CN109929036A - A kind of antibody screening method that epitope is special and the antibody screened - Google Patents

Abstract

A kind of antibody screening method that epitope is special, it is low to solve the problems, such as to have in screening of phage antibody library technology medicative functional antibodies screening efficiency, include the steps that being screened with function epitope peptide and living cells screens；It additionally provides and screens a kind of phage displaying antibody library complete anti-PD-1 monoclonal antibody of source of people obtained with this method, for specific functional epitope, PD-1 and the combination of its ligand can effectively be blocked, and have the characteristics that affinity is high, EC50 is low, therapeutic effect can be generated to animal model under low dosage.The present invention substantially increases the screening efficiency of functional antibodies, and provides a kind of immunotherapy medicaments of potential safer economy for malignant tumour, immunological diseases patient.

Description

A kind of antibody screening method that epitope is special and the antibody screened

Technical field

The invention belongs to biomedicine fields, and in particular to a kind of antibody screening method that epitope is special and be screened Antibody.

Background technique

" immunologic escape " of cancer cell is considered as the main mechanism of tumorigenesis and anti-medicine.Tumour can be by straight It connects or inhibits the signal of T cell come the removing of protecting oneself to escape immune system indirectly.The immunologic test point therapy of tumour (immune chenkpoint therapy) is a kind of by a series of approach regulatory T-cells such as co-suppression or costimulatory signals Activity improves the treatment method of anti-tumor immune response.

PD-1(apoptosis albumen 1, full name in English Programmed Cell Death Protein 1, also referred to as It is one for a kind of cell surface receptor that CD279) is in the T cell and pre B cell (pro-B cells) for be expressed in activation Immunologic test point has negative regulation effect to immune system, promotes self tolerance by inhibiting T cell inflammatory activities, it is more specific and Speech, by promoting in lymph node, (Treg is also inhibition T to PD-1 for the apoptosis of T cells with antigenic specificity and reduction modulating T cell Cell) the double mechanism of apoptosis prevent the generation of autoimmunity.

PD-1 is encoded by PDCD1 gene, 288 amino acid of overall length, wherein before N-terminal 20 amino acid be signal peptide, at Excision after ripe, the 21-170 amino acid are extracellular fragment, and the 171-191 amino acid is transmembrane region, the 192-288 amino acid For intracellular tail.

PD-1 can be with 2 kinds of ligand bindings --- B7 family member PD-L1 and PD-L2.On mouse, PD-L1 wide expression On a variety of organs such as Yu Xin, lung, thymus gland, spleen, kidney and almost all of mouse tumor cell system, and in a variety of human tumors High expression on cell；And the expression of PD-L2 is more limited to, mainly Dendritic Cells and a small number of tumor cell lines.Therefore, PD-1 And its ligand PD-L1 becomes the target spot of anti-tumor immunotherapy.

Existing 2 anti-PD-1 target therapeutic agents go through to list at present, are Opdivo(common name Nivolumab respectively) With Keytruda(common name Pembrolizumab), the approved indication of Opdivo includes unresectable or metastatic Melanoma and metastatic squamous non-small cell lung cancer, the approved indication of Keytruda have unresectable or metastatic Melanoma and non-small cell lung cancer, head and neck squamous cell carcinoma, hodgkin lymphoma classical type, bladder transitional cell carcinoma, microsatellite High unstability cancer etc..

According to the clinical test results that U.S. FDA package insert discloses, Opdivo treats 120 at one and can not cut off Or in the clinical test of metastasis melanin tumor patient objective remission rate (ORR) be 32%(38 people/120 people), including 4 completely Alleviate (CR) and the patient of (PR) is alleviated in 34 parts；In another 117 metastatic squamous Patients with Non-small-cell Lung for the treatment of Clinical test in objective remission rate be 15%(17 people/117 people).Keytruda can not be cut off at one 834 or metastatic is black Random controls that melanoma patient participates in, opening, in multi-center clinical trial, two test groups (receive various dose respectively Keytruda treatment) ORR be respectively every three weeks administration 10mg/kg of 33%() and 34%(10mg/kg is administered every two weeks), wherein CR Rate is respectively that 6%, 5%, PR rate is respectively 27%, 29%, and the ORR of control group (anti-CTLA-4 monoclonal antibody Ipilimumab treatment) is 12%；In the random controls of another 305 Metastatic Nsclc patient participation, opening, multi-center clinical trial, The ORR of test group (Keytruda treatment) is 45%(wherein CR4%, PR41%), and the ORR of control group (chemotherapy) be 28%(wherein CR1%, PR27%).Although the above research as can be seen that compared with chemotherapy and anti-CTLA-4 are treated, Opdivo and Keytruda's Anti- PD-1 treatment is obviously improved on objective remission rate ORR, however the patient for still only having one third or so is benefited, and one Most patients cannot obtain disease amelioration.

The validity of anti-PD-1 Antybody therapy malignant tumour is mainly related to the blocking effect in conjunction with PD-1/PD-L1 with it. Combination epitope and its polymorphism of the anti-PD-1 antibody on PD-1 molecule, and the affinity combined are (general normal with balance dissociation Number KD values indicate), many factors such as specificity can all influence the barrier effect that anti-PD-1 antibody combines PD-1/PD-L1.

Phage displaying antibody library is a kind of technology of high flux screening antibody, at present in the industry often with the target of recombinant expression point Sub- extracellular region is as target antigen screening antibodies library.The extracellular region of these target molecules only has one or several generally by 5-15 amino Sour composition function epitope is responsible for and ligand binding, therefore only just has patent medicine potentiality for the antibody of specific function epitope.And For the extracellular domain fragment of recombinant expression for screening of phage antibody library in addition to the functional domain for playing biological function individually, there are also a large amount of Dominant antigen epitope, therefore screen the antibody majority obtained and only combine activity, but do not block in its biological function And activity, there is no patent medicine potentiality, need to be tested using a large amount of function assessment and Epitope Identification work can just be selected and make with treatment Functional antibodies.Also there is the case using the epitope peptide screening antibodies library synthesized, however due to the epitope peptide and day of synthesis The epitope of right conformation there may be difference, can with the antibody in conjunction with epitope peptide it is different surely with the antigen table of liver cell surface Position combines.In short, existing screening of phage antibody library technology is low to the functional antibodies hit rate for having the epitope of patent medicine potentiality special, seriously The efficiency for affecting high flux screening increases the cost of later period clone identification.

Summary of the invention

The present invention provides a kind of method of high frequency zone antibody library, existing screening of phage antibody library technology is able to solve to having into The low problem of the special functional antibodies hit rate of the epitope of medicine potentiality.This screening technique is used the present invention also provides a kind of The anti-PD-1 monoclonal antibody obtained, and Nivolumab, Pembrolizumab possess different combination epitopes, and affinity It is high, specific good, it can solve the problems, such as that some patientss are invalid to existing anti-PD-1 treatment.

Technical scheme is as follows:

A kind of screening technique for the functional antibodies that epitope is special, including following two screening step:

Step 1: for the epitope with biological function, synthetic antigen epitope peptide is simultaneously coupled on solid-phase media, from biting First run screening is carried out in phage display antibody library, is obtained and the bacteriophage of epitope peptide specific binding and amplification；

Step 2: obtaining or building is in the cell strain of cell surface height expression purpose antigen, is obtained with living cells from first run screening Bacteriophage in further bacteriophage of the screening in conjunction with cell surface antigen, to prepare functional antibodies.

It, can be Step 1: the sequential Cycle Screening 2-5 wheel of step 2, be carried out by the technical program for the specificity of enhancing screening The techniqueflow chart of antibody screening can refer to attached drawing 1.

In step 1, the amino acid sequence of the epitope with biological function can be lacked according to crystallization of protein, segment Mistake/point mutation obtains the methods of the affinity analysis of ligand/receptor, software prediction, the epitope peptide be by 10 to The cyclic peptide of 15 amino acid composition, the Amino acid profile of cyclic peptide meet the following conditions:

(1) have in cyclic peptide and only 1 cysteine, cyclic peptide passes through the sulfydryl covalent coupling of the cysteine to solid-phase media On；

(2) respectively there are one section of link peptide by 2 or 3 Amino acid profiles in the two sides of cysteine in cyclic peptide, constitute link peptide Amino acid can only be glycine or proline；

(3) epitope being made of containing one section 5-8 amino acid in cyclic peptide.

Preferably, one section of link peptide being connected in cyclic peptide with cysteine aminoterminal is made of glycine completely, with half Guang Contain 1 to 2 proline in one section of connected link peptide of propylhomoserin c-terminus.

Specifically, when being used to screen the functional epitope (amino acid sequence that anti-human PD-1 75-82 amino acids are constituted For QTDKLAAF) monoclonal antibody when, epitope peptide used in step 1 is cyclic annular and meets SEQ ID NO:13 Documented sequence；Cell strain used in step 2 is the cell strain in cell surface height expression people PD-1, it is preferred that this is thin Born of the same parents' strain is the cell strain for the expression external source people's PD-1 gene being transformed using gene recombination technology, and under the cell strain native state (i.e. before genetic modification) does not express PD-1, it is preferred that people PD-1 is that 10,000/cell arrives in the expression density of cell surface 500,000/cell.

A kind of antibody screened using the above method, combination people's PD-1 molecule 75-82 since N-terminal of energy specificity Amino acids (amino acid sequence QTDKLAAF), complementary determining region CDR1, CDR2, CDR3 of the heavy chain of the antibody are respectively provided with The amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, complementary determining region CDR1, CDR2 of light chain, CDR3 is respectively provided with the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

Preferably, heavy chain variable region has the amino acid sequence of SEQ ID NO:7, and light chain variable region has SEQ ID The amino acid sequence of NO:8.

Preferably, heavy chain is IgG4 hypotype, and the amino acid sequence with SEQ ID NO:9, light chain is with SEQ ID NO: 10 amino acid sequence.

Preferably, heavy chain is by the nucleotide sequence coded of SEQ ID NO:11, light chain by SEQ ID NO:12 nucleosides Sequences code.

The antibody is by Chinese hamster ovary cell (Chinese hamster ovary celI) secreting, expressing.

Reverse transcriptase test (embodiment 6) and point mutation test (embodiment 7) show the antibody and Nivolumab and Pembrolizumab has different combination epitopes, to cannot can also tie with the D85G point mutation PD-1 in conjunction with Pembrolizumab It closes.

The antibody has IC50 value (embodiment 5) lower compared with Nivolumab and Pembrolizumab, can enhance people's T lymph Cell can more effectively block the combination of PD-1 and PD-L1 to SEB stimulation responses ability (embodiment 8) under low dosage, and Therapeutic effect (embodiment 9) is played to malignant tumour under lower dosage.

The antibody can be used to prepare treating malignant tumor drug and immune system disease individually or with other drugs, auxiliary material combination Sick therapeutic agent.

Beneficial effects of the present invention include, can be by the function special to epitope using screening of phage antibody library method of the invention The hit rate of energy property antibody is increased to 30% or more, greatly reduces the quantity and cost of clone identification.Anti- PD-1 Dan Ke of the invention Grand antibody possesses epitope in conjunction with different people PD-1 from Nivolumab, Pembrolizumab, to cannot be with Pembrolizumab In conjunction with point mutation PD-1 can also combine, and it is high with PD-1 affinity, have lower IC50 value, under low dosage more effectively The combination of PD-1 and PD-L1 is blocked, and therapeutic effect played to malignant tumour under lower dosage, and low dosage antibody There is positive effect to the stimulation of body, reduction drug cost and price using to reduction drug.

Detailed description of the invention

The special functional antibodies screening technique route map of Fig. 1 epitope

The Competitive assays experimental result picture that the anti-PD-1 monoclonal antibody 5L12 of the full source of people of Fig. 2 embodiment 5 combines PD-1/PD-L1

Other Competitive assays of anti-PD-1 antibody PD-1 monoclonal antibody 5L12 anti-to full source of people in conjunction with PD-1 of Fig. 3 embodiment 6 Experimental result picture

7 ELISA of Fig. 4 embodiment tests the binding ability of the PD-1 of the anti-PD-1 monoclonal antibody 5L12 of full source of people and D85G mutation Experimental result picture

Survey of the anti-PD-1 monoclonal antibody 5L12 enhancing human T lymphocyte of the full source of people of Fig. 5 embodiment 8 to SEB stimulation responses ability Try experimental result picture

Inhibiting tumor assay high dose group result figure of the anti-PD-1 monoclonal antibody 5L12 of the full source of people of Fig. 6 embodiment 9 in animal model

Inhibiting tumor assay low dose group result of the anti-PD-1 monoclonal antibody 5L12 of the full source of people of Fig. 7 embodiment 9 in animal model Figure.

Specific embodiment

Embodiment 1, synthesis PD-1 epitope peptide screening phage displaying antibody library

(1) epitope peptide synthesis and magnetic bead are coupled

According to sequence chemical synthesis people's PD-1 ring-type epitope peptide (student on commission's work bioengineering (Shanghai) stock of SEQ ID NO:13 The synthesis of part Co., Ltd), which includes people PD-1 75-82 amino acids QTDKLAAF, and for the half of magnetic bead coupling Each link peptide being made of 3 amino acid on cystine M and its both sides, 1475 Da of molecular weight.By synthetic epitope peptide with 10mg/mL is dissolved in PBS(phosphate buffer, pH7.2, sodium ascorbyl phosphate containing 20mM, 150mM sodium chloride) in it is spare；

By the derivative magnetic bead of 2mL amino, (Thermofisher Scientific company, article No. 21352, every mL magnetic bead weight are 2.5g contains about 12 μm of ol amino) it is cleaned with 2mL PBS, it cleans three times, magnetic separation magnetic bead；

By Sulfo-SMCC(Thermofisher Scientific company, article No. 22122) it is dissolved in the concentration of 4.8mg/mL PBS takes 200 μ L to mix with above-mentioned magnetic bead, and 4 DEG C are reacted 2 hours；

Separation magnetic bead is simultaneously cleaned with PBS, magnetic bead is divided into two parts, portion is resuspended in PBST(of the 1mL containing 1%BSA containing 0.1% The PBS of Tween 20) in, it is denoted as magnetic bead A；5 mL ring-type epitope peptides are added in another, and 4 DEG C are reacted 2 hours, and Beads enrichment is used PBST cleaning, is resuspended in PBST of the 1mL containing 1%BSA, is denoted as magnetic bead B.

(2) phage displaying antibody library is screened

Each 20 mL of 50 healthy volunteer's blood is collected, is mixed, by the document " structure of natural humanized IgG Fab phage antibody library Build and diversity analysis " (China Immunology Journal, 2010,26 (11): 1007-1010) method building phage display it is anti- Body library.

The magnetic bead A and 1mL that step (1) is prepared with the PBST containing 1%BSA diluted phage displaying antibody library (5 × 10^12 PFU) mixing, rotation mixing 1 hour at room temperature, magnetic force removes magnetic bead A, adds the magnetic bead prepared in step (1) B, rotation mixing 1 hour, magnetic separation magnetic bead B, is cleaned magnetic bead B 3 times with PBST at room temperature, and 0.2 mol/L is added Glycine-HCl (pH 2.2) solution mildly vibrates 20 min and the bacteriophage on magnetic bead B is washed lower collection, then uses 1mol/L Tris-HCl(pH 9.1) it neutralizes, the phage-infect Escherichia coli (E. coli) of acquisition are expanded, secondary library is become, It stores for future use.

Embodiment 2, PD-1 high express living cells postsearch screening functional antibodies

(1) cell strain in cell surface height expression PD-1 is constructed

(PDCD1 gene, containing N-terminal signal coding sequence, particular sequence is shown in NCBI to Cloning of full length PD-1 encoding gene Reference Sequence:NM_005018.2), it is inserted into pcDNA3.1 expression vector, is expanded in Escherichia coli (E. coli) 293T cell (ATCC number CRL-3216) is transfected after increasing, overexpression cell line is screened, according to document " Flow cytometric detection and quantitation of the epidermal growth factor receptor in comparison to Scatchard analysis in human bladder carcinoma cell lines. Cytometry. 1994 Sep 1;17 (1): 75-83 " and " Antibody- dependent cellular cytotoxicity mediated by cetuximab against lung cancer cell lines. Clin Cancer Res. 2007 Mar 1;13 (5): the flow cytometry that 1552-61 " is recorded measures the PD-1 on each cell strain surface Density is expressed, therefrom chooses the cell strain that the highest number of expression density is 2H-3, after measured the surface cell strain 2H-3 PD-1 Expressing density is averagely 12000/cell, and cell is used 293 SFM II serum free medium (Thermofisher Scientific company, article No. 11686029) suspend culture, cell quantity is expanded to 5 × 10^6 or more.

(2) living cells screens anti-PD-1 antibody

The blank 293T cell that logarithmic phase is grown is resuspended in 1mL DMEM culture solution with 1 × 10^6 cell/mL density, is added Enter embodiment 1(2) in screening obtain bacteriophage (being resuspended in after centrifugation in 1mL DMEM culture solution), after mixing 37 DEG C be incubated for 1 Hour, low-speed centrifugal removes cell, repeats the process 1 time, is screened with removing with the antibodies to "wrong" antigens of 293T cell combination, reduction Background value；Then embodiment 2(1 is added in the supernatant of centrifugation) in 5 × 10^ of 2H-3 cell of high expression PD-1 for preparing It 5, is incubated for 1 hour for 37 DEG C after mixing, low-speed centrifugal harvests cell, will with 1.5 times of 76 mM citric acid solutions (pH2.5) of volume Bacteriophage is washed down from cell, is then neutralized with 0.5 times of volume Tris-HCl(pH7.4), will obtain phage-infect large intestine bar Bacterium (E. coli) is expanded.

By embodiment 1(2) and embodiment 2(2) screening technique to previously obtained bacteriophage again successively carry out two-wheeled sieve Choosing carries out monoclonal to the bacteriophage finally obtained and expands, and selects 60 clones using ELISA method and carries out bacteriophage pair The identification of the compatibility and combination epitope of PD-1 is (with reference to " Journal of Immunology the 6th phase of volume 28 in June, 2012: 489-496 " Phage-ELISA identification method), people's PD-1 extracellular fragment full-length proteins (20-170 amino acids) of recombinant expression are respectively adopted 96 orifice plates are coated with chemically synthesized people's PD-1 75-82 amino acids (sequence QTDKLAAF), and with chemically synthesized sequence 96 orifice plates are coated with as negative control for small peptides such as LDSPDR, LAPKAQ, RSQPGQ, then addition bacteriophage and HRP are marked anti- Phage antibody is added the detection of HRP substrate chromogenic reaction and combines situation；Using flow cytometry identification bacteriophage to living cells The compatibility of the surface 2H-3 PD-1；It obtains 22 ELISA and flow cytometry identification is positive in conjunction with PD-1 and combines PD- The clone of 1 defined epitope (75-82 amino acids QTDKLAAF), positive rate is up to 36.67%.

Embodiment 3, the complete anti-PD-1 monoclonal antibody expression system of source of people of building

(1) identification of heavy chain and light chain variable region

From 22 bacteriophage positive colonies that embodiment 2 finally obtains, picks out 5 and identify that compatibility is higher through ELISA Clone is sequenced, and the clone that preferably out 1 number is 5L12 is further developed.Sequencing result shows the heavy chain of 5L12 Variable region has the amino acid sequence of SEQ ID NO:7, and light chain variable region has the amino acid sequence of SEQ ID NO:8, into One step antibody sequence analysis shows, complementary determining region CDR1, CDR2, CDR3 of heavy chain are respectively provided with SEQ ID NO:1, SEQ ID The amino acid sequence of NO:2, SEQ ID NO:3, complementary determining region CDR1, CDR2, CDR3 of light chain are respectively provided with SEQ ID The amino acid sequence of NO:4, SEQ ID NO:5, SEQ ID NO:6.

(2) building and transfection of expression vector

Using the light and weight chain variable region gene sequence of 5L12 as template, the heavy chain gene of human antibody is synthesized by over-lap PCR (IgG4 type), light chain gene, wherein the coded sequence of heavy chain is the nucleotide sequence of SEQ ID NO:11,5 ' end signal of heavy chain The coded sequence of peptide is the nucleotide sequence of SEQ ID NO:14, and the coded sequence of light chain is the nucleotide of SEQ ID NO:12 Sequence.The coded sequence of heavy chain, light chain is inserted into pcDNA3.1 (+) carrier (Invitrogen company), constructs the anti-PD- of full source of people The expression vector pcDNA3.1-anti-PD-1 of 1 monoclonal antibody, is carried out with Lipofectamine2000Reagent kit It transfects CHO-K1 cell (ATCC number CRL-9618), with G418 and Zeocin screening positive clone, then takes in cell culture Clear liquid screens overexpression cell line with ELISA method, and transfection, the specific experiment process screened are according to patent CN201010125241 The embodiment of (Authorization Notice No. CN102167742B) carries out.

(3) a small amount of expression and purifications

The overexpression cell line that screening is obtained is expanded with Chinese hamster ovary celI serum free medium to be cultivated, with Protein A affinity column (GE Products) isolate and purify human antibody 5L12, and the antibody of purifying is dialysed with PBS, finally with ultraviolet suction Receive standard measure.

The determination of the anti-PD-1 monoclonal antibody 5L12 affinity of embodiment 4, full source of people

The anti-PD-1 monoclonal antibody 5L12 of full source of people prepared using Surface Plasmon Resonance (SPR) measurement embodiment 3 is to people The affinity of PD-1.Using BIACORE3000 system, with purified 5L12 antibody coupling sensing chip, according to the behaviour of system It explains book to be operated, detecting the PD-1 extracellular fragment full-length proteins that 5L12 and Chinese hamster ovary celI recombinantly express respectively, (NCBI refers to sequence Arrange NM_005018.2 20-170 amino acids) and chemically synthesized PD-1 75-82 amino acids (sequence QTDKLAAF) with And sequence is the affinity of the small peptide of LDSPDR, LAPKAQ, RSQPGQ, testing result is shown in Table 1,5L12 to PD-1 extracellular fragment overall length And the affinity of epitope peptide QTDKLAAF is up to 10^-11M grades.

1 SPR method of table measures the result of full source of people anti-PD-1 monoclonal antibody 5L12 and different peptide fragment compatibilities

The anti-PD-1 monoclonal antibody 5L12 of embodiment 5, full source of people tests the Competitive assays that PD-1/PD-L1 is combined

The human PD-L 1 born of the same parents for being recombinantly expressed albumen PD-L1(CHO cell to be marked with the carbonate buffer solution of 0.025M pH9.5 Outer segment) it is diluted to 1%(mass volume ratio) concentration, it is fitted into bag filter.FITC is made into the molten of 0.1mg/ml with same buffer Liquid is contained in small beaker, is immersed in bag filter in FITC solution, and stirring is protected from light at 4 DEG C for 24 hours.Marking fluid in bag filter is taken out, Column is crossed with Sephadex G-50, free fluorescein is removed, it is spare to collect fluorescence antibody FITC-PD-L1.

By the genetic recombination 293T cell of surface expression people PD-1 with 1 × 10⁶Cell/ml density is resuspended in pH7.2's In PBS solution；By the fluorescent marker protein FITC-PD-L1 of fixed sub-saturated concentration respectively and with 4 × not marking of being serially diluted Remember antibody purification 5L12, Nivolumab(Opdivo), Pembrolizumab(Keytruda), human IgG (control) mixing, addition Into cell suspension, in 4 DEG C of 60 min of incubation, cell is harvested by centrifugation, washes 2 times with 1% FCS-PBS buffer, flow cytometer It detects cell fluorescence intensity and is analyzed with Cellquest software.Each concentration of competition antibody sets 3 multiple pipes, calculates half suppression Concentration IC50 value processed, maximum fluorescence intensity indicate the average fluorescent strength obtained in no competition antibody.

Experimental result is shown in Fig. 2, show that antibody 5L12 can block fluorescent labeled antibody FITC-PD-L1 and surface expression completely The combination of the 293T cell of PD-1, IC50 value are about 30 ng/mL, are the 1/ of Nivolumab, Pembrolizumab respectively 20,1/2, show that 5L12 can more effectively block PD-1 and the combination of its ligand, the minimum dose that can play effect is lower.

The competition suppression of embodiment 6, other anti-PD-1 antibody PD-1 monoclonal antibody 5L12s anti-to full source of people in conjunction with PD-1 System experiment

FITC label is carried out to 5L12 with the method for embodiment 5, preparation FITC-5L12 fluorescence antibody is spare.

By the genetic recombination 293T cell of surface expression people PD-1 with 1 × 10⁶Cell/ml density is resuspended in pH7.2's In PBS solution；By the fluorescent marker protein FITC-5L12 of fixed sub-saturated concentration respectively and with 10 × not marking of being serially diluted Remember antibody Nivolumab(Opdivo), Pembrolizumab(Keytruda), human IgG (control) mixing, be added to cell hang In liquid, in 4 DEG C of 60 min of incubation, cell is harvested by centrifugation, is washed 2 times with 1% FCS-PBS buffer, flow cytomery cell Fluorescence intensity is simultaneously analyzed with Cellquest software.Each concentration of competition antibody sets 3 multiple pipes, and maximum fluorescence intensity indicates The average fluorescent strength obtained when not competing antibody.

Experimental result is shown in Fig. 3, the results showed that Pembrolizumab makees the Competitive assays that are combined with of 5L12 and people PD-1 With, and Nivolumab does not have apparent competitive inhibitory effect to 5L12, this illustrate combination epitope of the 5L12 on people PD-1 with Nivolumab(is 25LDSPDR30, bibliography: An unexpected N-terminal loop in mainly in combination with epitope PD-1 dominates binding by nivolumab. Nature Communications, 2017,8:14369) no Together, but and Pembrolizumab(mainly in combination with epitope be Pro83~Gly90, bibliography: High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the 2016,6, Article number:35297 of human PD-1. Scientific Reports.) it is close, or The combination of Pembrolizumab and PD-1 spatially hinders the combination of 5L12 and PD-1.

Ability of the anti-PD-1 monoclonal antibody 5L12 of embodiment 7, full source of people in conjunction with the PD-1 that D85G is mutated

It by the 85th Aspartic acid mutations of people PD-1 is glycine (D85G mutation) by the method for genetic recombination, by people PD-1 born of the same parents Outer segment and people PD-1 extracellular fragment (D85G) encoding gene are inserted into pcDNA3.1 carrier and transfection CHO cell respectively, carry out instantaneous table It reaches, purify.

By the concentration of purifying be 1 mg/mL people PD-1 extracellular fragment and people PD-1 extracellular fragment (D85G) albumen with 100 μ L/ Hole is coated with 96 orifice plates respectively, and 96 orifice plates are added with 100 holes μ L/, so in the carry out 10 × be serially diluted since 1 mg/mL by 5L12 The rabbit anti-human igg of HRP label is added afterwards, carries out chromogenic reaction, reads data with microplate reader.ELISA result is shown in Fig. 4, as a result table Bright 5L12 can be combined with people PD-1 extracellular fragment and people PD-1 extracellular fragment (D85G), and Pembrolizumab report cannot be with The PD-1 of D85G mutation combines (bibliography: Structural basis for blocking PD-1-mediated immune suppression by therapeutic antibody pembrolizumab. Cell Research 2017. 27:147-150.), illustrate that 5L12 there are different binding sites from Pembrolizumab.

The anti-PD-1 monoclonal antibody 5L12 enhancing human T lymphocyte of embodiment 8, full source of people is to SEB stimulation responses ability Test

Peripheral blood from healthy volunteer is diluted in cell culture medium with 1: 10, whole blood bed board (every 150 μ L of hole) arrives In 96- orifice plate, with 10 × be serially diluted full source of people anti-PD-1 monoclonal antibody 5L12 preincubate 30-60 minutes, then it is added 1 The staphylococcal enterotoxin B (SEB) of μ g/mL is cultivated 2-4 days, and supernatant is collected, and measures wherein IL-2 content with ELISA, as a result See Fig. 5, shows that 5L12 can improve IL-2 of the whole blood cells under SEB stimulation and generate, and the additive of IL-2 level and 5L12 Measurer has correlation.

Inhibiting tumor assay of the anti-PD-1 monoclonal antibody 5L12 of embodiment 9, full source of people in animal model

1, the building of human PBMC's immunologic reconstitution mouse

User's lymphocyte separation medium, separation comes from health donors human PBMC (peripheral blood mononuclear cells), with 8 × 10⁷/mL It is resuspended in spare in RPMI-1640 culture medium；Select the NOD/SCID adult mice of the μ of mouse IgG < 5 g/mL in serum, tail vein note Penetrate 2 × 10⁷PBMC cell, took 250 μ L of peripheral blood from mouse orbit rear vein beard at the 0th, 14,28,42,56 day respectively, make Mouse anti human CD 19 monoclonal antibody combination flow cytomery CD3+T with the mouse antibody human CD3 monoclonal antibody of PE label, FITC label is thin Born of the same parents, CD19+B cell content, using human IgG content in ELISA method measurement mice serum, to determine immunologic reconstitution effect.

2, it constructs mouse tumor model and is treated using anti-human PD-1 antibody 5L12

Non-small cell lung carcinoma cell strain HCC827(high expresses PD-L1) with 1 × 10⁶Cell/only dose subcutaneous be implanted to people PBMC immunologic reconstitution mouse, to tumour growth to 250mm after implantation²After start to give anti-PD-1 treatment.Mouse is divided into high agent Amount group and low dose group, high dose group are given the 5L12(of 3 mg/kg respectively and are dissolved in PBS with the concentration of 1 mg/mL), Nivolumab, Pembrolizumab are administered once for every 10 days, are administered 4 times, measure tumor size with caliper every 1 week；It is low The dosage of dosage group is adjusted to 0.5 mg/kg, other test methods are constant；And use PBS as control.

Experimental result is shown in Fig. 6 and Fig. 7, show that 5L12 can play apparent inhibition to tumour growth under low dosage and make With effective dose is significantly lower than Nivolumab, Pembrolizumab.

Sequence table

<110>Taizhou Mai Botaike pharmaceutcal corporation, Ltd

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1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

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

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg

<210> 9

<211> 444

<212> PRT

<213>artificial sequence ()

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Tyr Pro Ser Thr Gly Tyr Thr Glu Tyr Ser Glu Asn Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 10

<211> 219

<212> PRT

<213>artificial sequence ()

<400> 10

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

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

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 11

<211> 1332

<212> DNA

<213>artificial sequence ()

<400> 11

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgcggctg 60

tcctgcgccg cctccggcta caccttcacc tcctactgga tgctgtgggt gcggcaggcc 120

cccggcaagg gcctggagtg ggtgtcctac atctacccct ccaccggcta caccgagtac 180

tccgagaact tcaaggaccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc ccggggcgtg 300

cggtactact tcgactactg gggccagggc accctggtga ccgtgtcctc cgcctccacc 360

aagggcccct ccgtgttccc cctggccccc tgctcccggt ccacctccga gtccaccgcc 420

gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaactcc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtcctc cggcctgtac 540

tccctgtcct ccgtggtgac cgtgccctcc tcctccctgg gcaccaagac ctacacctgc 600

aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggagtc caagtacggc 660

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gcccctccgt gttcctgttc 720

ccccccaagc ccaaggacac cctgatgatc tcccggaccc ccgaggtgac ctgcgtggtg 780

gtggacgtgt cccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 840

gtgcacaacg ccaagaccaa gccccgggag gagcagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc ctccatcgag aagaccatct ccaaggccaa gggccagccc 1020

cgggagcccc aggtgtacac cctgcccccc tcccaggagg agatgaccaa gaaccaggtg 1080

tccctgacct gcctggtgaa gggcttctac ccctccgaca tcgccgtgga gtgggagtcc 1140

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggactc cgacggctcc 1200

ttcttcctgt actcccggct gaccgtggac aagtcccggt ggcaggaggg caacgtgttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

tccctgggca ag 1332

<210> 12

<211> 657

<212> DNA

<213>artificial sequence ()

<400> 12

gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgggtgacc 60

atcacctgca agtcctccca gtccctgctg gactcctcca cccggaagaa ctacctggcc 120

tggtaccagc agaagcccgg caaggccccc aagctgctga tctactgggc ctccacccgg 180

gagtccggcg tgccctcccg gttctccggc tccggctccg gcaccgactt caccctgacc 240

atctcctccc tgcagcccga ggacttcgcc acctactact gcaagcagtc ctacaacctg 300

tggaccttcg gccagggcac caaggtggag atcaagcgga ccgtggccgc cccctccgtg 360

ttcatcttcc ccccctccga cgagcagctg aagtccggca ccgcctccgt ggtgtgcctg 420

ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

tccggcaact cccaggagtc cgtgaccgag caggactcca aggactccac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600

gtgacccacc agggcctgtc ctcccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 13

<211> 15

<212> PRT

<213>artificial sequence ()

<400> 13

Cys Pro Pro Ala Gln Thr Asp Lys Leu Ala Ala Phe Ala Ala Ala

1 5 10 15

<210> 14

<211> 66

<212> DNA

<213>artificial sequence ()

<400> 14

atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatatcc 60

agagga 66

Claims (10)

1. a kind of screening technique for the functional antibodies that epitope is special, characterized in that including following two screening step:

Step 1: synthetic antigen epitope peptide is simultaneously coupled on solid-phase media, and first run screening is carried out from phage displaying antibody library, It obtains and the bacteriophage of epitope peptide specific binding and amplification；

Step 2: obtaining or building is in the cell strain of cell surface height expression purpose antigen, is obtained with living cells from first run screening Bacteriophage in further bacteriophage of the screening in conjunction with cell surface antigen.

2. according to the method described in claim 1, it is characterized in that, epitope peptide described in step 1 is by 10 to 15 amino The cyclic peptide of acid composition, the Amino acid profile of cyclic peptide meet the following conditions:

Have in cyclic peptide and only 1 cysteine, cyclic peptide pass through on the sulfydryl covalent coupling to solid-phase media of the cysteine；

Respectively there are one section of link peptide by 2 or 3 Amino acid profiles in the two sides of cysteine in cyclic peptide, constitute the amino of link peptide Acid can only be glycine or proline；

The epitope being made of containing one section 5-8 amino acid in cyclic peptide.

3. according to the method described in claim 2, it is characterized in that, one section of link peptide being connected in cyclic peptide with cysteine aminoterminal It is made of completely glycine, 1 to 2 proline is contained in one section of link peptide being connected with cysteine c-terminus.

4. a kind of screening technique of the special anti-human PD-1 functional antibodies of epitope, characterized in that using described in claim 1 Method, and epitope peptide used in step 1 is the cyclic peptide with amino acid sequence documented by SEQ ID NO:13, Cell strain used in step 2 is the cell strain of cell surface height expression PD-1, and PD-1 is in the expression quantity of cell surface 10,000/cell is to 500,000/cell.

5. a kind of antibody screened using method described in claim 1, can specificity combination people's PD-1 molecule it is extracellular Section, characterized in that complementary determining region CDR1, CDR2, CDR3 of heavy chain be respectively provided with SEQ ID NO:1, SEQ ID NO:2, The amino acid sequence of SEQ ID NO:3, complementary determining region CDR1, CDR2, CDR3 of light chain be respectively provided with SEQ ID NO:4, The amino acid sequence of SEQ ID NO:5, SEQ ID NO:6.

6. antibody according to claim 5, characterized in that heavy chain variable region has the amino acid sequence of SEQ ID NO:7 Column, light chain variable region have the amino acid sequence of SEQ ID NO:8.

7. antibody according to claim 5, characterized in that heavy chain has the amino acid sequence of SEQ ID NO:9, light chain Amino acid sequence with SEQ ID NO:10.

8. antibody according to claim 5, characterized in that heavy chain is nucleotide sequence coded by SEQ ID NO:11's, gently Chain is nucleotide sequence coded by SEQ ID NO:12's.

9. a kind of composition contains monoclonal antibody as claimed in claim 6 and pharmaceutically acceptable carrier.

10. antibody described in claim 6-9 is in preparing treating malignant tumor drug and disease of immune system therapeutic agent Purposes.