CN108314734A - Anti- PD-1 monoclonal antibodies and its application - Google Patents
Anti- PD-1 monoclonal antibodies and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Abstract
The present invention relates to a kind of variable region sequences of new 1 monoclonal antibodies of anti-PD and the antibody, belong to biotechnology.The present invention is using 1 albumen of recombined human PD (rhEPD 1) as immunogene, immune BAL b/c mouse take immune Mouse spleen cells and merge it with myeloma cell sp2/0 Ag14, the hybridoma cell strain of 1 antibody of anti-PD can be expressed by obtaining, this cell strain being capable of the single monoclonal antibody of stably excreting.The present invention has cloned the H chains, L chains variable region nucleotide and amino acid sequence of 1 monoclonal antibodies of anti-PD simultaneously.1 monoclonal antibodies of anti-PD of the present invention can be specifically bound with PD 1, blocked the combination of PD1 and PD L1, restored the function of T cell.Immunologic test point inhibitor is can be used as, the treatment for cancer and infectious diseases.
Description
Technical field：
The present invention relates to a kind of monoclonal antibodies that can specifically bind PD-1, belong to biotechnology.It is specifically related to
The preparation method of the monoclonal antibody, variable region sequences and its application.
Background technology：
Hybridoma technology (hybridoma technique), also known as monoclonal antibody technique, Koehler in 1975 and
Milstein is proved, myeloma cell is merged with immune animal splenocyte, can be formed for the special of a certain antigen
The strong monoclonal antibody of property.Myeloma cell, which is utilized, in hybridoma technology continuous passage and lymphocyte can secrete spy in vitro
The characteristic of heterogenetic antibody or the factor merges two kinds of cells, then the cell of successful fusion can be with the master of both cells
Want characteristic.
PD-1/PD-L1 immunotherapies are to improve immune response by blocking the interaction between the two, have treatment more
The potentiality of type tumour and infectious diseases.Programmed death receptor -1 (programmed death-1, PD-1) is CD28
Superfamily member is a kind of important immunosuppression molecule, and main expression is in the T cell and B cell of activation.PD-1 matches with it
Body PD-Ls (programmed death-1 ligands, mainly PD-L1 and PD-L2) combinations can inhibit the increasing of T cell
It grows, activate and the secretion of relevant cell factor, make attack of the body from self immune system.But in the tumor microenvironment of body
In, after the PD-1 on the expression of tumour cell height PD-L1, with T cell is combined, the failure of inducing T cell inhibits T cell
Function, can not effective activating immune system, cause the immunologic escape of tumour cell.And for the monoclonal antibody of PD-1, PD-L1
The combination that PD-1 and PD-L1 can be blocked, makes T cell rejuvenate, to enhance immune response.Currently, more plants of anti-PD-1 and
The antibody of PD-L1 has listed, and the treatment of kinds of tumors is used for, such as Nivolumab (anti-PD-1 antibody), Keytruda (anti-PD-L1
Antibody) etc..
The present invention is prepared into using recombined human PD-1 extracellular region (rhEPD-1) albumen as antigen by hybridoma technology screening
To the anti-PD-1 monoclonal antibody specifics of high-affinity, and pass through inside and outside experimental verification its high-performance bio activity.
Invention content
Goal of the invention
It is an object of the present invention to provide a kind of anti-PD-1 method for preparing monoclonal antibody；
The second purpose of the present invention is to provide a kind of anti-PD-1 monoclonal antibodies；
The three of the object of the invention are to provide the variable region amino acid sequence and nucleic acid sequence of anti-PD-1 monoclonal antibodies.
Technical solution
The present invention is using the recombined human PD-1 albumen that laboratory is prepared early period as immunogene, multiple immune Bal b/c
Mouse waits for that its serum titer reaches fusion and requires, takes its splenocyte to carry out cell fusion with sp2/0-Ag14 myeloma cell, lead to
It crosses HAT selective mediums to screen to obtain the hybridoma cell strain of the energy anti-PD-1 antibody of stably excreting, through subcloning and expansion
After culture, antibody weight is carried out on a molecular scale, chain variable region gene is transferred；Monoclonal antibody can be by thin by hybridoma
Born of the same parents are injected to mouse peritoneal, are obtained through Protein A/G affinity chromatography column purifications after taking ascites to collect.
Advantageous effect
The present invention is successfully prepared anti-PD-1 monoclonal antibodies, and the antibody specificity is good, and Percentage bound is high, can block PD-1 with
PD-L1 bioconjugations.Lymphocyte activity can effectively be restored in vitro, human peripheral lymphocyte (PBMC) is promoted to secrete IFN-
γ and IL-2, and tumor inhibition effect of the monoclonal antibody to tumor-bearing mice is shown in vivo experiment.It tests inside and outside
It has been shown that, this anti-PD-1 monoclonal antibody is the potential drug of immunotherapy of tumors.
Description of the drawings：
Fig. 1 .SDS-PAGE detect Protein A/G affinity column purified product results.Swimming lane M is albumen Marker；
Swimming lane 1 is the antibody protein in eluent；Figure 1A are non-reduced SDS-PAGE electrophoresis results；Figure 1B are reproducibility SDS-PAGE
Electrophoresis result.
Fig. 2 monoclonal antibody subtype identification ELISA result schematic diagrams.
Fig. 3 Flow cytometries antibody and the natural PD-1 antigen binding situations of THP-1 cell surfaces.
The specific binding situation of Fig. 4 and Fig. 5 Flow cytometry antibody.Fig. 4 are detection antibody and Chinese hamster ovary celI
The combination of strain；Fig. 5 detection antibody and the combination for stablizing the Chinese hamster ovary celI (CHO-PD-1) for expressing PD-1.
The combination situation of Fig. 6 Flow cytometry PD-L1 albumen and THP-1 cell surfaces PD-1.
The anti-PD-1 monoclonal antibodies blocks PD-L1 of Fig. 7 Flow cytometries is combined with the PD-1 of cell surface.
Fig. 8 .ELISA experiments show the binding curve of antibody and PD-1 antigen proteins.
The concentration dependent of Fig. 9 Flow cytometry monoclonal antibody Pi and HEK293F-PD-1 cell combinations.
Figure 10 measure the parent of monoclonal antibody Pi and antigen by biomembrane interference technique detection molecules Interaction Force
And power.
Figure 11 .PCR amplification antibody weight, chain variable region gene product agarose electrophoresis are as a result, swimming lane M is DNA
Ladder；Swimming lane 1 and 2 is PCR amplification antibody heavy chain variable region gene product；Swimming lane 3 is PCR amplification antibody light chain variable region base
Because of product.
Figure 12 and Figure 13 detect influence of the anti-PD-1 monoclonal antibodies to PBMC cytokine secretions.Figure 12 are detection
The variation of PBMC secretion of gamma-IFN；Figure 13 are the variation for detecting PBMC secretions IL-2.
Pharmacodynamic experiment in Figure 14 bodies.Figure 14 A. are tumour growth curve；Figure 14 B. change for different group mouse weights
Curve graph；Figure 14 C. are different group mouse knurl weight results.
Specific implementation mode：
The present invention is described in further detail below in conjunction with embodiment.It should be appreciated, however, that these embodiments are only
Illustration is played, and is not for limiting the present invention.It is anti-that prepared by monoclonal to the present invention under the concept thereof of the present invention
The simple transformation of body and preparation method thereof, belongs to the scope of protection of present invention.
Experimental method used in following embodiments enumerated is conventional method unless otherwise specified.
1 animal immune of embodiment
Prepared using laboratory early period recombined human PD-1 albumen (be dissolved in PBS buffer solution, carried out Preliminary Identification, it is non-
This patent emphasis, does not do and is unfolded) mixed in equal volume with Quick Antibody after, intramuscular injection to 6-8 week old BAL b/c mouse
In vivo, 50 μ g/200 μ l/.Immunologic process is carried out according to Quick Antibody specifications.Altogether carry out 3-5 time be immunized, often
After secondary immune a period of time, takes capillary blood taking method to carry out eye socket to immune mouse and take blood (50 μ l), using indirect ELISA,
Utilize measurement mice serum antibody titer after confining liquid gradient dilution serum.Select specific antibody titres in serum highest small
Mouse is used for cell fusion.Final detection result shows immune serum potency up to 1: 100,000 or more.
The preparation of 2 hybridoma cell strain of embodiment
1. cell fusion
Cell fusion uses polyethylene glycol method.Concrete operations are as follows：
1) it merges the last week, recovery sp2/0-Ag14 myeloma cell, cell state can be adjusted with guanozola.
Cell fusion a few days ago, expands culture sp2/0-Ag14, and observation state makes it, close to exponential phase, melt in fusion the previous day
It closes the previous day and changes liquid processing；
2) generally immune mouse detection serum titer, which reaches 100,000, to merge.Select PD-1 specificity in serum anti-
Body potency highest mouse carries out impact for 3-4 days and is immunized before fusion, abdominal cavity or tail vein injection PD-1 albumen；
3) preparation (Turnover of Mouse Peritoneal Macrophages) of feeder cells：The previous day paving feeder cells are merged, week old is selected
The ICR mouse of larger (more than e.g.8 week old), the neck that breaks are put to death, and peritoneal macrophage is extracted under aseptic condition.Cell is diluted to
1×105Bed board after a/ml is put into 37 DEG C, 5%CO per 100 μ l of hole after marking2Cell incubator in overnight incubation.
4) 1 hour before progress cell fusion, sp2/0-Ag14 cells is anticipated, are centrifuged after sp2/0-Ag14 cells are resuspended
1,200rpm × 5min, serum-free DMEM are counted after cell is resuspended, and general total cell number range is 0.6-2 × 107A, 4 DEG C temporarily
It puts spare.
5) mouse to be fused is subjected to eye socket and takes blood, serum is collected in ibid operation, and temporarily -20 DEG C or -80 DEG C preservations, can do
Positive control after fusion.Disconnected neck puts to death mouse, is soaked in 75% alcohol, is transferred to cell room.It takes spleen to be ground, passes through
Single cell suspension is made in 70 μm of screen filtrations, centrifuges 2,000rpm × 5min, abandons supernatant.Erythrocyte cracked liquid is added in cell precipitation
Processing reacts centrifugal treating after 5min, is used in combination serum-free DMEM to wash once, is finally resuspended, viable count.
6) according to count results, sp20-Ag14 myeloma cell and splenocyte is mixed in 1: 5 ratio, is centrifuged after mixing thorough
Bottom exhausts supernatant.The PEG1450 (SIGMA) for taking 650 37 DEG C of pre-temperatures of μ l, is slowly dropped in cell precipitation, acts on 90s, immediately
The plasma-free DMEM medium of 37 DEG C of pre-temperatures is added, centrifuges 1 after mixing, 200 × 5min, cell precipitation is resuspended in containing HAT
(SIGMA) it in 20%FBS-DMEM culture mediums, is taped against in 96 orifice plates for being covered with feeder cells, 200 μ l/ are per hole, in 37
DEG C, 5%CO2Cell incubator in cultivate.
7) routine observation cell state after merging.In fusion the 4th day, liquid is partly changed, i.e., with fresh 20%FBS-HAT-
DMEM culture mediums, which swap out, cultivates the culture medium (100 μ l) of half in orifice plate；The 7-10 days after fusion, use instead containing HT
(SIGMA) 20%FBS-DMEM culture mediums are cultivated.
2. screening and the subcloning of positive fused cell
The case where cell secretory antibody in each hole being detected by ELISA.Coating is screened using rhEPD-1 albumen as ELISA
Antigen, the Goat anti Mouse IgG of HRP labels select the ELISA positives to be worth high hole as detection antibody, under microscope
If observation has the hybridoma or cell mass survived, it is marked, picking positive hole will be thin in hole using limiting dilution assay
Born of the same parents carry out cloning, the hybridoma cell strain of stably excreting monoclonal antibody are established after being subcloned twice, and be enlarged
Culture.
The preparation and purifying of 3 anti-PD-1 monoclonal antibodies of embodiment
1. ascites is collected
It takes in intraperitoneal injection 500 μ l paraffin oils (SIGMA) to 8-10 week old female Bal b/c Mice Bodies.After a week, abdomen
Chamber injection inoculation 1 × 106A good hybridoma of growth conditions.In inoculation 7-10 days or so of hybridoma, mouse
Abdomen obviously bloats, and acquires ascites at this time, 4 DEG C, 12,000rpm centrifugation 5min remove cell fragment, collect supernatant for follow-up
Purifying.
2. antibody purification
Antibody purification is carried out using ProteinA/G column packings (Abmart).Concrete operations：Ascites supernatant need to use Binding
Buffer is diluted to after certain volume and filtration sterilization loading again.It is cleaned, is washed away non-with Binding Buffer after loading
The impurity of specific binding, is finally eluted, and the elution that 5-10ml Elution Buffer carry out destination protein is added, and is received
Collect eluent and Neutralization Buffer are added immediately keeps pH value partial neutral, column packing clear with Binding Buffer
After washing, 20% ethyl alcohol, 4 DEG C of preservations.
3. antibody purity and molecular weight determination
The eluent of collection SDS-PAGE electrophoresis Preliminary Identification antibody, such as Figure 1A and Figure 1B.
The CHARACTERISTICS IDENTIFICATION of 4 monoclonal antibody of embodiment
1. the identification of antibody subtype
Using Mouse monoclonal antibody subtype identification kits (Proteintech), come the anti-PD-1 Dan Ke identified
The hypotype of grand antibody, it is pre-coated for mouse IgG 1, IgG2a, IgG2b, IgG2c, IgG3, IgM, kappa on ELISA Plate
Kit specification is shown in the specific antibody of light chain, lambda light chain, specific experiment operation.As a result such as
The heavy chain subgroup of Fig. 2, the anti-PD-1 monoclonal antibodies that we obtain are IgG1, light chain subtype Kappa.
The antibody of the present invention can be used as other homotypes to recombinantly express, such as IgG2, IgG3, IgG4, IgM and IgA.
2. the combination situation of PD-1 on Flow cytometry antibody and cell membrane
It selects human monocytic leukaemia's cell strain (THP-1) of cell surface expression people PD-1 and stablizes expression PD-1's
Chinese hamster ovary cell (CHO) CHO-PD-1, the specificity of anti-PD-1 monoclonal antibodies is determined by flow cytometry.It will give birth to
The good THP-1 of long status is counted after being resuspended with PBS, and cell density is adjusted to 1-2 × 106A/ml, packing to 1.5ml EP are managed
In, often isometric cell suspension is added in pipe, and general 100-300 μ l centrifuge 800g × 5min, abandon supernatant, and 100 μ l are added and dilute
Embodiment 3 in obtained anti-PD-1 monoclonal antibodies Pi, 4 DEG C of incubation 30min, PBS is washed 2-3 times after taking-up, adds AF647
Sheep anti mouse fluorescence secondary antibody, after 4 DEG C are protected from light incubation 30min, washing operation is the same, is eventually adding appropriate PBS and cell precipitation is resuspended, on
Flow cytometer is detected.The same THP-1 of detection method of CHO-PD-1 cells detects antibody and CHO and CHO-PD-1 respectively
The combination of cell illustrates its specific binding with PD-1.
From the point of view of flow cytometry combination situation (Fig. 3), anti-PD-1 monoclonal antibodies can preferably with human cell surface
PD-1 is combined, and Percentage bound is up to 89.9%；Fig. 4 shows that Pi is not bound with the Chinese hamster ovary celI without transfection, and Fig. 5 illustrates the Dan Ke
Grand antibody Pi can be combined specifically with the Chinese hamster ovary celI (CHO-PD-1) for stablizing expression PD-1.
3. monoclonal antibody Pi blocks PD-L1 to be combined with the PD-1 of cell surface
PD-L1 is the ligand of PD-1, and PD-L1 albumen can be combined with the PD-1 of cell surface, flow cytometer detection such as Fig. 6 institutes
Show, the Percentage bound of PD-L1 albumen and THP-1 cells is 83.1%.On this basis, it is detected using the method for flow cytometry anti-
The combination of PD-1 monoclonal antibodies blocks PD-L1 and cell surface PD-1.The processing of THP-1 cells is the same, every group of 5 μ g PD- of addition
L1 albumen, while anti-PD-1 monoclonal antibodies Pi being added by the concentration of 0,1,10 and 50 μ g/ml successively and is incubated altogether, fluidic cell
Art detects the combination situation of PD-L1 albumen and THP-1 cells.
From the point of view of streaming result (Fig. 7), the anti-PD-1 monoclonal antibodies blocks effects of 1 μ g/ml up to 50%, 10 μ g/ml and
50 μ g/ml can block PD-L1 protein bindings to cell surface completely.
The EC50 that 4.ELISA detections monoclonal antibody Pi is combined with PD-1
Using recombined human PD-1 albumen as solid phase antigen, overnight, the closing 2 of 5% skim milk of next day is small for 10 μ g/ml coatings
When, the gradient dilution series of monoclonal antibody is then added, reacts 2 hours at room temperature, the goat-anti of HPR labels is added after washing
Mouse secondary antibody detects OD450Value carries out data processing and mapping analysis using 7 softwares of GraphPad Prism, by fitting, obtains
To anti-PD-1 monoclonal antibodies to the binding curve and EC50 values of recombined human PD-1 albumen, Fig. 8 is as a result seen, the results showed that anti-PD-1
Monoclonal antibody can be combined with recombined human PD-1 protein-specifics, EC50=0.46nmol/L.
5. the concentration dependent of Flow cytometry monoclonal antibody Pi and HEK293F-PD-1 cell combinations
HEK293F is human embryonic kidney cells, and cell surface does not express people's PD-1 antigens, we pass through the method for cell transfecting
Transfection recombination PD-1 recombinant plasmids, enable HEK293F cell surfaces to express people's PD-1 antigens, after cultivating 72 hours, PD-1's
Expression quantity reaches maximum value, and collection cell prepares single cell suspension and the monoclonal antibody Pi of gradient dilution carries out total incubation, leads to
Overflow-type cell art detects the combination situation of the Pi and HEK293F-PD-1 of various concentration, and FlowJo software calculations incorporateds are averaged
Fluorescence intensity (MFI) carries out data processing and mapping analysis using 7 softwares of GraphPad Prism, reflects itself and cell combination
Concentration accordance with tolerance.
Can be seen that Pi and HEK293F-PD-1 cells from the experimental result of Fig. 9 is combined with good concentration dependent.
6. the measurement of anti-PD-1 monoclonal antibodies and antigen affinity
Anti- PD-1 monoclonal antibodies and the affinity of PD-1 use ForteBio Octet bio-molecular interaction instrument, profit
It is measured with biomembrane interference technique (BLI).Antigen is subjected to biotinylation first, it is made to carry biotin label, selection
The biosensor of Streptavidin.According to this characteristic (SA), the antigen after biotinylation is anchored on probe, is resisted
The measurement of original antibody affinity.Successively press balance, the anchoring of antigen, balance, combination, dissociation sequence setting program, use
ForteBio Octet measure affinity.Processing analysis is carried out to data using Octet Data Analysis Software.
Can be seen that the affinity of anti-PD-1 monoclonal antibodies Pi and PD-1 that BLI is measured from the experimental result of Figure 10 is
3.66×10-10nmol/L。
The clone of 5 positive hybridoma cell strain heavy and light chain variable region gene of embodiment
1. anti-PD-1 monoclonal antibodies heavy and light chain variable region gene extraction, amplification and Preliminary Identification
The hybridoma cell strain for collecting exponential phase, carrying for total serum IgE is carried out with the RNAiso Plus of TaKaRa companies
It takes, product description is shown in concrete operations.70 μ l RNase-free water dissolutions precipitation is added, 10 μ l is taken to carry out nucleic acid electrophoresis,
200V/10min.Remaining temporarily puts -80 DEG C of synthesis for preserving or carrying out first cDNA chain.
First cDNA is synthesized by template reverse transcription of total serum IgE, reagent is purchased from Vazyme companies.Reaction system is shown in explanation
Book, reaction condition, which is placed in PCR instrument, to specifications carries out first cDNA synthetic reaction.Product can carry out PCR amplification immediately
Reaction temporarily puts -20 DEG C of preservations.
PCR amplification weighs light variable domains gene, can be changed according to hybridoma strain antibody heavy and light chain in bibliography
Degenerate primer designed by area upstream and downstream gene sequence, primer sequence are as follows：
5’MH1：5’-ctt ccggaa ttc SAR GTN MAG CTG SAG SAG TC-3’
5’MH2：5’-ctt ccggaa ttc SAR GTN MAG CTG SAG SAG TCW GG-3’
3’VH：5’-ggaaga tct CTT GAC CAG GCA TCC TAG AGT CA-3’
5’Kappa：5’-Gggag ctc GAY ATT GTG MTS ACM CAR WCT MCA-3’
3’Kappa：5’-Ggtgca tgc GGA TAC AGT TGG TGC AGC ATC-3’
(degenerate codon explanation：R=a, g；Y=c, t；M=a, c；S=c, g；W=a, t).
50 μ l of reaction system, reaction condition are：94℃ 5min；94℃ 30s；57℃ 30s；72 DEG C of 45s, cycle 30
It is secondary, 72 DEG C of 7min.It takes 10 μ l PCR products to run nucleic acid electrophoresis to be identified, if Figure 11 is shown, PCR amplification obtains 450bp or so
Heavy light variable domains gene.Purified to PCR product with plastic recovery kit (raw work biology), operates same kit
Specification.
Because that utilized in pcr amplification reaction step is Taq Plus DNA Polymerase (Vazyme), PCR product
3 ' end band adenines (A), so PCR product is without carrying out 3 ' ends plus A reactions, can Direct Cloning to carrier T.PCR product is connected
It is connected to18-T carriers, take 1 μ l carrier Ts, after PCR product 4 μ l or 2 μ l (distilled water supplies volume to 4 μ l) mixing, add
Enter 5 μ l Solution I, 16 DEG C of connections are overnight.
Connection product is converted into competence JM109 using calcium chloride transformation, conversion operation：Take 10 μ l connection products
It is transformed into 100 μ l competent cells JM109, stands 30min, heat shock 2min in 42 DEG C of water-baths after mixing in ice-water bath, stand
Ice bath 1min is taken out, the 1ml non-resistant LB culture mediums of 37 DEG C of pre-temperatures are often added in pipe, 37 DEG C, 150rpm slowly shakes 1h.It takes
Centrifuge 4,000rpm × 5min after going out, coating ammonia benzyl resistant panel after precipitation is resuspended with 100 μ l LB culture mediums, upside down in
After cultivating 16-20h in 37 DEG C of constant incubators, result is observed.
It is cloned on picking tablet, the overnight incubation in the LB culture mediums of the resistance of benzyl containing ammonia.Take 5 μ l bacterium solutions, 12,000rpm ×
Supernatant is abandoned in 2min, suction, carries out bacterium colony PCR using bacterial sediment as template, operation is the same.10 μ l bacterium colony PCR products are taken to carry out core
Sour electrophoresis verification, picking have the positive colony of tape to send to sequencing.
2. gene sequencing and the analysis of anti-PD-1 monoclonal antibodies weight light variable domains
Sequencing result is shown, successfully obtains hybridoma cell strain constant region for immunoglobulin sequence gene.By on the websites NCBI
Database (https://www.ncbi.nlm.nih.gov/igblast/) the anti-PD-1 monoclonal antibodies weight light chain variable region of analysis
Gene, analysis result show：Hybridoma cell strain heavy chain of antibody variable domains VHNucleotide sequence and amino acid sequence such as
SEQ ID NO：1 and SEQ ID NO：Shown in 2；Hybridoma cell strain light variable domains VLNucleotide sequence and amino acid
Sequence such as SEQ ID NO：9 and SEQ ID NO：Shown in 10；Heavy-chain variable domains VH includes hypervariable region CDRH1, CDRH2 successively
And CDRH3, the nucleotide sequence are followed successively by SEQ ID NO：3,5,7, amino acid sequence is followed successively by SEQ ID NO：4、6、
8；Light variable domains VL includes hypervariable region CDRL1, CDRL2 and CDRL3 successively, and the nucleotide sequence is followed successively by SEQ
ID NO：11,13,15, amino acid sequence is followed successively by SEQ ID NO：12、14、16.
Influence of the 6 anti-PD-1 monoclonal antibodies of embodiment to PBMC cytokine secretions
96 orifice plates, 2 μ g/ml CD3 antibody of coating in advance, per 50 μ l of hole, 37 DEG C be incubated 4 hours after take out PBS wash 3 times it is standby
With.PBMC is isolated using lymphocyte separation medium (being purchased from CEDARLANE) from the blood of physical examination of healthy population, is operated according to production
Product specification method carries out.According to every hole 2 × 10 after counting5A cell is added in above-mentioned 96 processed orifice plates, 100 μ of volume
l.Add 5 μ l CD28 antibody (mother liquid concentration is 20 μ g/ml) stimulation per hole simultaneously.Experiment packet is as follows：(1) only CD3 antibody and
The PBMC of CD28 antibody stimulation；(2) CD3 antibody and the stimulation of CD28 antibody, and the PBMC that PD-L1 albumen is incubated altogether is added；(3)
CD3 antibody and the stimulation of CD28 antibody, are added PD-L1 albumen and the PBMC of Pi, three multiple holes of every group of setting.Cell at 37 DEG C and
After being cultivated 5 days under 5%CO2, take out culture medium supernatant from every hole and be used for cytokine assay, measure according to commercially available people's IFN-γ and
IL-2 detection kits (connection section biology) operation carries out.
As a result show (Figure 12 and Figure 13), PD-L1 can inhibit the activity of lymphocyte, reduce cell factor IFN-γ and
The secretion of IL-2.The PD-1 of anti-PD-1 monoclonal antibodies blocks PD-L1 and lymphocytic cell surface is combined, and is lived to restore cell
Power promotes the secretion of IFN-γ and IL-2.
Tumor-inhibiting action of the 7 anti-PD-1 monoclonal antibodies of embodiment in bearing mouse model
The female Bal b/c mouse of selection 6-8 week old are randomly divided into three groups.Every group of 5 mouse, grouping include：(1)
Control group, (2) antibody low dose group, (3) antibody high dose group.Control group is administered using Isotype control IgG, antibody height
Dosage group gives the anti-PD-1 monoclonal antibodies Pi of 10mg/kg and 5mg/kg respectively.It was administered, connects for the first time at the 0th day within the 1st day
Tumor, mouse is subcutaneously injected in left upper extremity proximal part oxter has surely turned the CT26-PD-L1 mouse colonic cells of human PD-L 1,2 ×
105A cell/mouse, the PD-L1 expressed on the CT26 cells of transfection have proven to preferably be combined with antibody.Weekly administration two
Secondary, tail vein injection administration observes tumor volume change, waits for that diameter of tumor reaches 1mm or more, can be carried out with vernier caliper daily
I.e. recordable tumor volume data is measured, gross tumor volume is calculated according to ab2/ 2 (a is major diameter, and b is minor axis) calculate, and work as control
Group gross tumor volume reaches 1000mm3When above, be euthanized to mouse, stripping tumor weigh take pictures result show such as Figure 14 A, according to
The results show that in CT26-PD-L1 mouse tumor models, according to tumor growth curve, it can be seen that administration group is compared with control group pair
Tumour growth has certain inhibiting effect, and high dose group inhibition is better than low dose group, and Figure 14 B are shown between Mice Body recombination
Without significant difference in group.Stripping tumor weighing results are shown in Figure 14 C after mouse is put to death, though administration group low dose group and high dose group with
Control group compares, significant difference (P ＜ 0.01 and P ＜ 0.05).
Claims (10)
1. the variable region sequences of anti-PD-1 monoclonal antibodies, it is characterised in that the antibody includes heavy-chain variable domains VHWith
Light variable domains VL；Wherein, heavy-chain variable domains VHNucleotides sequence be classified as SEQ ID NO：1, amino acid sequence is
SEQ ID NO：2；Heavy-chain variable domains VHInclude hypervariable region CDRH1, CDRH2 and CDRH3, the nucleotide sequence successively
It is followed successively by SEQ ID NO：3,5,7, the amino acid sequence is followed successively by SEQ ID：4、6、8；Light variable domains VLNucleosides
Acid sequence is SEQ ID NO：9, amino acid sequence is SEQ ID NO：10；Light variable domains VLInclude hypervariable region successively
CDRL1, CDRL2 and CDRL3, the nucleotide sequence are followed successively by SEQ ID NO：11,13,15, the amino acid sequence according to
Secondary is SEQ ID：12、14、16.
2. a kind of antibody or antigen-binding fragment or polypeptide, can be combined with PD-1, it is characterised in that it includes institutes in claim 1
Any amino acid sequence stated, including SEQ ID：2、4、6、8、10、12、14、16.
3. a kind of antibody or antigen-binding fragment or polypeptide, can be combined with PD-1, the sequence described in sequence and claim 2 is extremely
Less with 85% sequence similarity.
4. a kind of genetic engineering antibody, which is characterized in that the heavy chain and light-chain variable sequence that it is included and claim 1 institute
The variable region sequences stated are consistent or at least 85% similitude；The genetic engineering antibody includes but not limited to：People-mouse is chimeric
Antibody (chimeric antibody)；Either humanized antibody (humanized antibody, HAb)；Or antibody
Functional fragment Fab (Fragment of antigen binding, Fab)；Either single-chain antibody (Single-chain
Variable fragment, ScFv)；The either antibody functional segment V of heavy chain variable region and Whole light chains fusionH-L；Or
Person is bispecific antibody；The either arrangement of one or more CDR of heavy chain and light chain, the antibody work(connected or be composed
It can property segment；Either above-mentioned antibody and antibody functional fragment and other various albumen or polypeptide be attached, splice, merge and
Functional fusion protein of obtained class antibody.
5. a kind of nucleic acid, including the nucleotide sequence described in claim 1, including SEQ ID：1、3、5、7、9、11、13、15.
6. a kind of composition, it includes the antibody or antibody binding fragment or polypeptide or core described in any one of claim 1-5
Acid and at least one pharmaceutically acceptable carrier.
7. any antibody or antigen-binding fragment or polypeptide or nucleic acid or composition are in oncotherapy in claim 1-6
Using.
8. any antibody or antigen-binding fragment or polypeptide or nucleic acid or composition are in infectious diseases in claim 1-6
Application.
9. the antibody described in any one of a kind of method, including claim 1-6 of enhancing T cell function, antigen-binding fragment
Or polypeptide or nucleic acid or composition, and its in application medically.
10. any one of a kind of method, including claim 1-4 of detection PD-1 antibody or antigen-binding fragment or polypeptide, and
Its application in diagnosis.
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