A kind of anti-PD-1 human antibody
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of anti-PD-1 human antibody.
Background technique
The activation of T cell is the important step that immune system plays a role, and antigen presenting cell (APC) is not only needed to be mentioned
The first signal supplied, while the also adjusting by costimulatory signal (second signal).Costimulatory signal is a kind of cell surface
Memebrane protein plays the role of synergic adjustment T cell proliferation, activation and differentiation.
Apoptosis receptor 1 (programmed death 1, PD-1), also referred to as CD279, which is that one kind is important, to exempt from
Epidemic disease inhibits 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 cells, monocyte and bone marrow cell.PD-1 has
Two known ligands, PD-L1 and PD-L2.Currently, the study found that PD-1 can conduct inhibition in conjunction with its ligand PD-L1
Signal, lower lymph node CD8+T cell hyperplasia, and PD-1 can also by adjust Bcl-2 gene, control lymph node in
The accumulation of T cells with antigenic specificity.And the combination of PD-1 and PD-L1 are blocked, it can effectively prevent T lymphocyte from inhibiting signal
It generates, so that peripheral immune tolerance of the break immune system to autologous tissue, promotes the activation and proliferation of T lymphocyte, and
Cytokine-expressing.Therefore it is the target spot of cancer immunotherapy by PD-1, becomes a current research hotspot.
Bristol Myers Squibb in 2011 has listed Yervoy (Ipilimumab), goes through as first immune targeted drug
Treatment for advanced melanoma.Yervoy is a kind of monoclonal of anti-cell poison T lymphocyte antigen 4 (CTLA-4)
Antibody blocks the combination of CTLA-4 and its ligand B7.The prescription medicine effective percentage only 10%.Recently, Bristol Myers Squibb is developed
For the monoclonal antibody Opdivo (Nivolumab) of PD-1, clinical effectiveness shows that the curative effect of single medicine has been more than Yervoy, has
Efficiency has reached 35%, and to previously using the patient of Yervoy equally effective.In addition to the treatment in metastasis melanin tumor
Upper to have outside remarkable effect, Opdivo, which also carries out in the treatments such as colorectal cancer, non-small cell lung cancer, prostate cancer or kidney, to be faced
Bed test.
Therefore novel anti-PD-1 monoclonal antibody is researched and developed, for the immunization therapy of cancer, it is secondary makes it have lower poison
Effect, more preferably clinical drug effect, becomes current research hotspot, more medicament selections will be also provided to patient.
Summary of the invention
In view of prior art described above, it is an object of the invention to screen the novel of high-affinity by human antibody library
The human antibody of anti-PD-1, and its correlation function is verified, for solving the problems of the prior art.
In order to achieve the above objects and other related objects, the present invention provides a kind of anti-PD-1 human antibody, heavy chain of antibody
The amino acid sequence of variable region is SEQ ID NO:2 or its conservative series of variation, the amino acid sequence of antibody's light chain variable region
It is classified as SEQID NO:4 or its conservative series of variation.
The anti-PD-1 human antibody can be the full length sequence of antibody, be also possible to the segment of anti-PD-1 human antibody,
The segment be Fab, Fab ', F (ab ')2, Fv or scFv etc..
Preferably, the anti-PD-1 human antibody is source of people.
It is furthermore preferred that the anti-PD-1 human antibody is IgG1、IgG2Or IgG4Type antibody.
The present invention further provides the derivative of the anti-PD-1 human antibody, the derivative is PD-1 human antibody
Segment, antibody/antibody fragment-factor fusion protein, antibody/antibody fragment-chemical coupling thing;The anti-PD-1 human antibody
Segment be Fab, Fab ', F (ab ')2, Fv or scFv etc..
Antibody/antibody fragment-the factor fusion protein is specially antibody-factor fusion protein or antibody fragment-factor
Fusion protein.
Antibody/antibody fragment-the chemical coupling thing is specially antibody-chemical coupling thing or antibody fragment-chemical coupling
Object.
Second aspect of the present invention provides a kind of isolated DNA molecular, encode the anti-PD-1 human antibody heavy chain and/or
The variable region of light chain or full length amino acid.
Third aspect present invention provides a kind of construct, contains the isolated DNA molecular.
Preferably, the construct by the isolated DNA molecular be inserted into expression vector multiple cloning sites building and
At.
Expression vector in the present invention refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell disease
Poison, mammalian cell virus such as adenovirus, retrovirus or other carriers.
It is furthermore preferred that the expression vector is selected from one of pHLX101, pEE14.4 or pcDNA3.1 or a variety of groups
It closes.
Fourth aspect present invention provides a kind of expression system of monoclonal antibody, is transfected into host cell by the construct
It is built-up.
Host cell in the present invention can be prokaryotic cell, such as bacterial cell;Or low eukaryocyte, as yeast is thin
Born of the same parents;Or higher eucaryotic cells, such as mammalian cell.
Preferably, the host cell is selected from Chinese hamster ovary (Chinese Hamster Ovary (CHO)) cell
System, a variety of COS cell lines, HeLa cell line, myeloid cell series such as SP2/0 cell line, NS0 cell line, YB2/0 cell line etc.,
And B- cell or one of hybridoma or a variety of combinations of conversion.
Fifth aspect present invention provides the preparation method of the anti-PD-1 human antibody, includes the following steps: in suitable table
Up under conditions of the antibody, the expression system of the monoclonal antibody is cultivated, so that the monoclonal antibody is given expression to,
It is isolated and purified with the monoclonal antibody.
Host cell used in the present invention is the prior art, can be directly acquired by commercial sources, used in culture
Culture medium be also various conventional mediums, those skilled in the art can rule of thumb select applicable culture medium, be suitable for place
It is cultivated under conditions of chief cell growth.After host cell growth is to cell density appropriate, with suitable method (such as temperature
Degree conversion or chemical induction) promoter that induces selection, cell is further cultured for a period of time.Recombination in the above methods is more
Peptide can be expressed in cells, or on the cell membrane, or secreted outside the cell.If desired, can using its physics, it is chemical and
Other characteristics are separated by various separation methods and purify the albumen of recombination.These methods are well known to those skilled in the art
's.The example of these methods includes but is not limited to: conventional renaturation process, with protein precipitant processing (salting-out method), from
The heart permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, efficient liquid phase
Chromatograph the combination of (HPLC) and various other liquid chromatography technologies and these methods.
The present invention screens the gene order for obtaining purpose antibody from the cell strain of Colony Culture, to construct eukaryon table
Up to carrier, the activity of antibody can be rebuild after expression, obtain anti-PD-1 Humanized monoclonal antibodies.
Sixth aspect present invention provides the anti-PD-1 human antibody and is preparing the purposes on PD-1 molecule blocking medicine.
Preferably, the purposes prepared on PD-1 molecule blocking medicine is specially to prepare oncotherapy or diagnosing tumor class
Purposes on drug.
It is furthermore preferred that the tumour is selected from melanoma, colorectal cancer, non-small cell lung cancer, prostate cancer or kidney.
It is further preferred that the tumour is advanced melanoma.
A kind of pharmaceutical composition of seventh aspect present invention, the anti-PD-1 human antibody including therapeutically effective amount.
Human antibody of the invention can by any of mode compounding pharmaceutical composition in this field come using.It is this
Composition using the human antibody as active constituent, in addition one or more pharmaceutically acceptable carriers, diluent, filler,
Bonding agent and other excipient, this depends on administration mode and designed dosage form.It is controlled known to the branch art personnel of this field
Treat inert inorganic or organic carrier include but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, wax,
Fatty, more antelope based compound such as polyethylene glycol, water, sucrose, ethyl alcohol, glycerol, such, various preservatives, divide lubricant
Powder, corrigent.Moisturizing cuts to pieces, antioxidant, sweetener, colorant, stabilizer, salt, buffer is such that it is added
In, these substances be used to help as needed formula stability or help to improve activity it biological effectiveness or in mouth
Acceptable mouthfeel or smell are generated in the case where clothes, inhibitor can be used in such a composition can be its original chemical
The form of itself, or the form of its pharmaceutically acceptable salt is optionally used, human antibody of the invention can be administered alone,
Or with various combination medicine-feedings, and the combining form administration together with other healing potions.The composition so prepared is as needed
Any mode appropriate well known by persons skilled in the art may be selected to be administered inhibitor.
Anti- PD-1 human antibody provided by the present invention expression quantity with higher in mammalian cells, and it is right
The ability that PD-1 has apparent affinity and ligand-receptor is inhibited to combine.
Detailed description of the invention
Fig. 1 is the SDS-PAGE analysis for the PD-1-His recombinant protein expressed and purified.
Fig. 2 is the SDS-PAGE analysis for the PD-1-AP-His recombinant protein expressed and purified.
Fig. 3 is the affinity capacity experimental of anti-PD-1 antibody.
Fig. 4 is the ligand-receptor combination Inhibition test of anti-PD-1 antibody.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment;It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention;In description of the invention and claims, unless in text
In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001;Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates;the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego;Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998;METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999;With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
Expression and Purification of Human PD-1 extracellular region recombinant protein PD-1-His
It synthesizes people PD-1-AP-His fusion (being synthesized by Nanjing Genscript Biotechnology Co., Ltd.), wherein PD-1
Gene order is that SEQ ID NO:5, AP-His gene order is SEQ ID NO:6, and two sections of sequence connections are PD-1-AP-His
Fusion, for constructing PD-1-His recombinant protein expression vector, design forward and reverse primer is respectively as follows:
Forward primer:
5'-ATCCGCCGGCAAGCCGCCACCATGCAGATCCCACAGGCGCCCTGG-3'(SEQ ID NO:7);
Reverse primer:
5’-TCACTACGTATCAGTGGTGGTGGTGGTGGTGTTGGAACTGGCCGGCTGGCCTGG-3’(SEQ ID
NO:8), the gene of coding PD-1-His fusion protein is obtained for expanding, introduces restriction enzyme respectively in the upstream and downstream of gene
Enzyme site Ngo MIV and Sna BI, PCR condition is 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35 circulations, 72 DEG C of extensions
10min, archaeal dna polymerase are TAKARA Products.PCR product recycles (QIAquick Gel Extraction Kit Omega through Ago-Gel
Bio-tek Products) after purification, restriction enzyme Ngo MIV and Sna BI is added and carries out digestion, digestion products are through DNA
Reclaim reagent (Omega bio-tek Products) after purification with the carrier pHLX101 of identical digestion with restriction enzyme (by
Shanghai Henlius Biotech Co. Ltd.'s building, referring to Chinese patent 201210211812.1) it is attached reaction.Connection
Product is transformed into DH5 α Escherichia coli, is coated on the 2YT agar medium containing 50 μ g/ml carbenicillins.By acquisition
Positive colony is cultivated in the 2YT fluid nutrient medium containing 50 μ g/ml carbenicillins, is tested by the sequencing of Invitrogen company
After card, positive colony plasmid is extracted with the big pumping kit of plasmid (Omega bio-tek Products).
Using the Neon system of Invitrogen company, by the positive plasmid DNA electrotransfection of linearisation to Chinese hamster ovary celI.Turn
Chinese hamster ovary celI after dye is diluted colonized culture, is containing 50 μm of ol methionine imino groups for sulfone (methionine
Sulfoximine, MSX) 94113 culture mediums (IrvineScientific Products) in screened, to obtain list
Cloned cell line.By the monoclonal cell of acquisition tie up to containing 50 μm of ol methionine imino groups in 94113 culture mediums of sulfone into
Row shaking flask culture, expression time are usually 7-14 days, harvest cell culture supernatant when viable cell density is lower than 30%.It will be thin
Born of the same parents' culture solution supernatant is purified with nickel affinity column, finally obtains people PD-1 extracellular region recombinant protein PD-1-His (Fig. 1).
Embodiment 2
Expression and Purification of Human PD-1 extracellular region recombinant protein PD-1-AP-His
Using the PD-1-AP-His fusion synthesized in embodiment 1 as template, design forward and reverse primer is respectively as follows:
5 '-ATCCGCCGGCAAGCCGCCACCATGCAGATCCCACAGGCGCCCTGG-3 ' (SEQ ID NO:7) and 5 '-TCACTAC
GTATCAGTGGTGGTGGTGGTGGTGGCCTGAACC-3 ' (SEQ ID NO:9) obtains coding PD-1-AP-His for expanding
The gene of fusion protein introduces restriction endonuclease sites Ngo MIV and Sna BI, PCR condition in the upstream and downstream of gene respectively
For 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 60s, 35 circulations, 72 DEG C of extension 10min, archaeal dna polymerase is TAKARA Products.
PCR product recycles (QIAquick Gel Extraction Kit is Omega bio-tek Products) after purification through Ago-Gel, is added
Restriction enzyme Ngo MIV and Sna BI carries out digestion, and through DNA reclaim reagent, (Omega bio-tek company produces digestion products
Product) reaction is attached with the carrier pHLX101 of identical digestion with restriction enzyme after purification.Connection product is transformed into DH5 α
Escherichia coli are coated on the 2YT agar medium containing 50 μ g/ml carbenicillins.The positive colony of acquisition is being contained
It is cultivated in the 2YT fluid nutrient medium of 50 μ g/ml carbenicillins, it is big with plasmid after Invitrogen company sequence verification
It takes out kit (Omega bio-tek Products) and extracts positive colony plasmid.
Using the Neon system of Invitrogen company, by the positive plasmid DNA electrotransfection of linearisation to Chinese hamster ovary celI.Turn
Chinese hamster ovary celI after dye is diluted colonized culture, is containing 50 μm of ol methionine imino groups for sulfone (methionine
Sulfoximine, MSX) 94113 culture mediums (IrvineScientific Products) in screened, to obtain list
Cloned cell line.By the monoclonal cell of acquisition tie up to containing 50 μm of ol methionine imino groups in 94113 culture mediums of sulfone into
Row shaking flask culture, expression time are usually 7-14 days, harvest cell culture supernatant when viable cell density is lower than 30%.It will be thin
Born of the same parents' culture solution supernatant is purified with nickel affinity column, finally obtains people PD-1 extracellular region recombinant protein PD-1-AP-His (Fig. 2).
Embodiment 3
The building and expression of anti-PD-1 human antibody carrier for expression of eukaryon
The amino acid sequence of antibody heavy chain variable region is SEQ ID NO:2, DNA sequences encoding used in the present embodiment
For SEQ ID NO:1;
The amino acid sequence of antibody's light chain variable region is SEQ ID NO:4, DNA sequences encoding used in the present embodiment
For SEQ ID NO:3;(heavy chain and light-chain variable region gene are synthesized by Nanjing Genscript Biotechnology Co., Ltd.)
The DNA sequences encoding of above-mentioned light chain variable region and heavy chain variable region is expanded using PCR reaction, in antibody weight
Chain variable region and the gene both ends of light chain variable region introduce restriction enzyme site appropriate, wherein holding in antibody heavy chain variable region gene 5 '
And 3 ' end introduce Ngo MIV and Sna BI restriction enzyme site respectively, forward and reverse primer sequence is respectively as follows: 5 '-ACCTGCCGGC
AAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3 ' (SEQ ID NO:10) and 5 '-GCTCTACGTATCATTT
ACCTGGAGACAGGGAGAGGC-3 ' (SEQ ID NO:11) is introduced respectively at the end of antibody chain variable region gene 5 ' and 3 ' ends
Ngo MIV and SnaB I, forward and reverse primer sequence are respectively as follows: 5 '-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAG
CCTCATCTTGCTCTTC-3 ' (SEQ ID NO:10) and 5 '-GCATCTTACGTATTATTATGAACATTCCGTAAG-3 '
(SEQ ID NO:12).It is reacted using PCR, condition is 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 60s, 35 circulations, 72 DEG C of extensions
10min, archaeal dna polymerase are TAKARA Products.After PCR amplification, by PCR product through agarose gel electrophoresis recovery purifying.
Light-chain variable region gene is added restriction enzyme Ngo MIV and SnaB I and carries out digestion, and limitation is added in heavy chain variable region gene
Property restriction endonuclease Ngo MIV and Sna BI carry out digestion, purified after digestion through DNA purification kit, and with it is identical restricted
The carrier pHLX101 of endonuclease digestion.Connection product is transformed into DH5 α Escherichia coli, is coated on containing 50 μ g/ml carboxylic benzyl moulds
On the 2YT agar medium of element.The positive colony of acquisition is trained in the 2YT fluid nutrient medium containing 50 μ g/ml carbenicillins
It supports, after Invitrogen company sequence verification, extracts positive colony plasmid with the big pumping kit of plasmid.It utilizes
The Neon system of Invitrogen company, by the plasmid DNA transfection of linearisation to Chinese hamster ovary celI.Chinese hamster ovary celI after transfection carries out dilute
Colonized culture is released, is containing 50 μm of ol methionine imino groups for the 94113 of sulfone (methionine sulfoximine, MSX)
It is screened in culture medium (IrvineScientific Products), to obtain monoclonal cell system.
The monoclonal cell of acquisition is tied up to containing 50 μm of ol methionine imino groups for shaking in 94113 culture mediums of sulfone
Bottle culture, expression time is usually 7-14 days, harvests cell culture supernatant when viable cell density is lower than 30%.Utilize goat-anti
Human IgG Fd (Meridian Products) and goat-anti people's constant region of light chain of horseradish peroxidase-labeled carry out the double fastener heart
The content of antibody in supernatant is expressed in ELISA method detection, and using untransfected supernatant as negative control, the IgG sterling of people is as standard
Product.Experimental result shows that the destination protein for expressing acquisition can be identified there is human IgG antibody's feature, and structure by two antibody
The human antibody expression quantity built is in 200~500 μ g/ml, expression with higher.Using Protein A affinity column
Purpose antibody is isolated and purified from cells and supernatant.
Embodiment 4
Anti- PD-1 human antibody Function Identification
Affinity of antibody identification: by the PD-1-His recombinant protein being prepared in embodiment 1 by 2 μ g/ml (embodiments 1
Middle preparation) into ELISA Plate (working volume 30ul), 4 DEG C stand overnight coating.With the PBS of the Tween 20 containing 0.05%
(PBST) it washes 3 times.It is closed 1 hour with 0.1% BSA room temperature.It is washed 3 times with PBST, the antibody that embodiment 3 is prepared is prepared
At 200 μ g/ml concentration, and 3 times of gradient dilutions are carried out, totally 8 gradients, every hole is added 30 μ l, is stored at room temperature 1 hour.Use PBST
It washes 3 times, the ELIAS secondary antibody (Millipore Products) of the anti-human Fc of the diluted coupling HRP of 30ul 1:4000, room temperature is added
Stand 1 hour.It is washed 4 times with PBST, TMB colour developing is added, and with the H of 2M2SO4Terminate reaction.It is read at 450 nm with microplate reader.
It can be seen that from result, the antibody screened has apparent affinity, and its ability and positive control to PD-1
Nivolumab close (Fig. 3).
Ligand-antibody combination Inhibition test: by 2 μ g/ml B7-H1 (justice sticks up Divine Land Products) coating into ELISA Plate,
4 DEG C stand overnight.It is washed 3 times with PBST.It is closed 1 hour with 0.1% BSA room temperature.It is washed 3 times with PBST, the preparation of embodiment 3 is obtained
The antibody obtained is configured to 200 μ g/ml concentration, and carries out 3 times of gradient dilutions, and totally 8 gradients, are prepared with embodiment 2
22.5 μ g/ml PD-1-AP-His are mixed by 2:1, are added in ELISA Plate, are stored at room temperature 1 hour.It is washed 4 times, is added with PBST
PNPP colour developing, is read at 405nm with microplate reader.From the results, it was seen that the antibody that screening obtains can inhibit PD-1 to match with it
The combination of body B7-H1, and its ability is close with Nivolumab (Fig. 4).
PBS formula are as follows: potassium dihydrogen phosphate (KH2PO4): 0.27g, disodium hydrogen phosphate (Na2HPO4): 1.42g, sodium chloride
(NaCl): 8g, potassium chloride (KCl): 0.2g adds deionized water about 800mL that dissolution is sufficiently stirred, and concentrated hydrochloric acid tune pH then is added extremely
7.4, last constant volume to 1L.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.