CN108203464A

CN108203464A - High-affinity, high specific, more antigen recognizing epitopes the anti-human PD-1 antibody with higher function

Info

Publication number: CN108203464A
Application number: CN201611212475.2A
Authority: CN
Inventors: 殷刘松; 张贵斌; 蒋忻坡; 林峰; 张辉辉; 覃喜建; 赵涛
Original assignee: Nanjing Jinsirui Science and Technology Biology Corp
Current assignee: Nanjing Jinsirui Science and Technology Biology Corp
Priority date: 2016-12-25
Filing date: 2016-12-25
Publication date: 2018-06-26
Anticipated expiration: 2036-12-25
Also published as: CN108203464B

Abstract

The present invention disclose a kind of high-affinity, high specific, more antigen recognizing epitopes 1 antibody of anti-human PD with higher function.1 monoclonal antibodies of PD of the present invention are capable of being combined with PD L1, and can be effectively blocked the combination of 1 albumen of PD L1 and PD for specificity, specifically release the immune negative regulator of PD 1, activate T cell secrete cytokines.Above-mentioned function is close to or up to the level of current 1 targeted drug Keytruda (Pembrolizumab) of PD, and there are one the epitopes that antibody is different from Keytruda, have the diversity of bigger.

Description

High-affinity, high specific, more antigen recognizing epitopes have the anti-of higher function People's PD-1 antibody

Technical field

The invention belongs to tumour immunotherapies and molecular immunology field, and in particular to a kind of high-affinity, high specific, The anti-human PD-1 antibody with higher function of more antigen recognizing epitopes.

Background technology

Immunotherapy of tumors has become a most important means of oncotherapy, only after cancer cell is identified, is immunized System can attack cancer cell, canceration be effectively that normal somatic cell loses normal cell regulatory function, and gene mutation adds up To the result (Tian et al., 2011) of certain Cheng Du, still, cancer cell can disguise oneself as oneself normal somatic cell, ingenious The attack for avoiding immune system.Immunotherapy of tumors enables T cell preferably to identify by enhancing the immune function of human body Cancer cell, so as to play the role of killing cancer cell.

Immune system be body defence allogene invasion most effective mechanism, by multiple immune organs, tissue, cell and Molecule cooperates, and mutual containing protects body to infect from external and safeguard homeostasis to reach, this to cooperate, phase Mutual containing needs the coordination of numerous immunologic test point albumen, and the defence of irritation immunologic test point albumen enhancing immune system is anti- Should, inhibition immunologic test point albumen controls too strong immune system, prevent autoimmune response (Chen et al., 2013).Immunotherapy of tumors is exactly to promote antitumor immunocompetence, and the amplification to stop before going too far and the function of strengthening T cell make Autoimmune disease will not be generated by obtaining the enhancing of this immune function.T cell plays a leading role in cell immune response, development Ripe T cell is distributed to each immune organ, and under the induction of antigen, T cell activation is proliferated and breaks up, formed different Effector T cell and memory T cell, the activation of T cell need the synergistic effect of dual signal and cell factor.The of t cell activation One stage signal is completed by the specific binding of the antigen with the TCR and pMHC on its surface presentation, however, T cell swashs Work is not that only can just be completed by first order signal function, it is also necessary to which second level signal, the second level signal of T cell is anti- Former dependent/non-dependent, the second level signal of T cell not of the same race is also had nothing in common with each other, the second level signal of t cell activation come from The receptor or ligand of its cell surface and the signal after the corresponding collaboration stimulating factor interaction expressed on APC.This two The stimulation of a signal is related to different immunologic test point albumen, different according to the effect of generation, immunologic test point albumen can minute For irritation immunologic test point albumen and inhibition immunologic test point albumen.In the curative drug of research and development immunologic test point albumen When, to irritation immunologic test point albumen, agonist or irritation antibody are developed, it, to inhibition immunologic test point albumen Develop inhibitor or inhibiting antibody.

PD-1 (Programmed cell death protein 1 or CD279) is expression in T cell, ancestral's B cell, quilt Membrane receptor (Bennett et al.2003) in the B cell and bone marrow cell of activation, it belongs to b cell immunoglobulin A member in supergene family, with CD28, CTLA-4, ICOS and BTLA belong to CD28 membrane receptors family.PD-1 has two Known ligand, PD-L1 and PD-L2, PD-1 play inhibition immunoregulation effect, when PD-1 and it ligand (PD-L1 or When PD-L2) combining, by Information Conduction, it inhibits the proliferation of T cell, the secretion of cell factor and function, current research It was found that the mRNA of PD-L1 has expression in all tissues, but in tumour cell, PD-L1 is high expression, by thin with T PD-1 on born of the same parents combines, the activation of inhibiting tumour cells T cell and proliferation so that tumour cell being capable of immunologic escape.PD-1 The important link in immunotherapy of tumors is become with the relationship of PD-L1 or PD-L2, between PD-1/PD-L1 or PD-1/PD-L2 The generation and treatment of signal path and tumour have direct relation, and research and development being capable of specific inhibition PD-1 and PD-L1 or PD-1 and PD- L2 combine monoclonal antibody drug, be used to block PD-1 and PD-L1 with reference to or PD-1 and PD-L2 with reference to and generate To T cell down regulation, enhancing T cell carries out immunotherapy of tumors to the immune response of various antigens, is one non- The therapy of Chang Youyong, this patent develop new anti-human source PD-1 functional monoclonal antibodies, are used to block PD-1 With the combination of PD-L1 or PD-1 and PD-L2.

Invention content

The object of the present invention is to provide a kind of people PD-1 monoclonal antibodies and its applications.

Another object of the present invention is to provide the encoding gene of above-mentioned people PD-1 monoclonal antibodies.

Another object of the present invention is to provide the preparation method of above-mentioned people PD-1 monoclonal antibodies.

Another object of the present invention is to provide a kind of anti-tumor agent.

The purpose of the present invention can be achieved through the following technical solutions：

A kind of people PD-1 monoclonal antibodies, the protein sequence of the monoclonal antibody contain heavy chain variable region and light chain variable Area, the monoclonal antibody are selected from any one in following (1)~(3)：

(1) heavy chain variable region described in has such as SEQ ID NO：Amino acid sequence shown in 1, the light chain variable region With such as SEQ ID NO：Amino acid sequence shown in 3；

(2) heavy chain variable region described in has such as SEQ ID NO：Amino acid sequence shown in 5, the light chain variable region With such as SEQ ID NO：Amino acid sequence shown in 7；

(3) heavy chain variable region described in has such as SEQ ID NO：Amino acid sequence shown in 9, the light chain variable region With such as SEQ ID NO：Amino acid sequence shown in 11.

The encoding gene of above-mentioned people PD-1 monoclonal antibodies.The encoding gene is selected from any one in following (4)~(6) Kind：

(4) containing such as SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody heavy variable region shown in 2, with And such as SEQ IDNO：The nucleotide sequence of the coding monoclonal antibody light chain variable region shown in 4；

(5) containing such as SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody heavy variable region shown in 6, with And such as SEQ IDNO：The nucleotide sequence of the coding monoclonal antibody light chain variable region shown in 8；

(6) containing such as SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody heavy variable region shown in 10, And such as SEQ IDNO：The nucleotide sequence of the coding monoclonal antibody light chain variable region shown in 12.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned encoding gene.

Above-mentioned recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium is preparing people's PD-1 monoclonal antibodies In application.

A kind of method for preparing people's PD-1 monoclonal antibodies is felt being transfected comprising the recombinant expression carrier of above-mentioned encoding gene By state cell, and cultivated to obtain people's PD-1 monoclonal antibodies.

Invention technician has been prepared by the following the uniqueness of clone 1H10C4D4,2E5E4D11,154G5A5B7 V- Region Nucleotides/protein sequence：

1) mouse is immunized with people's PD-1 extracellular regions of recombinant expression, obtains the immune response for people PD-1；

2) spleen cell of mouse in step 1) is taken to be merged, and the hybridoma of acquisition is screened, is obtained Specific recognition people PD-1, and the protein bound positive female clones of PD-1 and PD-L1 can be blocked；

3) the female clone of the positive obtained in step 2) is subcloned, obtains stable hybridoma cell strain；

4) it is sequenced with the hybridoma cell strain obtained in step 3), obtains the variable region coding of antibody light chain and heavy chain Sequence.

The functional human PD-L 1 monoclonal of recombinant antibodies production is carried out with the variable region encoding sequences obtained in step 4) to resist Body.

Monoclonal antibody of the present invention can specifically bind people PD-1, can block PD-1 and PD-L1 albumen knots It closes, and the immune negative regulator of PD-1 can be released, activate T cell secrete cytokines.

Above-mentioned functional people PD-1 monoclonal antibody application in preparations of anti-tumor drugs.

A kind of anti-tumor agent, it includes above-mentioned functional people PD-1 monoclonal antibodies.

Beneficial effects of the present invention

The PD-1 monoclonal antibodies of the present invention are capable of being combined with PD-L1, and can be effectively blocked PD-L1 for specificity And the combination of PD-1 albumen specifically releases the immune negative regulator of PD-1, activates T cell secrete cytokines.Above-mentioned function connects Level that is near or reaching current PD-1 targeted drugs Keytruda (Pembrolizumab), and be different from there are one antibody The epitope of Keytruda has the diversity of bigger.

Description of the drawings

Fig. 1：Mouse serum titer detection after immune

Fig. 2：Monoclonal antibody purification can specifically bind people's PD-1 recombinant proteins

PD-1 antibody clonings are tested with PD-1-Fc recombinant protein combinations ELISA, Keytruda, EC₅₀=9.15ng/ml； 1H10C4D4, EC₅₀=6.81ng/ml；2E5E4E11, EC₅₀=4.65ng/ml；154G5A5B7, EC₅₀=8.72ng/ml.

Fig. 3：Monoclonal antibody purification can specifically bind the cell line of expression people PD-1

PD-1 antibody clonings with expression PD-1 CHO-K1/GLP1/G α 15 stablize cell combination (peak 2) without with mother cell CHO combines (peak 1).

Fig. 4：Monoclonal antibody purification can block the combination of PD-1 and PD-L1 albumen

The combination of recombinant protein PD-1 and recombinant protein PD-L1 are inhibited by PD-1 antibody clonings.Keytruda, IC₅₀ =0.2241 μ g/ml；1H10C4D4, IC₅₀=0.4934 μ g/ml；2E5E4E11, IC₅₀=0.4289 μ g/ml；154G5A5B7, IC₅₀=0.3662 μ g/ml.

Fig. 5：Monoclonal antibody purification can release the immune negative regulator of PD-1, and stimulation T cell secretes interleukin-22

A.CD4⁺The dendritic cells of T cell and allogeneic co-culture, and the PD- of various concentration is added in co-culture system 1 antibody cloning, PD-1 antibody clonings block PD-1 and expression PD-Ll on dendritic cells of the expression on CD4+T cells With reference to leading to the activation of T cell, IL-2 secretions increase, Keyruda:EC₅₀=0.1444 μ g/ml；1H10C4D4:EC₅₀= 0.0640μg/ml；2E5E4E11:EC₅₀=0.9291 μ g/ml；154G5A5B7:EC₅₀=1.123 μ g/ml.

B. the effectiveness cell of expression PD-1 and the target cell of expression PD-L1 co-culture, and relative light unit (RLU) is for weighing The index of effectiveness cell-stimulating.The PD-1 antibody clonings of various concentration are added in co-culture system, PD-1 antibody clonings block The combination of the PD-Ll of PD-1 and expression on target cell on effectiveness cell is expressed, leads to the activation of effectiveness cell, RLU readings Increase, Keytruda:EC₅₀=0.3273 μ g/ml；1H10C4D4:EC₅₀=1.131 μ g/ml；2E5E4E11:EC₅₀=1.008 μ g/ml；154G5A5B7:EC₅₀=1.422 μ g/ml.

Specific embodiment

The present invention relates to one kind to have functional people PD-1 antibody, below in conjunction with embodiment to the embodiment party of the present invention Case is described in detail.Unless otherwise indicated, the technical and scientific term used in the present invention with it is of the art common The meaning that technician is generally understood is identical.Unless otherwise indicated, the method for embodiment as described below and material be can be with The conventional products obtained by market purchase.Fields technician of the present invention will be understood that, method described below and material, It is only exemplary, and should not be taken as limiting the scope of the invention.

Embodiment 1：The acquisition of people's PD-1 hybridoma cell strains and the preparation of monoclonal antibody

1) animal immune

Antigen uses the recombinant protein PD-1-Fc for being fused to people's PD-1 extracellular domains of Fc sections of human IgG1 (GenScript, Z03370).With the 200 μ l Freund's complete adjuvants (Sigma-Aldrich) containing 50 μ g PD-1-Fc fusion proteins 1:1 lotion subcutaneous inoculation female Balb/c and C57bl/6 mouse.Then, every two weeks abdominal cavities/subcutaneous alternate injection contains 25 μ g The 1 of the incomplete Freund's adjuvant (Sigma-Aldrich) of PD-1-Fc:1 lotion is for up to 3 times, so as to carry out strengthening exempting to mouse Epidemic disease.10 mice serum potency reach 10 after exempting from three⁵More than.4 days before myeloma fusion, highest antibody titer is shown Two mouse (No. 834 and No. 837) of (Fig. 1) receive the abdominal cavity booster immunization of 25ug PD-1-Fc (without adjuvant).

2) hybridoma fusion and screening

Extraction spleen simultaneously homogenizes to generate single cell suspension, while it is unicellular to prepare myeloma cell (SP2/0) Suspension.Using electro' asion by 8.9 × 10⁷A splenocyte and 4.1 × 10⁷A SP2/0 murine myeloma cells are merged.It will melt The cell of conjunction is resuspended in the DMEM/ of 100ml selective agents containing hybridoma thymus nucleoside pyrimidine, hypoxanthine and aminopterin-induced syndrome In 10%FBS culture mediums, and moved in 50 × 96 orifice plates with the volume of 100 μ l with pipette.By plate in 6%CO at 37 DEG C₂ Middle incubation.After the incubation of 7 days, ELISA described below is begun to use to combine, ELISA competitions and FACS are with reference to carrying out testing needle To the antibody of PD-1-Fc there are situations.

ELISA combination detection methods：Indirect ELISA is for assessing in supernatant antibody for the binding ability of PD-1-Fc. The recombination PD-1-Fc of 0.5 μ g/ml or human IgG1 in the PBS in 100 μ l/ holes of elisa plate (Nunc) are coated with overnight at 4 DEG C. Plate is washed, and its PBST containing 1%BSA with 200 μ l/ holes is closed 0.5 hour at 37 DEG C with PBS-T (0.05% tween). Confining liquid then is discarded, 100 μ l Hybridoma Cell Culture supernatants is added in each plate, is then incubated at room temperature 1 hour.It will Plate is washed three times with PBST, and the goat anti-mouse IgG (Fab- specificity) of the conjugated horseradish peroxidase with 100 μ l/ holes (GenScript) it is incubated 0.5 hour for 37 DEG C.Plate with PBST is washed five times, then adds in TMB developing solutions (GenScript) simultaneously It is incubated 15 minutes in the dark at room temperature.Reaction is terminated by the 1MHCl terminate liquids (Sigma) for adding in 50 μ l.Use enzyme mark Instrument read plate at 450 nm.

ELISA race detection methods：Competitive ELISA be used to assess the antibody of supernatant kind for PD-1 and its ligand The blocking ability of the combination of PD-1 albumen.By the recombined human PD-1 of 0.5 μ g/ml in the PBS in 100 μ l/ holes of elisa plate (Nunc) Albumen is coated with overnight at 4 DEG C.Wash plate with PBS-T (0.05% tween), and by it with 200 μ l/ holes containing 1%BSA's PBST is closed 0.5 hour at 37 DEG C.Confining liquid is then discarded, each instrument connection adds in 50 μ l supernatants to be measured, and control wells add in The 50 unrelated supernatants of μ l.Then the PD-1-Fc (a concentration of 0.15 μ g/ml) of 50 μ l biotin labelings is added per hole, is incubated at 37 DEG C It educates 1 hour.Plate is washed three times with PBST, and with the streptavidin HRP in 100 μ l/ holes (SA-HRP, GenScript it) is incubated 10 minutes for 37 DEG C.Finally plate with PBST is washed five times, then adds in TMB developing solutions (GenScript) And it is incubated 15 minutes in the dark at room temperature.Reaction is terminated by the 1MHCl terminate liquids (Sigma) for adding in 50 μ l.Use enzyme Mark instrument read plate at 450 nm.

FACS detection methods：FACS Binding experiments be used for assess supernatant in antibody for Chinese hamster ovary celI film surface table The binding ability of the PD-1 reached.It collects for the Chinese hamster ovary celI of expression PD-1 of detection and the mother cell of negative control, it is clear with PBS It washes 3 times.2.5X10 is added in 96 orifice plates⁵A detection cell and supernatant 100ul to be measured, 4 DEG C are incubated 1 hour.Then use PBS Clean cell 3 times, the goat anti-mouse IgG of addition 100 μ l iFluor labels, 4 DEG C are incubated 45 minutes.Finally cleaned carefully with PBS Born of the same parents 3 times read signal with FACS BD Calibur.

3) Hybridoma Subclones

It is subcloned using limiting dilution assay.Using haemocytometer and in the thymus gland of selective agent containing hybridoma Cell is serially diluted in the DMEM/10%FBS culture mediums of nucleoside pyrimidine, hypoxanthine and aminopterin-induced syndrome to determine cell Quantity, until cell density reaches 5-15 cell/ml.For each hybridoma, the cell solution of 200 μ l is moved with pipette Into 96 holes, density is 1-3 cells/well.By culture in 5%CO at 37 DEG C₂After middle culture 1 week, supernatant is carried out Above-mentioned ELISA is combined, ELISA competitions and FACS binding tests, come assess for PD-1-Fc antibody there are situations.

Embodiment 2：The variable region sequencing of monoclonal antibody and antibody recombinant production

Using Rapid ELISA mouse antibodies hypotype identification kit (Clonotyping System-HRP, SouthernBiotech after) carrying out hypotype identification to monoclonal antibody, using TRIzol (Ambion) from 3 × 10⁶-5×10⁶It is a Hybridoma extracts total serum IgE, and utilizes antibody subtype specific primer and universal primer (PrimeScriptTM 1stStrand cDNA Synthesis Kit, Takara) by its reverse transcription be cDNA.Then pass through RACE PCR (GenScript) expand rat immune globulin heavy chain and light chain V- region segments, and by the PCR fragment of gained be subcloned to In pMD18-T carrier systems (Takara), and Insert Fragment is sequenced using vector-specific primers.It is finally obtained gram Unique V- Region Nucleotides/protein sequence of grand 1H10C4D4,2E5E4D11,154G5A5B7.

1H10C4D4 heavy chain variable amino acid sequences：SEQ ID NO:1

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

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

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

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

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

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

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

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

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

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

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

The DNA fragmentation with heavy chain variable region+constant region comprising light chain variable region+constant region is respectively synthesized, it is inserted respectively Enter in pTT5 expression vectors, form expression plasmid.

By above-mentioned plasmid co-transfection HEK293-6E cells, and after being cultivated 10 days in 37 DEG C of shaking flasks, supernatant is collected for resisting Body purifies.Before purifying, by pipeline and albumin A column 0.2M NaOH pyrogen removals.Column is used and contains 0.05M Tris and 1.5M The buffer solution of NaCl (pH=8.0) rebalances.Then by the cell culture supernatant of harvest, using 2 × above-mentioned buffer solution 1:1 dilution and filtration sterilization.The supernatant of filtering and albumin A column are incubated at room temperature 2 hours, with and 1 × above-mentioned buffer solution wash After column, IgG is eluted using sterile 0.1M sodium citrates (pH3.5), has collected eluent and with the sterile 1M of 1/9th volumes Tris-HCl (pH=9.0) is neutralized.Aseptically, it is the product buffer-exchanged is any to remove for PBS (pH7.4) Elution buffer and concentrate the sample.After concentration, pass through OD280nm pairs using 1.43 extinction coefficient Ec (0.1%) Antibody is quantified.

The antibody of purifying is divided by BioRad electrophoresis systems with 10% pre-prepared colloid (GenScript) by SDS-PAGE Analysis.The gel is dyed with Estain2.0 (GenScript) and by comparing colored zone and Protein Ladder (GenScript) estimate molecular size and purity.

Embodiment 3：Combination of the monoclonal antibody to people's PD-1 recombinant proteins

Indirect ELISA is used to assess binding ability of the antibody purification for PD-1-Fc.By elisa plate (Nunc) with 100 μ The recombination PD-1-Fc of 0.5 μ g/ml or human IgG1 are coated with overnight at 4 DEG C in the PBS in l/ holes.It is washed with PBS-T (0.05% tween) Plate is washed, and its PBST containing 1%BSA with 200 μ l/ holes is closed 0.5 hour at 37 DEG C.Then discard confining liquid, Xiang Shoukong Add in the 100 μ l of antibody purification of 10 μ g/ml, and according to 3 times of gradient dilutions, altogether 11 test concentrations gradients.Then in room temperature It is lower to be incubated 1 hour.Plate is washed 3 times with PBST, and the goat anti-mouse IgG of the conjugated horseradish peroxidase with 100 μ l/ holes (Fab- specificity) 37 DEG C of (GenScript) incubation 0.5 hour.Plate with PBST is washed five times, then adds in TMB developing solutions (GenScript) it and is incubated 15 minutes in the dark at room temperature.It is terminated by the 1MHCl terminate liquids (Sigma) for adding in 50 μ l Reaction.Use microplate reader read plate at 450 nm.Such as Fig. 2, PD-1 antibody clonings and PD-1-Fc recombinant protein combinations ELISA realities It tests, the EC respectively cloned₅₀It is as follows：Keytruda:EC₅₀=9.15ng/ml；1H10C4D4:EC₅₀=6.81ng/ml；2E5E4E11: EC₅₀=4.65ng/ml；154G5A5B7:EC₅₀=8.72ng/ml；Compared with Keytruda, the clone of these tests reaches Or more than to its antigen binding capacity.

Embodiment 4：Combination of the monoclonal antibody to the cell line of expression people PD-1

It collects for the Chinese hamster ovary celI of expression PD-1 of detection and the mother cell of negative control, is cleaned 3 times with PBS.In 96 holes 2.5X10 is added in plate⁵A detection cell and 5 μ g/ml antibody purifications 100ul, 4 DEG C are incubated 1 hour.Then cell is cleaned with PBS 3 times, the goat anti-mouse IgG of addition 100 μ liFluor labels, 4 DEG C are incubated 45 minutes.Finally cell is cleaned with PBS 3 times, use FACS BD Calibur read signal.Such as Fig. 3, in all figures the combination of clone and cell line all show very strong combination energy Power, FACS offset all 1 log even more than.

Embodiment 5：The combination of monoclonal antibodies block PD-1 and PD-L1 albumen

Elisa plate (Nunc) was coated with the recombined human PD-1 albumen of 0.5 μ g/ml in the PBS in 100 μ l/ holes at 4 DEG C Night.Plate is washed, and its PBST containing 1%BSA with 200 μ l/ holes is small in 37 DEG C of closings 0.5 with PBS-T (0.05% tween) When.Confining liquid is then discarded, first instrument connection adds in 50 μ g/ml, 50 μ l of antibody purification to be measured, and according to 3 times of gradient dilutions, common Count 11 test concentrations gradients.Then the PD-L1-Fc (a concentration of 0.15 μ g/ml) of 50 μ l biotin labelings is added per hole, 37 DEG C be incubated 1 hour.Plate is washed three times with PBST, and with the streptavidin HRP in 100 μ l/ holes (SA-HRP, GenScript it) is incubated 10 minutes for 37 DEG C.Finally plate with PBST is washed five times, then adds in TMB developing solutions (GenScript) And it is incubated 15 minutes in the dark at room temperature.Reaction is terminated by the 1MHCl terminate liquids (Sigma) for adding in 50 μ l.Use enzyme Mark instrument read plate at 450 nm.Such as Fig. 4, the IC respectively cloned₅₀It is as follows, Keytruda, IC₅₀=0.2241 μ g/ml；1H10C4D4, IC₅₀=0.4934 μ g/ml；2E5E4E11,IC₅₀=0.4289 μ g/ml；154G5A5B7,IC₅₀=0.3662 μ g/ml；3 plants grams Grand blocking ability is very close to positive drug Keytruda.

Embodiment 6：Monoclonal antibody Epitope Identification

Competitive ELISA is used to assess the epitope of antibody purification.It will be in PBS of the elisa plate (Nunc) with 100 μ l/ holes The recombination PD-1-Fc of 0.5 μ g/ml is coated with overnight at 4 DEG C.Plate is washed with PBS-T (0.05% tween), and by it with 200 μ l/ The PBST containing 1%BSA in hole is closed 0.5 hour at 37 DEG C.Confining liquid then is discarded, a pair of (one of them is separately added into per hole Marked biotin) for the test antibodies of competitive assay, each 100 μ l of antibody purification (10 μ g/ml).Then it is incubated at 37 DEG C It educates 1 hour.Plate is washed three times with PBST, and with the streptavidin HRP in 100 μ l/ holes (SA-HRP, GenScript it) is incubated 10 minutes for 37 DEG C.Plate is washed five times with PBST, then add in TMB developing solutions (GenScript) and It is incubated 15 minutes in the dark at room temperature.Reaction is terminated by the 1MHCl terminate liquids (Sigma) for adding in 50 μ l.Use microplate reader Read plate at 450 nm.1H10C4D4,2E5E4E11 and 154G5A5B7 are same epitope with positive control drug, but The epitope of 23F12A9 and other all clones are all different.

Embodiment 7：The functional detection of monoclonal antibody

In mixed lymphocyte reaction (MLP), by kit (Miltenyl Biotec), from the peripheral blood mononuclear cells of people It detaches and purifies and obtain CD⁴⁺The monocyte of T cell and allogeneic.Induced monocyte becomes dendritic cells.Contain per hole 10⁵A CD⁴⁺T cell and 10⁴The monocyte of a allogeneic, final working volume are 200 μ l.The antibody samples of various concentration It is added in each hole.Nonreactive body opening is as ground control, and 4 antibody of human IgG is as negative control, pyridine aldoxime methyliodide (PAM) monoclonal antibody The positive control of (Pembrolizumab (Keytruda), Merck＆Co., Inc) as PD-1 antibody.37 DEG C and 5%CO₂Item Under part, after being incubated 72 hours, 100 μ l supernatants is taken to detect IL-2 contents (detection kit of Cisbio) from every hole.

In the antibody functional test experience of cellular level, the PD-1 of people and by NFAT core transcriptional response elements (NFAT- RE) the fluorescent reporter gene stable transfection of control is in effectiveness cell, PD-L1 and cell surface protein antigen polypeptide/master of people Histocompatibility complex (MHC) stable transfection is wanted, as artificial antigen presenting cell, to express the effect of PD-1 in target cell It is co-cultured with the target cell of cell and expression PD-L1, relative light unit (RLU) is for weighing the index of effectiveness cell-stimulating. In co-culture system add in various concentration PD-1 antibody clonings, PD-1 antibody clonings block expression on effectiveness cell PD-1 and The combination of the PD-l on target cell is expressed, leads to the activation of effectiveness cell, RLU readings increase.As shown in figure 5, with Keytruda is compared, and the clone of these tests meets or exceeds its function.

SEQUENCE LISTING

<110>Nanjing Genscript Biotechnology Co., Ltd.

<120>High-affinity, high specific, more antigen recognizing epitopes the anti-human PD-1 antibody with higher function

<130> 2016

<160> 12

<170> PatentIn version 3.3

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Ala Ser Gln Asp Val Gly Ser Val 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 Phe Thr

85 90 95

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

100 105 110

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu

115 120 125

Glu Leu Lys

130

<210> 4

<211> 393

<212> DNA

<213>Artificial sequence

<400> 4

atgggcatca agatggagac acattctcag gtctttgtat acatgttgct gtggttgtct 60

ggtgttgaag gagacattgt gatgacccag tctcacaaat tcatgtccac atcagtagga 120

gacagggtca gcatcacctg caaggccagt caggatgtgg gttctgttgt agcctggtat 180

caacagaaac cagggcaatc tcctaaacta ctgatttact gggcatccac ccggcacact 240

ggagtccctg atcgcttcac aggcagtgga tctgggacag atttcactct caccattagc 300

aatgtgcagt ctgaagactt ggcagattat ttctgtcagc aatatagtag ctatccgctc 360

acgttcggtt ctgggaccaa gctggagctg aaa 393

<210> 5

<211> 138

<212> PRT

<213>Artificial sequence

<400> 5

Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly

1 5 10 15

Val Leu Cys Glu Val Lys Leu Val Glu Ser Gly Gly Val Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu

50 55 60

Glu Trp Val Ala Tyr Ile Ser Gly Gly Gly Gly Asp Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Gly Asp Thr Ala Met

100 105 110

Tyr Tyr Cys Ala Arg His Gly Tyr Asp Ala Ala Cys Phe Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ala

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

1. a kind of people PD-1 monoclonal antibodies, it is characterised in that：The protein sequence of the monoclonal antibody contain heavy chain variable region and Light chain variable region, the monoclonal antibody are selected from any one in following (1)~(3)：

(1) heavy chain variable region described in has such as SEQ ID NO：Amino acid sequence shown in 1, the light chain variable region have Such as SEQ ID NO：Amino acid sequence shown in 3；

(2) heavy chain variable region described in has such as SEQ ID NO：Amino acid sequence shown in 5, the light chain variable region have Such as SEQ ID NO：Amino acid sequence shown in 7；

(3) heavy chain variable region described in has such as SEQ ID NO：Amino acid sequence shown in 9, the light chain variable region have Such as SEQ ID NO：Amino acid sequence shown in 11.

2. the encoding gene of people PD-1 monoclonal antibodies described in claim 1.

3. encoding gene according to claim 2, it is characterised in that the encoding gene is selected from appointing in following (4)~(6) Meaning is a kind of：

(4) containing such as SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody heavy variable region shown in 2, Yi Jiru SEQ IDNO：The nucleotide sequence of the coding monoclonal antibody light chain variable region shown in 4；

(5) containing such as SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody heavy variable region shown in 6, Yi Jiru SEQ IDNO：The nucleotide sequence of the coding monoclonal antibody light chain variable region shown in 8；

(6) containing such as SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody heavy variable region shown in 10 and Such as SEQ IDNO：The nucleotide sequence of the coding monoclonal antibody light chain variable region shown in 12.

4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing encoding gene described in claim 3.

5. the recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium described in claim 4 are preparing people PD-1 Dan Ke Application in grand antibody.

A kind of 6. method for preparing people's PD-1 monoclonal antibodies, it is characterised in that encoding gene described in Claims 2 or 3 will be included Recombinant expression carrier transfection competent cell, and cultivated to obtain people's PD-1 monoclonal antibodies.

7. people PD-1 monoclonal antibody application in preparations of anti-tumor drugs described in claim 1.

8. a kind of anti-tumor agent, it is characterised in that：It includes people PD-1 monoclonal antibodies described in claim 1.