CN104479020A - Anti-PD-1 (programmed cell death-1) humanized antibody - Google Patents
Anti-PD-1 (programmed cell death-1) humanized antibody Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and in particular relates to an anti-PD-1 humanized antibody. The anti-PD-1 humanized antibody is characterized in that the amino acid sequence of a variable region of a heavy chain of the antibody is shown as SEQ ID NO: 2 or is a conserved variation sequence; the amino acid sequence of a variable region of a light chain of the antibody is shown as SEQ ID NO: 4 or is a conserved variation sequence. The anti-PD-1 humanized antibody shows relatively high expression quantity in mammalian cells, and is obvious in affinity with PD-1 and able to inhibit the combination of a ligand and a receptor.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of anti-PD-1 human antibody.
Background technology
The activation of T cell is the important step that immunity system plays a role, and not only needs the first signal that antigen presenting cell (APC) provides, and is also subject to the adjustment of costimulatory signal (second signal) simultaneously.Costimulatory signal is the membranin of a class cell surface, the effect played synergic adjustment T cell propagation, activation and break up.
Programmed cell death acceptor 1 (programmed death 1, PD-1), also claims CD279, is a kind of important immunosuppression molecule, contactin CD28 family member, and molecular weight is the I type transmembrane glycoprotein of 50 ~ 55kD.PD-1 persistent expression is on the surface of T cell, B cell, natural killer cell, monocyte and medullary cell.PD-1 has two known ligands, PD-L1 and PD-L2.At present, research finds, PD-1 is combined with its part PD-L1, can conduct the signal of inhibition, lowers the hyperplasia of lymphoglandula CD8+T cell, and PD-1 by adjustment Bcl-2 gene, can also control the accumulation of T cells with antigenic specificity in lymphoglandula.And block the combination of PD-1 and PD-L1, can effectively stop T lymphocyte to suppress the generation of signal, thus the peripheral immune tolerance of break immune system against its own organism, promote the lymphocytic activation and proliferation of T and cytokine-expressing.Therefore be the target spot of cancer immunotherapy by PD-1, become a current study hotspot.
Bristol Myers Squibb in 2011 has gone on the market Yervoy (Ipilimumab), is approved for the treatment of advanced melanoma as first immune targeted drug.Yervoy is the monoclonal antibody of a kind of anti-cell poison T lymphocyte-associated antigen 4 (CTLA-4), blocks the combination of CTLA-4 and its part B7.This prescription medicine efficient only 10%.Recently, the monoclonal antibody Opdivo for PD-1 (Nivolumab) of Bristol Myers Squibb exploitation, the curative effect that its clinical effectiveness shows single medicine has exceeded Yervoy, efficiently reaches 35%, and to previously using the patient of Yervoy effective equally.Except having except remarkable effect in the treatment of metastasis melanin tumor, Opdivo also carries out clinical trial in the treatments such as colorectal cancer, nonsmall-cell lung cancer, prostate cancer or kidney.
Therefore research and develop novel anti-PD-1 monoclonal antibody, for the immunotherapy of cancer, make it have lower toxic side effect, better clinical drug effect, becomes current study hotspot, also will provide more medicament selection to patient.
Summary of the invention
Prior art in view of the above, the object of the invention is to the human antibody of the novel anti-PD-1 by human antibody library screening high-affinity, and verifies its correlation function, for solving the problems of the prior art.
For achieving the above object and other relevant objects, the invention provides a kind of anti-PD-1 human antibody, the aminoacid sequence of its antibody heavy chain variable region is SEQ ID NO:2 or its conservative form variation sequence, and the aminoacid sequence of its antibody chain variable region is SEQID NO:4 or its conservative form variation sequence.
Described anti-PD-1 human antibody can be the full length sequence of antibody, and also can be the fragment of anti-PD-1 human antibody, described fragment be Fab, Fab ', F (ab ')
2, Fv or scFv etc.
Preferably, described anti-PD-1 human antibody is people source.
Preferred, described anti-PD-1 human antibody is IgG
1, IgG
2or IgG
4type antibody.
The present invention further provides the derivative of described anti-PD-1 human antibody, described derivative is fragment, antibody/antibody fragment-factor fusion protein, the antibody/antibody fragment-chemical coupling thing of PD-1 human antibody; The fragment of described anti-PD-1 human antibody is Fab, Fab ', F (ab ')
2, Fv or scFv etc.
Described antibody/antibody fragment-factor fusion protein is specially antibody-factor fusion rotein or antibody fragment-factor fusion protein.
Described antibody/antibody fragment-chemical coupling thing is specially antibody-chemical coupling thing or antibody fragment-chemical coupling thing.
Second aspect present invention provides a kind of DNA molecular of separation, the encode described anti-heavy chain of PD-1 human antibody and/or the variable region of light chain or full length amino acid.
Third aspect present invention provides a kind of construct, the DNA molecular containing described separation.
Preferably, the multiple clone site structure that described construct is inserted into expression vector by the DNA molecular of described separation forms.
Expression vector in the present invention refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral as adenovirus, retrovirus or other carriers.
Preferred, described expression vector is selected from one or more the combination in pHLX101, pEE14.4 or pcDNA3.1.
Fourth aspect present invention provides a kind of expression system of monoclonal antibody, is transfected into constructing host cell forms by described construct.
Host cell in the present invention can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.
Preferably, described host cell is selected from Chinese hamster ovary (Chinese Hamster Ovary (CHO)) clone, multiple COS clone, HeLa clone, myeloid cell series are as SP2/0 clone, NS0 clone, YB2/0 clone etc., and transform B-cell or hybridoma in one or more combination.
Fifth aspect present invention provides the preparation method of described anti-PD-1 human antibody, comprise the steps: under the condition of the described antibody of applicable expression, the expression system of the monoclonal antibody described in cultivation, thus give expression to described monoclonal antibody, be isolated and purified with described monoclonal antibody.
Host cell used in the present invention is prior art, directly obtain by commercial sources, substratum used in cultivation is also various conventional medium, and those skilled in the art rule of thumb can select the substratum be suitable for, and cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in cell or on cytolemma.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The present invention screens the gene order obtaining object antibody from the cell strain of Colony Culture, in order to build carrier for expression of eukaryon, can rebuild the activity of antibody after expression, obtain anti-PD-1 Humanized monoclonal antibodies.
Sixth aspect present invention provides the purposes of described anti-PD-1 human antibody on preparation PD-1 molecule blocking medicine.
Preferably, the purposes on described preparation PD-1 molecule blocking medicine is specially the purposes on preparation oncotherapy or diagnosing tumor class medicine.
Preferred, described tumour is selected from melanoma, colorectal cancer, nonsmall-cell lung cancer, prostate cancer or kidney.
Preferred further, described tumour is advanced melanoma.
A kind of pharmaceutical composition of seventh aspect present invention, comprises the described anti-PD-1 human antibody for the treatment of significant quantity.
Human antibody of the present invention can be used by any known mode compounding pharmaceutical composition in this area.This composition for activeconstituents, adds one or more medicine acceptable carriers, thinner, weighting agent, bonding agent and other vehicle with described human antibody, and this depends on administering mode and designed dosage form.Inorganic or the organic carrier of the treatment inertia that this area branch art personnel are known includes, but is not limited to lactose, W-Gum or derivatives thereof, talcum, vegetables oil, wax, fat, many antelopes based compound such as polyoxyethylene glycol, water, sucrose, ethanol, glycerine, like this, various sanitas, lubricant, dispersion agent, correctives.Moisturizing is cut to pieces, antioxidant, sweeting agent, tinting material, stablizer, salt, damping fluid is like this also can add wherein, these materials are as required for helping the stability of formula or contributing to improving activity or its biological effectiveness or produce acceptable mouthfeel or smell when oral, but the form of inhibitor its original chemical itself can be used in such a composition, or optionally use the form of the acceptable salt of its pharmacology, human antibody of the present invention can be individually dosed, or with various combination medicine-feeding, and combining form administration together with other healing potion.The composition of preparation like this can select any suitable mode well known by persons skilled in the art that inhibitor is carried out administration as required.
Anti-PD-1 human antibody provided by the present invention has higher expression amount in mammalian cell, and to the ability that PD-1 has obvious avidity and suppresses ligand-receptor to combine.
Accompanying drawing explanation
Fig. 1 is that the SDS-PAGE expressing the PD-1-His recombinant protein that also purifying obtains analyzes.
Fig. 2 is that the SDS-PAGE expressing the PD-1-AP-His recombinant protein that also purifying obtains analyzes.
Fig. 3 is the avidity capacity experimental of anti-PD-1 antibody.
Fig. 4 is that the ligand-receptor of anti-PD-1 antibody is in conjunction with Inhibition test.
Embodiment
Below by way of specific specific examples, embodiments of the present invention are described, those skilled in the art the content disclosed by this specification sheets can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this specification sheets also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention; In specification sheets of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
Embodiment 1
Expression and purification people PD-1 extracellular region recombinant protein PD-1-His
Synthesis people PD-1-AP-His fusion gene (being synthesized by Nanjing Genscript Biotechnology Co., Ltd.), wherein PD-1 gene order is SEQ ID NO:5, AP-His gene order is SEQ ID NO:6, two sections of sequences connections are PD-1-AP-His fusion gene, for structure PD-1-His expression of recombinant proteins carrier, design forward and reverse primer are respectively:
Forward primer:
5’-ATCCGCCGGCAAGCCGCCACCATGCAGATCCCACAGGCGCCCTGG-3’(SEQ ID NO:7)；
Reverse primer:
5 '-TCACTACGTATCAGTGGTGGTGGTGGTGGTGTTGGAACTGGCCGGCTGGCCTGG-3 ' (SEQ IDNO:8), the gene of coding PD-1-His fusion rotein is obtained for increasing, restriction endonuclease sites Ngo MIV and Sna BI is introduced respectively in the upstream and downstream of gene, PCR condition is 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35 circulations, 72 DEG C extend 10min, and archaeal dna polymerase is TAKARA Products.PCR primer reclaims after (reclaiming test kit is Omega bio-tek Products) purifying through sepharose, add restriction enzyme Ngo MIV and Sna BI to carry out enzyme and cut, digestion products reclaims after reagent (Omega bio-tek Products) purifying through DNA and carries out ligation with the carrier pHLX101 (being built by Shanghai Henlius Biotech Co. Ltd., see Chinese patent 201210211812.1) with identical digestion with restriction enzyme.Connect product conversion to DH5 α intestinal bacteria, be coated on the 2YT nutrient agar containing 50 μ g/ml Pyocianils.The positive colony of acquisition is cultivated in containing the 2YT liquid nutrient medium of 50 μ g/ml Pyocianils, after Invitrogen company sequence verification, takes out greatly test kit (Omega bio-tek Products) with plasmid and extract positive colony plasmid.
Utilize the Neon system of Invitrogen company, by linearizing positive plasmid DNA electrotransfection to Chinese hamster ovary celI.Chinese hamster ovary celI after transfection carries out dilution colonized culture, containing 50 μm of ol methionine(Met) imino-s for sulfone (methioninesulfoximine, MSX) screen in 94113 substratum (IrvineScientific Products), thus obtain monoclonal cell system.Tied up to by the monoclonal cell of acquisition and carry out shake-flask culture containing 50 μm of ol methionine(Met) imino-s in 94113 substratum of sulfone, expression time is generally 7-14 days, when viable cell density lower than 30% time harvested cell nutrient solution supernatant.Cell culture supernatant nickel affinity column is carried out purifying, finally obtains people PD-1 extracellular region recombinant protein PD-1-His (Fig. 1).
Embodiment 2
Expression and purification people PD-1 extracellular region recombinant protein PD-1-AP-His
With the PD-1-AP-His fusion gene of synthesis in embodiment 1 for template, design forward and reverse primer are respectively: 5 '-ATCCGCCGGCAAGCCGCCACCATGCAGATCCCACAGGCGCCCTGG-3 ' (SEQ ID NO:7) and 5 '-TCACTACGTATCAGTGGTGGTGGTGGTGGTGGCCTGAACC-3 ' (SEQ ID NO:9) obtains the gene of coding PD-1-AP-His fusion rotein for increasing, restriction endonuclease sites NgoMIV and Sna BI is introduced respectively in the upstream and downstream of gene, PCR condition is 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 60s, 35 circulations, 72 DEG C extend 10min, archaeal dna polymerase is TAKARA Products.
PCR primer reclaims after (reclaiming test kit is Omega bio-tek Products) purifying through sepharose, add restriction enzyme Ngo MIV and Sna BI to carry out enzyme and cut, digestion products reclaims after reagent (Omega bio-tek Products) purifying through DNA and carries out ligation with the carrier pHLX101 of identical digestion with restriction enzyme.Connect product conversion to DH5 α intestinal bacteria, be coated on the 2YT nutrient agar containing 50 μ g/ml Pyocianils.The positive colony of acquisition is cultivated in containing the 2YT liquid nutrient medium of 50 μ g/ml Pyocianils, after Invitrogen company sequence verification, takes out greatly test kit (Omega bio-tek Products) with plasmid and extract positive colony plasmid.
Utilize the Neon system of Invitrogen company, by linearizing positive plasmid DNA electrotransfection to Chinese hamster ovary celI.Chinese hamster ovary celI after transfection carries out dilution colonized culture, containing 50 μm of ol methionine(Met) imino-s for sulfone (methioninesulfoximine, MSX) screen in 94113 substratum (IrvineScientific Products), thus obtain monoclonal cell system.Tied up to by the monoclonal cell of acquisition and carry out shake-flask culture containing 50 μm of ol methionine(Met) imino-s in 94113 substratum of sulfone, expression time is generally 7-14 days, when viable cell density lower than 30% time harvested cell nutrient solution supernatant.Cell culture supernatant nickel affinity column is carried out purifying, finally obtains people PD-1 extracellular region recombinant protein PD-1-AP-His (Fig. 2).
Embodiment 3
The structure of anti-PD-1 human antibody carrier for expression of eukaryon and expression
The aminoacid sequence of antibody heavy chain variable region is SEQ ID NO:2, and the DNA sequences encoding used in the present embodiment is SEQ ID NO:1;
The aminoacid sequence of antibody chain variable region is SEQ ID NO:4, and the DNA sequences encoding used in the present embodiment is SEQ ID NO:3; (heavy chain and chain variable region gene synthesize by Nanjing Genscript Biotechnology Co., Ltd.)
PCR reaction is used to increase to the DNA sequences encoding of above-mentioned variable region of light chain and variable region of heavy chain, suitable restriction enzyme site is introduced at the gene two ends of antibody heavy chain variable region and variable region of light chain, wherein hold and 3 ' end introducing Ngo MIV and Sna BI restriction enzyme site respectively at antibody heavy chain variable region gene 5 ', forward and reverse primer sequences are respectively: 5 '-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3 ' (SEQ IDNO:10) and 5 '-GCTCTACGTATCATTTACCTGGAGACAGGGAGAGGC-3 ' (SEQ ID NO:11), hold and 3 ' end introducing Ngo MIV and SnaB I respectively at antibody chain variable region gene 5 ', forward and reverse primer sequences are respectively: 5 '-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3 ' (SEQ IDNO:10) and 5 '-GCATCTTACGTATTATTATGAACATTCCGTAAG-3 ' (SEQ ID NO:12).Adopt PCR reaction, condition is 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 60s, 35 circulations, and 72 DEG C extend 10min, and archaeal dna polymerase is TAKARA Products.After pcr amplification, PCR primer is reclaimed purifying through agarose gel electrophoresis.Chain variable region gene adds restriction enzyme Ngo MIV and SnaB I to carry out enzyme and cuts, heavy chain variable region gene adds restriction enzyme Ngo MIV and Sna BI to carry out enzyme and cuts, enzyme carries out purifying through DNA purification kit after cutting, and with the carrier pHLX101 of identical digestion with restriction enzyme.Connect product conversion to DH5 α intestinal bacteria, be coated on the 2YT nutrient agar containing 50 μ g/ml Pyocianils.The positive colony obtained is cultivated in containing the 2YT liquid nutrient medium of 50 μ g/ml Pyocianils, after Invitrogen company sequence verification, takes out greatly test kit extract positive colony plasmid with plasmid.Utilize the Neon system of Invitrogen company, by linearizing plasmid DNA transfection to Chinese hamster ovary celI.Chinese hamster ovary celI after transfection carries out dilution colonized culture, containing 50 μm of ol methionine(Met) imino-s for sulfone (methionine sulfoximine, MSX) screen in 94113 substratum (IrvineScientific Products), thus obtain monoclonal cell system.
Tied up to by the monoclonal cell of acquisition and carry out shake-flask culture containing 50 μm of ol methionine(Met) imino-s in 94113 substratum of sulfone, expression time is generally 7-14 days, when viable cell density lower than 30% time harvested cell nutrient solution supernatant.Utilize the goat-anti people constant region of light chain of goat anti-human igg Fd (Meridian Products) and horseradish peroxidase-labeled to carry out double sandwich-ELISA method and detect the content of expressing antibody in supernatant, using untransfected supernatant as negative control, the IgG sterling of people is as standard substance.Experimental result shows, and expressing the target protein obtained by two antibody recognition, can have human IgG antibody's feature, and the human antibody expression amount built is at 200 ~ 500 μ g/ml, has higher expression level.Adopt Protein A affinity column separation and purification object antibody from cells and supernatant.
Embodiment 4
Anti-PD-1 human antibody Function Identification
Affinity of antibody is identified: by the PD-1-His recombinant protein for preparing in embodiment 1 by 2 μ g/ml (preparing in embodiment 1) bag by (working volume 30ul) in enzyme plate, 4 DEG C of hold over night.3 times are washed with the PBS (PBST) of the Tween 20 containing 0.05%.BSA room temperature with 0.1% closes 1 hour.Wash 3 times with PBST, the antibody that embodiment 3 prepares is mixed with 200 μ g/ml concentration, and carries out 3 times of gradient dilutions, totally 8 gradients, every hole adds 30 μ l, and room temperature leaves standstill 1 hour.Wash 3 times with PBST, add the ELIAS secondary antibody (Millipore Products) of the anti-human Fc of the coupling HRP that 30ul 1:4000 dilutes, room temperature leaves standstill 1 hour.Wash 4 times with PBST, add TMB colour developing, and with the H of 2M
2sO
4termination reaction.With microplate reader reading at 450 nm.Can find out from result, screen the antibody obtained, to PD-1, there is obvious avidity, and its ability close with positive control Nivolumab (Fig. 3).
Part-antibodies Inhibition test: by 2 μ g/ml B7-H1 (justice sticks up Divine Land Products) bags by enzyme plate, 4 DEG C of hold over night.3 times are washed with PBST.BSA room temperature with 0.1% closes 1 hour.Wash 3 times with PBST, the antibody that embodiment 3 prepares is mixed with 200 μ g/ml concentration, and carries out 3 times of gradient dilutions, totally 8 gradients, the 22.5 μ g/ml PD-1-AP-His prepared with embodiment 2 mix by 2:1, join in enzyme plate, and room temperature leaves standstill 1 hour.Wash 4 times with PBST, add pNPP colour developing, with microplate reader reading under 405nm.As can be seen from the results, screen the combination that the antibody capable obtained suppresses PD-1 and its part B7-H1, and its ability close with Nivolumab (Fig. 4).
PBS formula is: potassium primary phosphate (KH2PO4): 0.27g, Sodium phosphate dibasic (Na2HPO4): 1.42g, sodium-chlor (NaCl): 8g, Repone K (KCl): 0.2g, add deionized water and be about the abundant stirring and dissolving of 800mL, then add concentrated hydrochloric acid and adjust pH to 7.4, last constant volume is to 1L.
In sum, the present invention effectively overcomes various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (12)
1. an anti-PD-1 human antibody, the aminoacid sequence of its antibody heavy chain variable region is SEQ ID NO:2 or its conservative form variation sequence, and the aminoacid sequence of its antibody chain variable region is SEQ ID NO:4 or its conservative form variation sequence.
2. anti-PD-1 human antibody as claimed in claim 1, is characterized in that, described antibody is people source.
3. anti-PD-1 human antibody as claimed in claim 1, is characterized in that, described antibody is IgG
1, IgG
2or IgG
4type antibody.
4. the derivative of anti-PD-1 human antibody as claimed in claim 1, described derivative is the fragment of described anti-PD-1 human antibody, antibody/antibody fragment-factor fusion protein or antibody/antibody fragment-chemical coupling thing.
5. the derivative of anti-PD-1 human antibody as claimed in claim 4, is characterized in that, the fragment of described anti-PD-1 human antibody is Fab, Fab ', F (ab ')
2, Fv or scFv.
6. the DNA molecular be separated, the anti-heavy chain of PD-1 human antibody described in coding claim 1-3 arbitrary claim and/or the variable region of light chain or full length amino acid.
7. a construct, containing, for example the DNA molecular of separation according to claim 6.
8. a kind of construct as claimed in claim 7, is characterized in that, the multiple clone site structure being inserted into expression vector by the DNA molecular of separation according to claim 5 forms.
9. an expression system for monoclonal antibody, is transfected into constructing host cell by the construct described in the arbitrary claim of claim 7-8 and forms.
10. the preparation method of the anti-PD-1 human antibody as described in claim as arbitrary in claim 1-3, comprise the steps: under the condition of the described antibody of applicable expression, cultivate the expression system of monoclonal antibody according to claim 9, thus give expression to described monoclonal antibody, be isolated and purified with described monoclonal antibody.
The purposes of anti-PD-1 human antibody as described in 11. claims as arbitrary in claim 1-3 on preparation PD-1 molecule blocking medicine.
12. 1 kinds of pharmaceutical compositions, comprise the described anti-PD-1 human antibody for the treatment of significant quantity.
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Application publication date: 20150401 Assignee: Shanghai Zuolin Biotechnology Co.,Ltd. Assignor: SHANGHAI HENLIUS BIOTECH, Inc. Contract record no.: X2023980037618 Denomination of invention: A Human Antibody Against PD-1 Granted publication date: 20190802 License type: Common License Record date: 20230705 |