention will be described in detail with reference to examples. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated, the methods and materials of the examples described below are all conventional products available on the market. Those skilled in the art to which the invention pertains will appreciate that the methods and materials described below are illustrative only and should not be taken as limiting the scope of the invention.

Example 1: acquisition of human PD-1 hybridoma cell strain and preparation of monoclonal antibody

1) Animal immunization

The antigen employed was the recombinant protein PD-1-Fc (GenScript, Z03370) fused to the extracellular domain of human PD-1 of human IgG1 Fc fragment. Female Balb/C and C57bl/6 mice were immunized subcutaneously with 200. mu.l Freund's complete adjuvant (Sigma-Aldrich) 1:1 emulsion containing 50. mu.g of PD-1-Fc fusion protein. Mice were then boosted by intraperitoneal/subcutaneous injections of up to 3 times a 1:1 emulsion containing 25 μ g PD-1-Fc in Freund's incomplete adjuvant (Sigma-Aldrich) every two weeks. Serum titers of 10 mice all reached 10 after triple immunization⁵The above. Two mice (no 834 and no 837) exhibiting the highest antibody titers (fig. 1) received intraperitoneal booster immunizations of 25ug PD-1-Fc (without adjuvant) 4 days prior to myeloma fusion.

2) Hybridoma fusion and screening

Spleens were extracted and homogenized to produce a single cell suspension, while myeloma cells (SP 2/0) were prepared as a single cell suspension. Use electrical fusion to mix 8.9X 10⁷Spleen cells and 4.1X 10⁷Individual SP2/0 mouse myeloma cells were fused. The fused cells were resuspended in 100ml DMEM/10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine and aminopterin and pipetted into 50X 96 well plates in a volume of 100. mu.l. Plates were incubated at 37 ℃ in 6% CO₂And (4) carrying out incubation. After 7 days of incubation, ELISA binding, ELISA competition and FACS binding as described below were startedAntibodies against PD-1-Fc were tested for the presence.

ELISA binding detection method: an indirect ELISA was used to assess the binding capacity of the antibody to PD-1-Fc in the supernatant. ELISA plates (Nunc) were coated overnight at 4 ℃ with 0.5. mu.g/ml recombinant PD-1-Fc or human IgG1 in 100. mu.l/well PBS. Plates were washed with PBS-T (0.05% Tween) and blocked with 200. mu.l/well of 1% BSA in PBST for 0.5 h at 37 ℃. The blocking solution was then discarded and 100. mu.l of hybridoma cell culture supernatant was added to each plate, followed by incubation at room temperature for 1 hour. The plates were washed three times with PBST and incubated with 100. mu.l/well of horseradish peroxidase-conjugated goat anti-mouse IgG (Fab-specific) (GenScript) for 0.5 h at 37 ℃. The plates were washed five times with PBST, then TMB color developing solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark. The reaction was stopped by adding 50. mu.l of 1MHCl stop buffer (Sigma). The plate was read using a microplate reader at 450 nm.

ELISA competition detection method: a competition ELISA was used to assess the blocking ability of the antibodies in the supernatant for the binding of PD-1 and its ligand PD-L1 protein. ELISA plates (Nunc) were coated overnight at 4 ℃ with 0.5. mu.g/ml recombinant human PD-1 protein in 100. mu.l/well PBS. Plates were washed with PBS-T (0.05% Tween) and blocked with 200. mu.l/well of 1% BSA in PBST for 0.5 h at 37 ℃. Blocking solution was then discarded and 50. mu.l of test supernatant was added to each test well and 50. mu.l of irrelevant supernatant was added to the control well. Then 50. mu.l biotin-labeled PD-1-Fc (concentration 0.15. mu.g/ml) was added per well and incubated at 37 ℃ for 1 hour. Plates were washed three times with PBST and incubated with 100. mu.l/well streptavidin-HRP (SA-HRP, GenScript) for 10 min at 37 ℃. The plates were finally washed five times with PBST, then TMB colour developing solution (GenScript) was added and incubated for 15 min at room temperature in the dark. The reaction was stopped by adding 50. mu.l of 1MHCl stop buffer (Sigma). The plate was read using a microplate reader at 450 nm.

FACS detection method: FACS binding experiments were used to assess the binding ability of antibodies in the supernatant to PD-1 expressed on the surface of CHO cell membranes. CHO cells expressing PD-1 and the mother cells of the negative control for detection were collected and washed 3 times with PBS. Adding into 96-well plate2.5X10⁵The test cells and test supernatants were incubated at 100ul for 1 hour at 4 ℃. The cells were then washed 3 times with PBS, 100. mu.l of iFluor-labeled goat anti-mouse IgG was added, and incubated for 45 min at 4 ℃. Finally, cells were washed 3 times with PBS and signals were read with FACS BD Calibur.

3) Hybridoma subcloning

Subcloning was performed using limiting dilution method. The cell number was determined using a hemocytometer and serial dilutions of cells in DMEM/10% FBS medium containing the hybridoma cell selection agents thymidine, hypoxanthine and aminopterin until the cell density reached 5-15 cells/ml. For each hybridoma, 200. mu.l of the cell solution was pipetted into 96 wells at a density of 1-3 cells/well. Cultures were incubated at 37 ℃ in 5% CO₂After 1 week of medium culture, supernatants were subjected to the above ELISA binding, ELISA competition and FACS binding tests to assess the presence of antibodies against PD-1-Fc.

Example 2: variable region sequencing of monoclonal antibodies and recombinant production of antibodies

After subtype identification of monoclonal antibodies using a Rapid ELISA mouse antibody subtype identification kit (cloning System-HRP, southern Biotech), TRIzol (Ambion) was used from 3X 10⁶-5×10⁶Total RNA was extracted from each hybridoma cell and reverse-transcribed into cDNA using antibody subtype specific primers and universal primers (PrimeScript 1stStrand cDNA Synthesis Kit, Takara). Murine immunoglobulin heavy and light chain V-region fragments were then amplified by RACE PCR (GenScript) and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara) and the inserts were sequenced using vector-specific primers. The unique V-region nucleotide/protein sequences of clones 1H10C4D4, 2E5E4D11, 154G5A5B7 were finally obtained.

1H10C4D4 heavy chain variable region amino acid sequence:SEQ ID NO 1

1H10C4D4 heavy chain variable region DNA sequence:SEQ ID NO 2

1H10C4D4 light chain variable region amino acid sequence:SEQ ID NO 3

1H10C4D4 light chain variable region DNA sequence:SEQ ID NO 4

2E5E4D11 heavy chain variable region amino acid sequence:SEQ ID NO 5

2E5E4D11 heavy chain variable region DNA sequence:SEQ ID NO 6

2E5E4D11 light chain variable region amino acid sequence:SEQ ID NO 7

2E5E4D11 light chain variable region DNA sequence: SEQ ID NO 8

154G5A5B7 heavy chain variable region amino acid sequence: SEQ ID NO 9

154G5A5B7 heavy chain variable region DNA sequence: SEQ ID NO 10

154G5A5B7 light chain variable region amino acid sequence: 11 SEQ ID NO

154G5A5B7 light chain variable region DNA sequence: SEQ ID NO 12

DNA fragments comprising the light chain variable region + constant region and the heavy chain variable region + constant region were synthesized separately and inserted into pTT5 expression vectors, respectively, to form expression plasmids.

The above plasmids were co-transfected into HEK293-6E cells and cultured in a 37 ℃ shake flask for 10 days, and then the supernatant was collected for antibody purification. Prior to purification, the tubing and protein A column were depyrogenated with 0.2M NaOH. The column was re-equilibrated with buffer containing 0.05M Tris and 1.5M NaCl (pH = 8.0). The harvested cell culture supernatant was subsequently diluted 1:1 with 2 × above buffer and filter sterilized. The filtered supernatant and the protein a column were incubated at room temperature for 2 hours, after washing the column with 1 × the above buffer, IgG was eluted using sterile 0.1M sodium citrate (pH 3.5), and the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH = 9.0). Under sterile conditions, the product buffer was exchanged for PBS (ph 7.4) to remove any elution buffer and concentrate the sample. After concentration, the antibody was quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

Purified antibodies were analyzed by SDS-PAGE with 10% pre-gel (GenScript) by a BioRad electrophoresis system. The gel was stained with estain2.0 (GenScript) and molecular size and purity were estimated by comparing the stained bands to Protein Ladder (GenScript).

Example 3: binding of monoclonal antibodies to human PD-1 recombinant proteins

An indirect ELISA was used to assess the binding ability of the purified antibody to PD-1-Fc. ELISA plates (Nunc) were coated overnight at 4 ℃ with 0.5. mu.g/ml recombinant PD-1-Fc or human IgG1 in 100. mu.l/well PBS. Plates were washed with PBS-T (0.05% Tween) and blocked with 200. mu.l/well of 1% BSA in PBST for 0.5 h at 37 ℃. Blocking solution was then discarded and 100. mu.l of purified antibody at 10. mu.g/ml was added to the first well and diluted in 3-fold gradients for a total of 11 concentration gradients tested. Then incubated at room temperature for 1 hour. The plates were washed 3 times with PBST and incubated with 100. mu.l/well of goat anti-mouse IgG conjugated to horseradish peroxidase (Fab-specific) (GenScript) for 0.5 h at 37 ℃. The plates were washed five times with PBST, then TMB color developing solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark. The reaction was stopped by adding 50. mu.l of 1MHCl stop buffer (Sigma). The plate was read using a microplate reader at 450 nm. As shown in FIG. 2, the PD-1 antibody clones were subjected to ELISA test for binding to PD-1-Fc recombinant protein, and EC of each clone was determined₅₀The following were used: keytruda EC₅₀=9.15 ng/ml; 1H10C4D4: EC₅₀=6.81 ng/ml; 2E5E4E11: EC₅₀=4.65 ng/ml; 154G5A5B7: EC₅₀=8.72 ng/ml, and the clones tested reached or exceeded their antigen binding capacity compared to Keytruda.

Example 4: binding of monoclonal antibodies to cell lines expressing human PD-1

CHO cells expressing PD-1 and the mother cells of the negative control for detection were collected and washed 3 times with PBS. Add 2.5X10 to 96-well plates⁵Each test cell was incubated with 100ul of 5. mu.g/ml purified antibody for 1 hour at 4 ℃. Cells were subsequently washed 3 times with PBS, 100 μ Ι fluor-labeled goat anti-mouse IgG added, and incubated for 45 min at 4 ℃. Finally, cells were washed 3 times with PBS and signals were read with FACS BD Calibur. As shown in FIG. 3, the binding of clones and cell lines showed strong binding ability in all the figures, and the FACS drift was 1 log or more.

Example 5: monoclonal antibody blocks binding of PD-1 and PD-L1 protein

ELISA plates (Nunc) were coated overnight at 4 ℃ with 0.5. mu.g/ml recombinant human PD-1 protein in 100. mu.l/well PBS.Plates were washed with PBS-T (0.05% Tween) and blocked with 200. mu.l/well of 1% BSA in PBST for 0.5 h at 37 ℃. Subsequently, the blocking solution was discarded, 50. mu.l of the purified antibody to be tested was added to the first test well and diluted in 3-fold gradient for a total of 11 test concentration gradients. Then 50. mu.l biotin-labeled PD-L1-Fc (concentration 0.15. mu.g/ml) was added per well and incubated at 37 ℃ for 1 hour. Plates were washed three times with PBST and incubated with 100. mu.l/well streptavidin-HRP (SA-HRP, GenScript) for 10 min at 37 ℃. The plates were finally washed five times with PBST, then TMB colour developing solution (GenScript) was added and incubated for 15 min at room temperature in the dark. The reaction was stopped by adding 50. mu.l of 1MHCl stop buffer (Sigma). The plate was read using a microplate reader at 450 nm. As shown in FIG. 4, IC of each clone₅₀Keytruda, IC as follows₅₀=0.2241 μg/ml; 1H10C4D4, IC₅₀=0.4934 μg/ml; 2E5E4E11, IC₅₀=0.4289 μg/ml; 154G5A5B7, IC₅₀=0.3662 μ g/ml, the blocking capacity of the 3 clones was very close to that of the positive drug Keytruda.

Example 6: monoclonal antibody epitope identification

Competition ELISAs were used to evaluate the epitopes of the purified antibodies. ELISA plates (Nunc) were coated overnight at 4 ℃ with 0.5. mu.g/ml recombinant PD-1-Fc in 100. mu.l/well PBS. Plates were washed with PBS-T (0.05% Tween) and blocked with 200. mu.l/well of 1% BSA in PBST for 0.5 h at 37 ℃. The blocking solution was then discarded and a pair (one of which was labeled biotin) of the test antibodies used in the competition assay was added to each well, 100. mu.l (10. mu.g/ml) of each purified antibody. Then incubated at 37 ℃ for 1 hour. Plates were washed three times with PBST and incubated with 100. mu.l/well streptavidin-HRP (SA-HRP, GenScript) for 10 min at 37 ℃. The plates were washed five times with PBST, then TMB color developing solution (GenScript) was added and incubated for 15 minutes at room temperature in the dark. The reaction was stopped by adding 50. mu.l of 1MHCl stop buffer (Sigma). The plate was read using a microplate reader at 450 nm. 1H10C4D4, 2E5E4E11 and 154G5A5B7 were all the same epitope as the positive control, but the epitope for 23F12A9 was different from that of all other clones.

Example 7: functional detection of monoclonal antibodies

CD was isolated and purified from human peripheral blood mononuclear cells in a mixed lymphocyte reaction by means of a kit (Miltenyl Biotec)⁴⁺T cells and allogeneic monocytes. Inducing monocytes to become dendritic cells. Each well containing 10⁵Personal CD⁴⁺T cells and 10⁴Individual allogeneic monocytes, the final working volume was 200 μ l. Antibody samples of different concentrations were added to each well. Wells without antibody as background control, human IgG4 antibody as negative control, pembrolizumab (keytruda), Merck&Co., Inc) as a positive control for PD-1 antibody. 37 ℃ and 5% CO₂Under these conditions, after 72 hours of incubation, 100. mu.l of supernatant was taken from each well to detect IL-2 content (Cisbio's assay kit).

In the test of functional detection of antibody at cellular level, human PD-1 and a fluorescent reporter gene controlled by NFAT nuclear transcription response element (NFAT-RE) are stably transfected in utility cells, human PD-L1 and cell surface protein antigen polypeptide/Major Histocompatibility Complex (MHC) are stably transfected in target cells as artificial antigen presenting cells, the utility cells expressing PD-1 are co-cultured with the target cells expressing PD-L1, and a Relative Light Unit (RLU) is used as an index for activation of the utility cells. Different concentrations of PD-1 antibody clones were added to the co-culture system, and the PD-1 antibody clones blocked the binding of PD-1 expressed on utility cells and PD-Ll expressed on target cells, resulting in activation of utility cells and increased RLU readings. As shown in FIG. 5, all of these tested clones achieved or exceeded their function compared to Keytruda.

SEQUENCE LISTING

<110> Nanjing Kinsrui Biotechnology Ltd

<120> anti-human PD-1 antibody with high affinity, high specificity, multiple antigen recognition epitopes and higher functionality

<130> 2016

<160> 12

<170> PatentIn version 3.3

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130 135

<210> 6

<211> 414

<212> DNA

<213> Artificial sequence

<400> 6

atgaatttcg ggctcagctt gattttcctt gtccttgttt taaaaggtgt cctgtgtgaa 60

gtgaagctgg tggagtctgg gggagtttta gtgcagcctg gagggtccct gaaactctcc 120

tgtgcagcct ctggattcac tttcagtagc tataccatgt cttgggttcg ccagactccg 180

gagaagaggc tggagtgggt cgcatacatt agtggtggtg gtggtgacac ctactatcca 240

gacactgtga agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg 300

caaatgaaca gtctgaagtc tggggacacg gccatgtatt actgtgcaag acatggttac 360

gacgctgcct gttttgctta ctggggccaa gggactctgg tcactgtctc tgca 414

<210> 7

<211> 131

<212> PRT

<213> Artificial sequence

<400> 7

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Phe Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

100 105 110

Gln Gln His Tyr Ser Ile Pro Trp Thr Val Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210> 8

<211> 393

<212> DNA

<213> Artificial sequence

<400> 8

atgggcatca agatggagtc acagattcag gcatttgtat tcgtgtttct ctggttgtct 60

ggtgttgacg gagacattgt gatgacccag tctcacaaat tcatgtccac atcagttgga 120

gacagggtca gcttcacctg caaggccagt caggatgtga atactgctgt ggcctggtat 180

caacaaaaac cagggcaatc tcctaaacta ctgatttact gggcatccac ccggcacact 240

ggagtccctg atcgcttcac aggcagtgga tctgggacag attatactct caccatcagc 300

agtgtgcagg ctgaagacct ggcgctttat tactgtcagc aacactatag cattccgtgg 360

acggtcggtg gaggcaccaa gctggaaatc aaa 393

<210> 9

<211> 135

<212> PRT

<213> Artificial sequence

<400> 9

Met Ala Val Leu Ala Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys

1 5 10 15

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

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Thr Val Tyr Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Met Ile Trp Gly Asp Gly Thr Thr Asp Tyr Asn Ser

65 70 75 80

Ala Leu Lys Ser Arg Leu Ile Ile Asn Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr

100 105 110

Tyr Cys Ala Arg Asp Asp Tyr Gly Thr Phe Leu Tyr Trp Gly Gln Gly

115 120 125

Thr Leu Val Thr Val Ser Ala

130 135

<210> 10

<211> 405

<212> DNA

<213> Artificial sequence

<400> 10

atggctgtcc tggcattact cttctgcctg gtaacattcc caagctgtat cctttcccag 60

gtgcagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcaca 120

tgcaccgtct cagggttctc attaaccgtc tatggtgtaa actgggttcg ccagcctcca 180

ggaaagggtc tcgagtggct ggggatgatt tggggtgatg gaaccacaga ctataattca 240

gctctcaaat ccagactgat catcaacaag gacaactcca agagccaagt tttcttaaaa 300

atgaacagtc tgcaaactga tgacacagcc aggtactact gtgccagaga tgactatggt 360

accttccttt actggggcca agggactctg gtcactgtct ctgca 405

<210> 11

<211> 132

<212> PRT

<213> Artificial sequence

<400> 11

Met Asp Ser Gln Ala Gln Val Leu Ile Leu Leu Leu Leu Trp Val Ser

1 5 10 15

Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala

20 25 30

Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg

65 70 75 80

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

85 90 95

Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr

100 105 110

Tyr Cys Lys Gln Ser Tyr Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys

115 120 125

Leu Glu Ile Lys

130

<210> 12

<211> 396

<212> DNA

<213> Artificial sequence

<400> 12

atggattcac aggcccaggt tcttatattg ctgctgctat gggtatctgg tacctgtggg 60

gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 120

atgagttgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 180

tggtatcagc agaaaccagg gcagtctcct aaactgctga tcttctgggc atccactagg 240

gaatctgggg tccctgatcg cttcacaggc agtgtatctg ggacagattt cactctcacc 300

atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttatactctt 360

cggacgttcg gtggaggcac caagctggaa atcaaa 396

Claims (8)

1. A human PD-1 monoclonal antibody characterized by: the protein sequence of the monoclonal antibody contains a heavy chain variable region and a light chain variable region, and the heavy chain variable region of the monoclonal antibody is represented by SEQ ID NO: 1, and the light chain variable region is SEQ ID NO: 3.

2. A gene encoding the human PD-1 monoclonal antibody of claim 1.

3. The encoding gene of claim 2, wherein: the coding gene contains the nucleotide sequence shown as SEQ ID NO: 2 and the nucleotide sequence shown as SEQ ID NO: 4, and the nucleotide sequence is used for coding the variable region of the light chain of the human PD-1 monoclonal antibody.

4. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the coding gene of claim 3.

5. Use of the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 4 for the preparation of human PD-1 monoclonal antibody.

6. A method for preparing a human PD-1 monoclonal antibody, comprising: transfecting competent cells with a recombinant expression vector comprising the coding gene of claim 2 or 3, and culturing to obtain a human PD-1 monoclonal antibody.

7. The use of the human PD-1 monoclonal antibody of claim 1 in the preparation of an anti-tumor medicament.

8. An anti-tumor formulation characterized by: comprising the human PD-1 monoclonal antibody of claim 1.

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