CN108299561A - A kind of PD-1 nano antibodies and its cloning expression method and application - Google Patents

Abstract

The invention discloses a kind of 1 nano antibodies of PD and its cloning expression method and applications.The 1 nano antibody molecular weight of PD is small, high specificity, affinity are high, can inhibit the interaction between 1 molecule of PD L1 and PD, and can specific recognition cell surface native conformation PD 1, block PD L1 to be combined to keep T cell activity with PD 1；With the identical binding ability with 1 monoclonal antibodies of PD (IgG whole antibodies)；And the state that activating T cell can be reversed to be inhibited by PD L1.Meanwhile compared with 1 monoclonal antibodies of PD, the relative activity higher of nano antibody of the present invention after same high temperature process, thermal stability is more preferable, has the potentiality for being developed into immunologic test point inhibitor.Meanwhile nano antibody cloning expression method yield of the invention is higher, obtains purity of protein up to 93% or more, preparation method is simple, of low cost, has a good application prospect in the preparation of antitumor drugs.

Description

A kind of PD-1 nano antibodies and its cloning expression method and application

Technical field

The invention belongs to biomedicine technical field, more particularly to a kind of PD-1 nano antibodies and its cloning expression method with Using.

Background technology

Tumour is one of Global mortality highest disease, seriously threatens human health；In addition to operation, radiotherapy, tumour Therapy further include：Chemotherapy, hormonotherapy, targeted drug therapy and tumour immunotherapy.From 2013《Science》 After tumour immunotherapy is classified as year most important scientific breakthrough by magazine, tumour immunotherapy is more concerned in recent years.

Targeting blocking immunity checkpoint tumour immunotherapy has been increasingly becoming the focus of attention of oncotherapy, from 2014, FDA successively ratifies the Keytruda of Mo Shadong and the Opdivo of Bristol Myers Squibb for since treating melanoma, having targeted journey The antibody of sequence death molecule 1 (programmed death-1, PD-1, CD279) clinically illustrates excellent, wide spectrum anti- Tumor promotion so that PD-1 inhibitor becomes the medicine of most foreground in current immunotherapy of tumors.After T cell is activated PD-1 expression is induced, with its ligand PD-L1 (programmed cell death 1ligand 1) or PD-L2 (programmed 1 ligand 2, PD-L2 of cell death) combine after, phosphorylation, Protein-tyrosine-phosphatase occur for the tyrosine in the areas ITSM Molecule is recruited so that and the effector molecule dephosphorylation in downstream activates PD-1/PD-Ls signal paths, negative regulator signal of transduceing, Inhibit T cell activation, lowers immune response.Under normal circumstances, which plays key work in terms of maintaining T cell stable state With.But in tumor microenvironment, tumor-infiltrating lymphocytes, tumor specific T cells and some relevant T cell surfaces of damage Height expression PD-1 molecules, after tumor surface PD-L1 is combined with T cell surface PD-1, transmit inhibition signal, keep T cell active It lowers, to inhibit immune response of the body to tumour, tumour is caused to generate immunologic escape.Since PD-1, PD-L1 widely join With to tumor immune escape, therefore, the activity of the releasable T Lymphocvte Killers tumour of PD-1/PD-Ls accesses is targeted and blocks, To effectively inhibit tumour progression.

At present listing or granted clinical test PD-1 antibody be monoclonal antibody (monoclonal anbibody, MAb) (Fig. 1), monoclonal IgG whole antibodies have that tissue permeability is weak, immunogenicity is stronger, the inadequate natural endowments such as of high cost, in addition, The whole antibody long half time for targeting PD-1, there is the potential risk for causing body excessive immune.

1993, Hamers Casterman etc. isolated natural deletions light chain, only containing heavy chain from camel serum Camel antibodies are named as heavy chain antibody (heavy-chain antibody, HCAb).And VHH (variable domain of Heavy chain of heavy-chain antibody) be on heavy chain antibody can molecule of the antigen binding structural domain, be logical The variable region for crossing technique for gene engineering clone's heavy chain antibody, obtains the single domain antibody being only made of a heavy chain variable region.Because VHH crystal diameters 2.5nm, long 4nm, so VHH is also referred to as nano antibody (Nanobody, Nb).Nano antibody has high parent With power, high stability, highly-water-soluble, high antigen binding, high expression, strong tissue permeability, low immunogenicity and half-life period Short feature has unique advantage in therapeutic antibodies course of drug development, and there has been no the PD-1 nanometers of tool functional activity at present The report of antibody drug.

Invention content

The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of new anti-PD-1 antibody (nano antibody).

Another object of the present invention is to provide the cloning expression methods of the PD-1 nano antibodies.

Another object of the present invention is to provide the application of the PD-1 nano antibodies.

The purpose of the invention is achieved by the following technical solution：

A kind of nano antibody of Programmed death 1 (PD-1 nano antibodies), including by framework region FR (framework Region) and complementary determining region CDR (complementarity-determining regions) composition variable region structure Domain, the Variable domain include CDR1, CDR2 and CDR3 selected from the following：

(1) CDR1 shown in SEQ ID No.1 or SEQ ID No.2,

(2) CDR2 shown in SEQ ID No.3,

(3) CDR3 shown in SEQ ID No.4 or SEQ ID No.5.

The Variable domain includes CDR1, CDR2 and CDR3 selected from the following：

(1) shown in CDR2 and SEQ ID No.4 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.3 CDR3；

(2) shown in CDR2 and SEQ ID No.5 shown in CDR1 shown in SEQ ID No.2, SEQ ID No.3 CDR3。

Described framework region FR FR1, FR2, FR3 and the FR4 selected from the group below：

(1) FR1 shown in SEQ ID No.6；

(2) FR2 shown in SEQ ID No.7 or SEQ ID No.8；

(3) FR3 shown in SEQ ID No.9；

(4) FR4 shown in SEQ ID No.10.

The amino acid sequence of the PD-1 nano antibodies is selected from shown in following any sequence：

QVQLQESGGGSVQAGGSLRLSCVASGFTHLRYSMGWFRQVLDKEREGVAAIGGDGRTSYADSVKGRFTIFKDNAKNT LYLDMNSLNPEDTAMYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTQVTVSS (SEQ ID No.11) or

QVQLQESGGGSVQAGGSLRLSCVASGLTYLRYSMGWFRQVPDKEREGVAAIGGDGRTSYADSVKGRFTIFKDNAKNT LYLDMNSLNPEDTAMYYCAAAVLLDGSFSLLAPLTPYKYDYWGQGTQVTVSS(SEQ ID No.12)；

Start on the left of above-mentioned amino acid sequence as aminoterminal, right side is terminated as c-terminus, and sequence is：No underscore Sequence is framework region respectively：FR1~FR4；Underscore is complementary determining region, CDR1~CDR3.

The PD-1 nano antibodies include and any amino acid sequence shown in SEQ ID No.11 or SEQ ID No.12 Row have at least 90%, preferably at least 95%, the more preferably at least amino acid sequence of 99% sequence identity.

Any amino acid sequence as shown in SEQ ID No.11 or SEQ ID No.12 is by substitution, missing or addition The amino acid sequence of one or several amino acid.

The substitution is preferably compared with any amino acid sequence shown in SEQ ID No.11 or SEQ ID No.12 Including one or more amino acid substitutions, preferably conserved amino acid replace.For example, including 1,2,3,4,5,6,7,8,9 or 10 Conserved amino acid replaces.

The nucleotide sequence of the coding PD-1 nano antibody amino acid sequences, (1) chosen from the followings, (2) or (3)：

(1) nucleotide sequence as shown in SEQ ID No.13 or SEQ ID No.14；

(2) different from nucleotide sequence shown in SEQ ID No.13 or SEQ ID No.14, but encode such as SEQ ID The nucleotide sequence of amino acid sequence shown in No.11 or SEQ ID No.12；

(3) with the nucleotide sequence of (1) or (2) any one of them nucleotide sequence complementation.

The PD-1 nano antibodies have at least one of following characteristics：

(1) PD-1 and PD-L1 is blocked to combine；

(2) T cell inactivated state caused by PD-L1 is reversed；

(3) inhibit tumour growth.

A kind of nucleic acid molecules encode the PD-1 nano antibodies.

A kind of recombinant expression carrier, the weight of the nucleotide sequence containing the coding PD-1 nano antibody amino acid sequences Group expression vector.

The expression vector be phagemid vector or can encode the PD-1 nano antibodies amino acid sequence other Bacterial expression vector and Yeast expression carrier, eukaryotic expression vector.

The phagemid vector is preferably phasmid pMECS.

A kind of host cell has converted and has incorporated the coding institute on the recombinant expression carrier or genome The host cell and its progeny cell of the nucleotide sequence for the PD-1 nano antibodies stated.

The host cell and its progeny cell refer to bacterial cell, fungal cell, zooblast or plant cell and The offspring of these host cells.

The host cell is preferably Escherichia coli；Further preferably E.coli WK6.

The cloning expression method of the PD-1 nano antibodies, includes the following steps：

The nucleotide sequence of the coding PD-1 nano antibodies is cloned into expression vector, structure recombinant expression carries Body；The recombinant expression carrier is transferred in expression system again and is expressed；Substep purifying is carried out to destination protein, finally To the PD-1 nano antibodies.

Application of the PD-1 nano antibodies in preparing prevention and/or treating cancer drug.

The prevention and/or treating cancer drug is preferably immunologic test point inhibitor medicaments.

The drug can be pharmaceutical composition, including pharmaceutically acceptable carrier.

The drug can also be applied in combination therapy, i.e., be combined with other medicaments.For example, combination therapy can wrap Include at least one other antitumor drugs of PD-1 nano antibodies joint of the present invention.

The present invention has the following advantages and effects with respect to the prior art：

1. the PD-1 nano antibody molecular weight small (about 18kDa) of the present invention, high specificity, affinity are high, PD-L1 can be inhibited With the interaction between PD-1 molecules, and can specific recognition cell surface native conformation PD-1, block PD-L1 and PD-1 to tie It closes to keep T cell active；With binding ability identical with PD-1mAb.It is of the invention in vitro in PBMC Activation models PD-1 nano antibodies can reverse the state that activating T cell is inhibited by PD-L1, raise the expression of IFN-γ.

2. compared with PD-1 monoclonal antibodies (present invention selects to be commercialized the anti-PD-1 monoclonal antibodies of mouse as a contrast), this The nano antibody of invention (is abbreviated as Nb_PD-1, the Nb2 and Nb3 as shown in SEQ ID No.11 or SEQ ID No.12) it is special Property is suitable therewith, and the relative activity higher of nano antibody of the invention after same high temperature process, thermal stability are more preferable； With the potentiality for being developed into immunologic test point inhibitor, material is provided for follow-up oncotherapy drug candidate.

3. the PD-1 nano antibody cloning expression methods of the present invention, after test tube or shaking flask culture, yield is up to 3.6mg/L ~4.5mg/L, purity of protein are more than 93%, and preparation method is simple, of low cost, and future can advanced optimize condition of culture and put Greatly, to obtain higher yield.

4. the present invention successfully constructs the eukaryon expression plasmid of encoding human PD-1 extracellular fragments (PD-1ECD) recombinant protein, lead to It is overexpressed purifying, purity is obtained and is more than 90%, the PD-1ECD recombinant proteins with functional activity, and as antigen, from great Rong Measure the nanometer that the small two strain molecule amounts that filtered out in natural bacteriophage nano antibody library, high specificity, affinity are high, thermal stability is good Antibody Nb2 and Nb3.

Description of the drawings

Fig. 1 is the sketch map of PD-1 antibody present Research in the prior art.

Fig. 2 is the affinity determination curve graph of PD-1 nano antibodies Nb2 and Nb3.

Fig. 3 is the thermal stability analysis figure of PD-1 nano antibody Nb2 and Nb3 and commercially available PD-1 monoclonal antibodies；Wherein, A： 25 DEG C of processing；B：37 DEG C of processing；C：60 DEG C of processing；D：90 DEG C of processing.

Fig. 4 is the interpretation of result figure of the competion experiment of PD-1 nano antibody Nb2 and Nb3 and PD-1mAb；Control For negative control.

Fig. 5 is the interpretation of result figure of the competion experiment of PD-1 nano antibody Nb2 and Nb3 and PD-L1；Control is Negative control.

Fig. 6 is PD-1 nano antibody Nb2 and Nb3 and HEK 293T/PD-1 cell membrane surface PD-1 molecular binding events point Analysis figure；Wherein, A：PD-1mAb groups；B：2 groups of Nb；C：3 groups of Nb；D：For PD-1mAb groups, the sample peak of 3 groups of 2 groups of Nb and Nb Graphic data overlap-add procedure Integrated comparative.

Fig. 7 is that PD-1 nano antibody Nb2 and Nb3 inhibits the active barrier effect analysis chart of T cell to PD-L1.

Specific implementation mode

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

The preparation of related reagent in embodiment：

1.RPMI 1640/FBS culture mediums：89mL RPMI 1640Medium, 10mL FBS, 1mL is dual anti-, 1 μ g Human IL-2 is mixed well, and 4 DEG C save backup, one week term of validity.

2.DMEM/FBS culture mediums：445mL DMEM, 50mL FBS, 5mL is dual anti-, mixes well, and 4 DEG C save backup.

3.WST-8 solution：1.8mg 1-methoxy PMS, 0.1158g WST-8,0.351g NaCl, are completely dissolved in In 40mL ultra-pure waters, 0.45 μm of membrane filtration, packing, -20 DEG C be kept in dark place it is spare.

4.PEG/NaCl solution：PEG6000 200g, NaCl 146.1g, ultra-pure water fully dissolve, and are settled to 1L, 121 DEG C Sterilize 20min, and 4 DEG C save backup.

5.TES solution：0.146g EDTA (final concentration 0.5mM), 171.15g sucrose (final concentration 0.5M), Tris-HCl (0.2M, pH 8.0) fully dissolves, and is settled to 1L, 0.22 μm of filter membrane degerming, 4 DEG C save backup.

Expression, purifying and identification of the 1 people's PD-1 Extracellular domain proteins of embodiment in eukaryocyte

According to PD-1 extracellular fragments (PD-1ECD) gene order (NCBI Gene ID:5133) and with reference to pCMV (it is purchased from Shanghai Green skies Bioisystech Co., Ltd) multiple cloning sites information, designs suitable primer and Nhe I, BamH I digestions position is added Point and 6XHis labels use TaKaRa using pEZ-M02-PD-1 (being purchased from Guangzhou FulenGen Co., Ltd.) as template PrimerSTAR HS DNA Polymerase carry out PCR reactions, PCR product is examined through 1% agarose gel electrophoresis, There is a clearly amplified band above 500bp, is consistent with expected size 546bp.Use the FastDigest of Fermentas companies Nhe I and BamH I carry out double digestion to pCMV carriers and the PD-1ECD genes amplified, are inserted into pCMV plasmids, structure coding The carrier for expression of eukaryon pCMV-PD-1ECD of PD-1ECD recombinant proteins.PCMV-PD-1ECD is imported into HEK by rotaring dyeing technology 293F cells (being purchased from Thermo Fisher, USA) transient expression PD-1ECD recombinant proteins, crude protein pass through the affine layers of Ni-NTA PD-1ECD recombinant proteins are obtained after analysis column (being purchased from GE Healthcare, USA) purifying, concentration.Pass through SEC-HPLC and ELISA Testing and appraisal PD-1ECD recombinant protein purity is more than 90%, and has in conjunction with activity with mouse PD-1mAb, PD-L1, can be used as antigen Albumen is for subsequently screening nano antibody work.

Embodiment 2PD-1 nano antibodies screen

1.VCSM13 helper phages prepare and titer determination

Take the VCSM13 (being purchased from Agilent Technologies) of different dilutions that the E.coli TG1 newly expanded are added In (purchased from Chinese plasmid vector bacterium cell gene collection) bacterium solution, it is incubated at room temperature 15min.Mixture respectively adds 3mL liquid Change the LB top agar (less than 50 DEG C), LB tablets are poured into after mixing, 37 DEG C are incubated overnight, and calculate plaque number and to obtain Single plaque.The E.coli TG1 bacterium solutions that the single plaque of picking is newly expanded to 5mL, after 37 DEG C of shaken cultivation 3h, transfer training It supports in object to 100mL 2YT fluid nutrient mediums (the 70 μ g/mL containing kanamycins), 37 DEG C of shaken cultivations are stayed overnight.Culture is collected by centrifugation 7%DMSO, -20 DEG C or -80 DEG C preservations are added in supernatant, centrifuging and taking supernatant after 70 DEG C of water-baths.

2. the natural bacteriophage nano antibody amplified library of large capacity

Taking 100 μ L large capacities natural bacteriophage nano antibody libraries, (phasmid used is pMECS, the nano antibody library Yan J, Wang P, Zhu M, Li G are had been disclosed in, E,Xiong S,Wan Y.Characterization and applications of Nanobodies against human procalcitonin selected from a novelNanobody phage display library.J Nanobiotechnology.2015May 6；13(1):33, Can be found in "VHH library construction " chapters and sections) it is added the E.Coli TG1 bacterium solutions that newly expand of 2mL, 37 After DEG C standing, adds 8mL 2 × YT/Amp-Glu fluid nutrient mediums, after 37 DEG C of shaken cultivation 30min, add 1mL helper phages Body VCSM13 shakes up, and is stored at room temperature 30min.Low speed room temperature centrifuges, and precipitates appropriate 2 × YT/Amp-Kan Liquid Cultures base weight Outstanding, 37 DEG C of shaken cultivations are stayed overnight.Next day recycles culture solution, PEG precipitating phages, through 0.22 μm of filter mistake after 1 × PBS resuspensions Filter, the fresh phage library preparation solution being prepared are tested for subsequent elutriation.

3. large capacity natural bacteriophage nano antibody library is eluriated

It is screened (6.68 × 10 in natural phage library using display technique of bacteriophage¹¹CFU/Total) with PD-1 extracellular fragments The bacteriophage of specific binding.

With 100mM NaHCO₃Dilute PD-1ECD antigens (i.e. PD-1ECD recombinant proteins made from embodiment 1, similarly hereinafter) extremely Suitable concentration is added in 96 hole elisa Plates, is coated with 4~6 holes, overnight in 4 DEG C.Next day is de- with 0.05%PBST board-washings 1 time, 2% After fat milk room temperature closes 2h, PBST board-washings 2 times.The phage library preparation solution that appropriate step 2 obtains is taken, is bitten with PBS dilutions Cell concentration is to 1 × 10¹²Phage/mL obtains phage library dilution.100 μ L phage library dilutions are added per hole, Phage library dilution is sucked after incubation at room temperature 1h, and (often wheel is eluriated to eluriate result by upper wheel and adjust and be washed for PBST board-washings 5~20 times Plate number).The 100mM triethylamine solutions of Fresh are added, eluent is sucked out after being stored at room temperature 10min, 1M Tris- are added HCl is neutralized, and obtains the phage antibody for being incorporated into solid phase.Take 600 μ L phage antibodies that the E.Coli TG1 that 2mL is newly expanded are added Bacterium solution mixing, 37 DEG C of standings, bacterium solution after being infected take out appropriate measurement CFU, 2 × YT/ of 8mL are added after infection in bacterium solution 1mL helper phages VCSM13 is added after 37 DEG C of shaken cultivations and shakes up, is stored at room temperature 30min for Amp-Glu culture mediums.800×g 10min is centrifuged, precipitation is resuspended in appropriate 2 × YT/Amp-Kan culture mediums, and 37 DEG C of shaken cultivations are stayed overnight.Third day recycles culture solution, PEG precipitating phages, 1 × PBS are filtered through 0.22 μm of filter after being resuspended, are tested for subsequent elutriation.Last wheel is eluriated, and 4 DEG C preserve calculate library size tablet (30 ＜ monoclonals ＜ 300) or a dilution tablet is spare thereon.

4.PD-1 extracellular fragment bacteriophage nano antibodies library enrichment verification (Phage-ELISA method)

PD-1ECD is stayed overnight with the holes 100ng/ coated elisa plate, with 100mM NaHCO₃As negative control, next day board-washing 1 It is secondary, after 2% skim milk room temperature closes 2h, PBST board-washings 2 times.Often wheel is eluriated the phage library solution prepared and is diluted with 1 × PBS To 2 × 10¹¹Phage/mL, with 2% skim milk 1:1 mixing, the mixed liquor as often taken turns.The mixed liquor often taken turns is with 100 holes μ L/ Antigen hole and its corresponding nonreactive foramen primum is added, is incubated at room temperature 1h.Mixed liquor is sucked out, abandons in bleaching liquid, after PBST board-washings 5 times Anti-M13 Bacteriophage antibody (HRP) (being purchased from Divine Land Yi Qiao, Beijing biotechnology company) are added to carry out instead It answers, is stored at room temperature 1h.Antibody diluent is sucked out, TMB colour developings are added after board-washing, sulfuric acid terminates, and microplate reader measures OD_450nm.With OD_450nm(antigen hole-nonreactive foramen primum) maps, it can be seen that light absorption value is gradually reinforced after often wheel is eluriated, that is, the bacteriophage for being directed to antigen receives Meter Kang Ti is enriched with.The results show that three-wheel specifically binds PD-1ECD bacteriophage after eluriating obviously is enriched with.

5.PD-1 extracellular fragment bacteriophage nano antibody library screening

1mL TB/Amp culture mediums are added per hole for 96 hole deep-well plates；It is selected at random per hole and last wheel guarantor in step 3 is added Deposit a monoclonal on tablet.Standby LB/Amp-Glu tablets, each corresponding region of clone, 37 DEG C are incubated overnight, 4 DEG C of guarantors It deposits.IPTG (final concentration 1mM) is added in 96 hole deep-well plates, 37 DEG C of shaken cultivation 3h after inoculation, and 28 DEG C of shaken cultivations are stayed overnight, simultaneously It is coated with two piece of 96 orifice plate, antigen hole and nonreactive foramen primum are fifty-fifty on same ELISA Plate, and 4 DEG C overnight.Next day, 0.05%PBST board-washings 1 It is secondary, close 2h with 2% skim milk room temperature.Supernatant is abandoned in 96 hole deep-well plates centrifugations, after TES is resuspended, 4 DEG C of oscillation 2h, then add per hole Enter 300 μ L TES/4,4 DEG C vibrate 2h, centrifuging and taking supernatant.96 orifice plates suck confining liquid, with 0.05%PBST board-washings 2 times.Deep hole 96 orifice plates of coating PD-1ECD antigens, 100 holes μ L/ are added in plate supernatant, while 100 holes μ L/ are also added in corresponding nonreactive foramen primum, It is incubated at room temperature 1h.Incubating Solution is sucked out, Anti-6X His tag antibody (HRP) (being purchased from Abmart) are added in PBST board-washings It is reacted, is incubated at room temperature 1h；Antibody diluent is sucked out, TMB colour developings are added after board-washing, microplate reader measures OD_450nm.Light absorption value More than 1, and higher than twice of same periplasmic extract nonreactive foramen primum or more, regard as positive colony；By ELISA Preliminary Identifications, 22 possible positive antibody clones are obtained altogether.The access 5mL LB/Amp cultures of positive colony corresponding region thalline on tablet In base, 37 DEG C of shaken cultivations to suitable concentration.Illustrate with reference to kit, mini-scale plasmid extraction process prepares pMECS-Nb_PD-1Plasmid, Inspection is sequenced.

The DNA sequence dna obtained after 22 positive antibody clones are sequenced, amino acid is obtained with Bioedit software translations Sequence.According to international immunogenetics information system (IMGT) to the framework region (framework region, FR) of protein sequence Distinguished with antibody identification region (complementarity-determining regions, CDR), CDR1, CDR2, The clone of CDR3 sequence all sames is considered as same antibody strain, and the different clone of CDR sequence is considered as different antibodies strain, is finally obtained 6 positive antibodies clone (number is Nb1~6), their CDR region amino acid sequence has differences, wherein 2 plants of nano antibodies Amino acid sequence feature is shown in Table 1.

The primary structural sequence feature of 1 anti-PD-1 extracellular fragments nano antibody of table

The expression and purification of embodiment 3PD-1 nano antibodies

1.E.coli WK6 electricity turns competence preparation

E.coli WK6 (purchased from Chinese plasmid vector bacterium cell gene collection) seed liquor of -80 DEG C of preservations is taken to connect After kind amplification, it is seeded in 250mL LB culture mediums in the ratio of 1% (v/v), 37 DEG C of shaken cultivations to bacterium solution OD₆₀₀=0.35 ~0.4, ice bath 30min.4 DEG C of centrifugations, precipitation are added sterile ultra-pure water and are resuspended, and repeated centrifugation is primary, and cold 10% nothings of 20mL are added Bacterial sediment is resuspended in bacterium glycerite.After 4 DEG C of centrifugations, supernatant is carefully sucked out, it is heavy that thalline is resuspended in cold 10% sterile glycerol solutions of 1mL It forms sediment, is dispensed into the sterilizing 1.5mL EP pipes of precooling, -80 DEG C save backup.

2.pMECS-Nb _PD-1Electrotransformation

Electric revolving cup is pre-chilled on ice, WK6 competent cells made from step 1 is taken out, in melting 10min on ice.Take 100ng pMECS-Nb _PD-1Plasmid storing solution (obtains in embodiment 1 " 5.PD-1 extracellular fragment bacteriophage nano antibodies library screening " pMECS-Nb_PD-1Plasmid), mixing in WK6 competence bacterium solutions is added, after turning according to the parameter electricity that electroporation is recommended, is added immediately Mixture is sucked out in 1mL SOC culture medium mixings, is placed in 37 DEG C of shaken cultivation 20min, and 1 μ L Amp storing solutions are added, continue to cultivate 20min.Appropriate bacterium solution is taken to be coated on LB/Amp agar plate surfaces, 37 DEG C of culture 12h~16h detect transfection efficiency.Next day, It chooses monoclonal amplification and freezes strain.

The WK6 expression of 3.PD-1 nano antibodies

The strain frozen is inoculated into 10mL LB-Amp culture mediums, in 37 DEG C of shaken cultivation 7h, as bacterium seed liquor. Bacterium seed liquor is seeded in the ratio of 1% (v/v) in TB/Amp-Glu-Mg Shake flask mediums, 37 DEG C, shaken cultivation to conjunction After suitable density, addition IPTG (final concentration 1mM), 28 DEG C, 200rpm overnight incubations.Next day, centrifugation are abandoned supernatant, are washed with 1 × PBS After twice of thalline, -20 DEG C of preservations of bacterial sediment.

4. permeability evolution extracts thalline periplasmic space total protein

Thalline thaw at RT 30min, the TES resuspension that step 3 obtains, 4 DEG C, 200rpm vibrates 1~6h.It adds twice The TES/4 of TES volumes continues 4 DEG C, and 200rpm vibrates 2h or stays overnight.11,300 × g, 4 DEG C of centrifugation 30min, takes supernatant.It is added 0.2M MgCl₂Solution, until final concentration of 1mM, obtains thalline periplasmic space total protein.

5.PD-1 nano antibodies purify

Ni Sepharose^TMExcel (being purchased from GE Healthcare) dress column, pillar specification is 1.6 × 20cm, column bed body Product is 5mL.After Ni-NTA affinity columns being balanced with 1 × PBS (pH 7.4), 1mL/min loadings (the thalline week that step 4 obtains Matter space total protein).After 1 × PBS (pH 7.4) multiple equilibria, albumen is eluted with 20mM imidazoles, collecting eluting peak, (PD-1 nanometers anti- Body).The molecular size range and purity of PD-1 nano antibodies are detected by 15%SDS-PAGE protein electrophoresis：SDS-PAGE electrophoresis knots Fruit is shown in 18kDa and nearby apparent band occurs, is consistent with expection；The inspection result of Western Blot proves near 18kDa The band of appearance can be combined with anti-His mAb, Nb_PD-1With His labels, it was demonstrated that Nb_PD-1By successful expression；Illustrate collection There is the enrichment of target protein in solution.6 plants of Nb in SDS-PAGE electrophoresis results_PD-1Sample lane band is more single after purification, says Bright Nb_PD-1Purity is higher；Peak sample is penetrated in the inspection result of Western Blot without there is band, is purified in equilibrium process No destination protein is eluted；Illustrate that purification effect is preferable.

6.PD-1 nano antibodies concentrate

With the Amicon-Ultra-15 super filter tubes (MWCO 3kDa) of Millipore, 1 × PBS (PH7.4) is slow as washing Fliud flushing, 4 DEG C, 4 000rpm centrifugal ultrafiltrations, removal imidazo, which is concentrated into, needs volume, is preserved after 0.22 μm of degerming.

BCA methods measure antibody protein concentration, SDS-PAGE and Western Blot verification nano antibody expression, SEC-HPLC Detect nano antibody purity.

2 plants of Nb_PD-1Engineering bacteria yield and acquisition purity of protein are as shown in table 2.

2 Nb of table_PD-1Protein yield and purity

From the data in table 2, it can be seen that 2 plants of Nb_PD-1Engineering bacteria yield is more than 3.6mg/L, and purity is between 93~97%.

7.Nb_PD-1Physicochemical property is predicted

By using bioinformatics website ExPASy to nanoprotein sequence carry out molecular weight, theoretical isoelectric point (PI) and Overall average hydrophily (GRAVY) is analyzed, shown in table 3.

3 Nb PD-1ECD physical and chemical parameters of table are analyzed

From the data in table 3, it can be seen that Nb_PD-1Molecular weight is each about 18.3kDa, illustrates this expressed 2 plants of Nb_PD-1It is small point Son amount albumen, meets the small feature of nano antibody molecular weight.Theoretical isoelectric point (PI), Nb_PD-1Theoretical isoelectric point is 6.3 left in PI It is right.Overall average hydrophily (GRAVY) is the average value of all amino acid pro water numbers in finger protein, by being carried out to each amino acid It scores and obtains, the strongest Protein G RAVY of hydrophily is -4.5, and the strongest Protein G RAVY of hydrophobicity is 4.5.From hydrophobic property From the point of view of, it is determined as hydrophilic protein when GRAVY is negative value.It can thus be appreciated that 6 plants of Nb_PD-1It is hydrophilic protein matter.

Embodiment 4Nb_PD-1Physicochemical property

1.ELISA methods detect Nb_PD-1Specificity

It is public that CTLA4, CD4, CD8a, CD28 and the CD80 used in the present embodiment is purchased from Divine Land Yi Qiao, Beijing biotechnology Department.

Involved PD-L1 is prepared as follows to obtain in the present embodiment：According to PD-L1 gene orders (Gene ID: 29126), and pCMV (the green skies Bioisystech Co., Ltd in Shanghai) multiple cloning sites information is referred to, design primer simultaneously introduces Nhe I/Xba I restriction enzyme sites, while 6XHis labels are added.Using pReceiver-M02-PD-L1 (being purchased from reactivation gene) as mould Plate carries out PCR reactions, PCR product 1% Ago-Gel electricity using the PrimerSTAR HS DNA Polymerase of TaKaRa Swimming is examined.Using FastDigest Nhe I and the Xba I of Fermentas companies to pCMV carriers and the PD-L1 bases amplified Because carrying out double digestion, it is inserted into pCMV plasmids, the carrier for expression of eukaryon pCMV-PD-L1 of structure coding PD-L1 recombinant proteins.Pass through PCMV-PD-L1 is imported HEK 293F cells (ThermoFisher, USA) transient expression PD-L1 recombinant proteins by rotaring dyeing technology, Crude protein obtains PD-L1 recombinant proteins after Ni-NTA affinity chromatographys column purification, concentration.

(unrelated protein compares PD-1ECD recombinant proteins, PD-L1, the Enbrel for being obtained embodiment 1 using coating buffer, purchase From Pfizer, USA), CTLA4, CD4, CD8a, CD28 and CD80 be configured to final concentration of 1 μ g/mL solution respectively, with 100 μ L/ Hole is added in 96 hole elisa Plates, overnight in 4 DEG C of coatings.Next day, 0.05%PBST board-washings 2 times, the closing of 5% 37 DEG C of skim milk 2h.Respectively by the positive control PD-1mAb of a concentration of 600ng/mL (Mouse anti-PD-1antibody, purchase after board-washing 2 times From abcam), PD-1 nano antibodies (Nb2, Nb3) are added in 96 orifice plates made from blank control PBS and embodiment 2,100 μ The holes L/, 37 DEG C of incubation 1h.Primary antibody (Mouse anti-HA tag antibody are purchased from Abmart) is added after board-washing 3 times to carry out Reaction, 37 DEG C of incubation 1h.Secondary antibody is added after board-washing 3 times, and (Goat anti-mouse IgG mAb are purchased from Cell Signaling Technology), 37 DEG C of incubation 1h.TMB colour developings are added after board-washing 5 times, sulfuric acid terminates, and microplate reader measures OD_450nmAnd OD_630nm。

Light absorption value intuitively reflects Nb_PD-1With the binding ability of PD-1.Nb_PD-1With PD-1 combine light absorption value with they and it is right It is compared according to the highest light absorption value of antigen binding, ratio is more than 3, and it is positive nano antibody to define the nano antibody.The results show that Nano antibody Nb 2 and Nb 3 and PD-1 specific binding capacities it is stronger and other 7 control antigens binding abilities it is weaker.

2.Nb_PD-1Affinity costant measures

The affinity costant of PD-1 nano antibodies is measured using non-competing enzyme immunoassay.

Compound concentration is the PD-1ECD of 4 kinds of various concentrations of 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL respectively Antigenic solution, with 100 holes μ L/ be added 96 hole elisa Plates in, in 4 DEG C coating overnight.Next day board-washing is simultaneously closed.It will be prepared PD-1 nano antibody degree be adjusted to 1*10^-6mol/L(Nb2：18.398ug/mL Nb3：18.37669ug/mL) horizontal, 3 times times Than dilution, sample is added in 96 hole elisa Plates after board-washing 2 times, 100 holes μ L/, 37 DEG C of incubation 1h by blank control PBS.It washes Primary antibody (Mouse anti-HA tag antibody are purchased from Abmart) is added after plate 3 times to be reacted, 37 DEG C of incubation 1h.It washes Secondary antibody (Goat anti-mouse IgG mAb are purchased from Cell Signaling Technology) is added after plate 3 times, 37 DEG C incubate Educate 1h.TMB is added after board-washing 5 times to develop the color 10min, 2.29% sulfuric acid is added and terminates, microplate reader measures OD_450nmAnd OD_630nm。 It is fitted " S " type curve, obtains the Nb corresponding to 4 half light absorption values_PD-1Concentration (EC₅₀), the results are shown in Figure 2.

Nano antibody is univalent antibody, by formula K_A=(n-1)/(nAb'-Ab) calculates affinity costant (Ab and Ab' in formula Indicate, when antigen concentration is Ag and Ag', to generate the antibody concentration (mol/L) of half light absorption value, n=Ag/Ag' respectively), obtain 6 A KA values calculate average value and standard deviation.The results are shown in Table 4, it is known that, Nb 2 and 3 affinity costants of Nb be respectively (2.28 ± And (4.57 ± 1.64) × 10 1.38)⁷L/mol。

4 Nb of table_PD-1KA values are solved to obtain after affinity determination curve simulation

3.Nb_PD-1Thermal stability

It is configured to the coating buffer of final concentration of 0.5 μ g/mL PD-1, is added in 96 hole elisa Plates with 100 holes μ L/, in 4 DEG C Coating is overnight.Next day, 0.05%PBST board-washings 2 times, 37 DEG C of closing 2h of 5%BSA.Sample is handled during closing：It will be a concentration of The positive control PD-1mAb and Nb of 500ng/mL_PD-1Be respectively placed in 25 DEG C, 37 DEG C, 60 DEG C, 90 DEG C of four kinds of different temperatures it is lower each 0min, 10min, 30min, 60min, 120min and 180min.It is immediately placed on ice.After board-washing 2 times, by what is handled well Sample is added in 96 hole elisa Plates, 100 holes μ L/, 37 DEG C of incubation 1h.Primary antibody (Mouse anti-HA are added after board-washing 5 times Tag antibody are purchased from Abmart) it is reacted (PBS is added in PD-1mAb groups), 37 DEG C of incubation 1h.Secondary antibody is added after board-washing (Goat anti-mouse IgG mAb are purchased from Cell Signaling Technology) reacts, and it is aobvious that TMB is added after board-washing Color, microplate reader measure OD_450nmAnd OD_630nm。

Using formula Relative activity%=Ab'/Ab × 100%, (Ab and Ab' is indicated at sample respectively in formula Generated light absorption value before reason and after processing) calculate thermostabilization relative activity, that is, antibodies bind antigen is relatively living after being heat-treated Property.

The results are shown in Figure 3, and after 60 DEG C of processing, PD-1mAb relative activities are remarkably decreased increase with time, Relative activity is close to 0% after 180min, and Nb 2 is maintained at 80% or more, Nb 3 and is maintained at 60% or more；It is handled by 90 DEG C Afterwards, PD-1mAb relative activities are remarkably decreased, and relative activity is reduced to 0% after handling 10min, and Nb 2 handles phase after 120min 0%, Nb, 3 relative activities are just down to after handling 60min to activity and drop to 0%；It can be seen that the Nb of the present invention_PD-1Relatively In common mouse monoclonal antibody, the stability in high-temperature process is better.

Embodiment 5Nb_PD-1Bioactivity research

1. protein labeling HRP experiments

The 250 μ L protein liquids to be marked are taken, 25 μ L reactions are added and start liquid, gently mixing.Horseradish mistake is added in mixed liquor Mixing in oxidizing ferment bottle is placed in 37 DEG C of incubator 2h.25 μ L terminator mixings are added, are placed in room temperature 1h.It is (final concentration of that glycerine is added 50%) it, saves backup for -20 DEG C.

2.Nb_PD-1Competitive assay

2.1Nb_PD-1With the competion experiment of PD-1mAb

It is configured to the coating buffer of final concentration of 0.5 μ g/mL PD-1, is added in 96 hole elisa Plates with 100 holes μ L/, in 4 DEG C Coating is overnight.Next day, 0.05%PBST board-washings 2 times, 5% 37 DEG C of skim milk close 2h.With 32000 times of mouse PD- of dilution 1mAb gradient dilutions Nb_PD-1, while using one plant of nano antibody (U.S. Abcam company ab218695) for rabbit igg as the moon Property control (Control), operation repetitive, nano antibody maximum concentration is 100 μ g/mL, dilutes 11 gradients, after board-washing 2 times plus Enter into 96 hole elisa Plates, 100 holes μ L/, 37 DEG C of incubation 1h.Secondary antibody (Goat anti-mouse IgG are added after board-washing 5 times MAb is purchased from Cell Signaling Technology), 37 DEG C of incubation 1h.TMB colour developings are added after board-washing 5 times, microplate reader measures OD _450nmAnd OD_630nm。

Nb 2 and Nb 3 distinguishes the half effective inhibition concentration for diluting 32000 times of PD-1mAb and 0.5 μ g/mL PD-1 0.55831μg/mL、0.20549μg/mL.As shown in Figure 4, with Nb_PD-1Concentration increases, and the binding force of PD-1mAb and PD-1 are in Reveal the trend successively decreased, illustrate that the combination epitope of this Nb 3 (Nb 2) and PD-1 is Chong Die or close with PD-1 epitopes with PD-1mAb, Either the affinity of Nb 3 and PD-1 is higher than the affinity between PD-1mAb and PD-1.Meanwhile as a result showing Nb 2 and Nb 3 In conjunction with the possible different or Nb 3 in region of PD-1 PD-1mAb and PD-1 affinity are likely lower than with PD-1 affinity.

2.2Nb_PD-1With the competion experiment of PD-L1

It is configured to the coating buffer of final concentration of 0.5 μ g/mL PD-1, is added in 96 hole elisa Plates with 100 holes μ L/, in 4 DEG C Coating is overnight.Next day, 0.05%PBST board-washings 2 times, 5%BSA close 2h.Nb is diluted with the PD-L1-HRP of 2.5 μ g/mL_PD-1(0 ~20 μ g/mL) at respective concentration, it is added in 96 hole elisa Plates after board-washing 2 times, 100 holes μ L/, 37 DEG C of incubation 1h；It is anti-with nanometer Body ab218695 is as negative control group (Control), operation repetitive.Secondary antibody (Goat anti-mouse are added after board-washing 5 times IgG mAb are purchased from Cell Signaling Technology), 37 DEG C of incubation 1h.TMB is added after board-washing 5 times to develop the color 10min, The termination of 2.29% sulfuric acid is added, microplate reader measures OD_450nmAnd OD_630nm。

As shown in Figure 5, as Nb 2 or 3 concentration of Nb increase, the binding force of PD-L1 and PD-1 show the trend successively decreased, Illustrate the combination epitope of this two plants of nano antibodies and PD-1 with PD-L1 is Chong Die or close with PD-1 epitopes or Nb 2, Nb 3 with The affinity of PD-1 is higher than PD-L1, and the PD-L1 that can be incorporated into the surfaces PD-1 is competed, i.e. Nb 2 or Nb 3 can effectively hinder The combination of disconnected PD-L1 and PD-1.

3.pCDH-CMV-MCS-EF1-Puro-PD-1 construction of recombinant plasmid

According to PD-1 gene orders and with reference to pCDH-CMV-MCS-EF1-Puro (be purchased from System Biosciences, USA) multiple cloning sites information, simultaneously Xba I/BamH I restriction enzyme sites are added in design primer, to clone PD-1 extracellular fragment genes Sequence；Pass through PCR amplification PD-1 genetic fragments；Xba I, BamH I double digestion PD-1 genetic fragments and pCDH-CMV-MCS- EF1-Puro；Then connection, conversion；Screening positive clone, sequence verification, last recombinant plasmid largely extract, and obtain containing PD-1 Recombinant plasmid.

4. lentivirus-mediated PD-1 genes transfect HEK 293T cells and stablize strain screening

It is based on 37 DEG C, 5%CO with DMEM/FBS cultures₂Culture HEK 293T cells (are purchased from Thermo in incubator Fisher, USA), when cell density is up to 70%~80%, change plasma-free DMEM medium.By 1.5mL Opti-MEM, respectively DNA solution (7.5 μ g psPAX2,5 μ g slow virus packaging plasmids pMD2.G (being purchased from Addgene, USA) and 10 weights of the μ g containing PD-1 Group plasmid is sufficiently mixed, and is stored at room temperature 5min.

DNA/PEI mixed liquors：1.5mL Opti-MEM and 112.5 μ g PEI are added in another EP pipes, are added after being sufficiently mixed Mixing in mixed liquor 1, is stored at room temperature 15min.It is 70~80% to be in that DNA/PEI mixed liquors, which are dropped evenly to cell density, The HEK 293T cells of exponential phase.After 37 DEG C of culture 12h, 10mL DMEM/FBS culture mediums are changed to, continue to cultivate, 48h After HEK 293T cell supernatants are collected by centrifugation, through 0.45 μm of membrane filtration, conventional PEG precipitate virus particle.DMEM/FBS is trained It supports base weight to hang in HEK 293T cells of the virion addition in exponential phase, 37 DEG C, 5%CO₂, cultivate 48h.Centrifugation is changed The DMEM/FBS medium cultures containing 10 μ g/mL puromycins are added in liquid.Visual cell's density changes liquid or passage, until being stablized HEK 293T/PD-1 cell strains.Western blot identify that HEK293T/PD-1 cells, control group HEK 293T do not occur Band, i.e., HEK 293T itself do not express PD-1, and specific band, i.e. HEK occur in the HEK 293T/PD-1 cells filtered out 293T/PD-1 expresses PD-1.

5. laser co-focusing identifies HEK 293T/PD-1 cells

HEK 293T/PD-1 and HEK the 293T cells of logarithmic growth phase are digested through 0.25% pancreatin, and piping and druming is resuspended extremely Single cell suspension, diluted concentration to 2 × 10⁵cells/mL.The burnt special capsule of copolymerization is added, culture is suitable to cell density.1× PBS is added 4% paraformaldehyde and fixes 15min after cleaning 2 times.1 × PBS is added 1%BSA after cleaning 2 times and is resuspended, 37 DEG C of closings 1h.Antibody (Mouse anti-PD-1antibody (PE) are purchased from BD companies) is incubated after 1 × PBS cleanings, 37 DEG C are protected from light incubation 1h.PBST is cleaned 3 times, and DAPI solution is added and is incubated 15min.PBST is cleaned 3 times and anti-fluorescence quencher is added.Confocal fluorescent is aobvious Micro mirror detects PD-1 expressions on HEK 293T cell membranes.Focusing results are shown altogether, compared to control group HEK 293T, HEK There is apparent fluorescence in cell film location in 293T/PD-1, and HEK 293T/PD-1 cell membranes have PD-1 molecules；Illustrate HEK 293T/ PD-1 stable cell lines screen successfully.

6. streaming verifies Nb_PD-1It is combined with PD-1 molecules on HEK 293T/PD-1 cell membranes

To stablize the HEK 293T/PD-1 cells of expression PD-1 molecules as the Nb of the experiment material analysis present invention_PD-1With it is thin The combination situation of after birth surface PD-1 molecules is known with the nano antibody of the analysis present invention with the PD-1 molecular specificities naturally expressed Not activity.

HEK 293T/PD-1 and HEK the 293T cells of logarithmic growth phase, through 0.25% pancreatin digest, piping and druming be resuspended at Single cell suspension.Take 5 × 10⁶Cells/ is managed, and 1 × PBS is cleaned 1 time.Each group HEK 293T/PD-1 are incubated antibody sequence：PD- 1mAb groups：Mouse anti-PD-1antibody+Goat anti-mouse IgG-FITC；2 groups of Nb：(Nb 2+Mouse anti-His antibody)+Goat anti-mouse IgG-FITC；3 groups of Nb：(Nb 3+Mouse anti-His antibody)+Goat anti-mouse IgG-FITC.Control group：HEK 293T cell+1%BSA+Goat anti-mouse IgG (FITC) 1 × PBS is added 4% paraformaldehyde and fixes 15min after cleaning 2 times.1%BSA weights are added in 1 × PBS after cleaning 3 times It is outstanding, 37 DEG C of closing 1h.It is incubated albumen/antibody by above-mentioned grouping after 1 × PBS cleanings, 37 DEG C are protected from light incubation 1h.It is incubated fluorescence antibody (Goat anti-mouse IgG-FITC) is added 100 μ L antibody diluents and is resuspended, and 37 DEG C of incubations are protected from light 1h.PBST cleanings 3 Time, 1 × PBS is resuspended, flow cytomery.

The results are shown in Figure 6, can be seen that from A, B, C, and PD-1mAb, Nb 2, Nb 3 can identify native conformation PD-1.Nb 2 and Nb, 3 groups of Mean values are respectively 50.7,46.9, essentially identical with PD-1mAb group Mean values 44.8, PD-1mAb groups Median Being worth (36.8), 2 groups of Median values (36.5) of Nb and 3 groups of Median values (37.5) of Nb, also almost the same (D in Fig. 6 is further PD-1mAb, Nb 2 in A, B, C, 3 data of Nb are overlapped comparison to present), that is, show that Nb 2 and Nb 3 has and mouse PD- 1mAb is identical, identifies the ability of cell surface PD-1, illustrates Nb_PD-1Middle Nb 2 and Nb 3 can specifically bind cell membrane surface PD-1 molecules.

7.PHA Activation In Vitro PBMC scale-model investigations Nb_PD-1Function

Using PHA (phytohemagglutin phytolectin) Activation In Vitro T cell, with IFN-γ Concentration Testing T cell activation situation, research should Nb in system_PD-1The active barrier effect of T cell is inhibited to PD-L1.

1 healthy volunteer 10~15mL of whole blood is taken, human PBMC's cell is detached using human lymphocyte separating liquid.Equilibrium liquid Twice of cleaning, RPMI 1640/FBS culture mediums are resuspended cell and count, with 5 × 10⁴/ hole is inoculated in 96 orifice plates, per 50 μ L of hole. Corresponding culture hole is added with 25 holes μ L/ in the PHA of gradient, until final concentration is respectively 0 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/ mL.The good PD-L1 to final concentration of 0 μ g/mL, 5 μ g/mL, 10 μ of gradient dilution is added with 25 holes μ L/ in cultivating system simultaneously G/mL, 20 μ g/mL, 30 μ g/mL and 40 μ g/mL are placed in 37 DEG C of cell incubator culture 72h.300 × g is centrifuged in 10min harvests Clearly, the expression of IFN-γ in supernatant is measured (purchased from connection section biology) using people's IFN-γ ELISA detection kit, it is final to select It is irritaiting concentration to select 5 μ g/mL PHA, and 10 μ g/mL PD-L1 are inhibition concentration, carry out Nb_PD-1Active appraisal experiment.

Same method separation human PBMC's cell is simultaneously inoculated in same cell density in 96 orifice plates.Configure optimal pH A Irritaiting concentration (5 μ g/mL) and PD-L1 inhibition concentrations (10 μ g/mL) are added in corresponding hole.RPMI 1640/FBS culture mediums Dilute antibody Nb_PD-1It is added to final concentration of 0 μ g/mL, 1.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL and 20 μ g/mL Corresponding reaction system, 37 DEG C of cell incubator culture 72h are harvested supernatant and are surveyed using people's IFN-γ ELISA detection kit Determine the expression of IFN-γ in supernatant.3 healthy volunteer's whole bloods are taken to repeat to test respectively, statistical data.

The results are shown in Figure 7, and in the above inhibition system, IFN-γ concentration is all returned after adding Nb 2 or Nb 3 respectively It rises, and in certain Nb_PD-1Under concentration, IFN-γ concentration is with Nb_PD-1Concentration is increased and is increased.Illustrate under certain condition, two plants Nb_PD-1The state that activating T cell can be reversed to be inhibited by PD-L1, and then raise the expression of IFN-γ.Meanwhile it is dense in Nb 2 When degree is 2.5 μ g/mL (PHA+PD-L1+Nb2 groups), IFN-γ concentration highest；(the PHA+PD-L1 in 3 a concentration of 5 μ g/mL of Nb 3 groups of+Nb), IFN-γ concentration highest.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications, Equivalent substitute mode is should be, is included within the scope of the present invention.

Sequence table

<110>Ji'nan University

<120>A kind of PD-1 nano antibodies and its cloning expression method and application

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody CDR1 amino acid sequences

<400> 1

Gly Phe Thr His Leu Arg Tyr Ser Met Gly

1 5 10

<210> 2

<211> 10

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody CDR1 amino acid sequences

<400> 2

Gly Leu Thr Tyr Leu Arg Tyr Ser Met Gly

1 5 10

<210> 3

<211> 7

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody CDR2 amino acid sequences

<400> 3

Ile Gly Gly Asp Gly Arg Thr

1 5

<210> 4

<211> 23

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody CDR3 amino acid sequences

<400> 4

Ala Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu

1 5 10 15

Val Pro Tyr Lys Tyr Asp Tyr

20

<210> 5

<211> 23

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody CDR3 amino acid sequences

<400> 5

Ala Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu

1 5 10 15

Thr Pro Tyr Lys Tyr Asp Tyr

20

<210> 6

<211> 25

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody FR1 amino acid sequences

<400> 6

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser

20 25

<210> 7

<211> 15

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody FR2 amino acid sequences

<400> 7

Trp Phe Arg Gln Val Leu Asp Lys Glu Arg Glu Gly Val Ala Ala

1 5 10 15

<210> 8

<211> 15

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody FR2 amino acid sequences

<400> 8

Trp Phe Arg Gln Val Pro Asp Lys Glu Arg Glu Gly Val Ala Ala

1 5 10 15

<210> 9

<211> 38

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody FR3 amino acid sequences

<400> 9

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

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Asn Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 10

<211> 11

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody FR4 amino acid sequences

<400> 10

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 11

<211> 129

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody amino acid sequences

<400> 11

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Ser Met Gly Trp Phe Arg Gln Val Leu Asp Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Phe Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Asp Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val

100 105 110

Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 12

<211> 129

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PD-1 nano antibody amino acid sequences

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Ser Met Gly Trp Phe Arg Gln Val Pro Asp Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Phe Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Asp Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 13

<211> 513

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>Encode the nucleotide sequence of PD-1 nano antibodies

<400> 13

atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc 60

atggcccagg tgcagctgca ggagtctggg ggaggctcgg tgcaggctgg agggtctctg 120

agactctcct gtgtagcctc agggttcacc cacttgcgct actccatggg gtggttccgc 180

caggttctag ataaggagcg cgagggggtc gcagctattg gcggtgatgg taggacaagc 240

tacgcagact ccgtaaaggg ccgattcacc atcttcaaag acaacgccaa gaatactctg 300

tatctggaca tgaacagcct gaaccccgag gacactgcca tgtactactg tgcggcagcg 360

gtactcctag atggtagctt ctcgctcctg gcccctctag taccatataa gtatgactac 420

tggggccagg ggacccaggt caccgtctcc tcagcggccg catacccgta cgacgttccg 480

gactacggtt cccaccacca tcaccatcac tag 513

<210> 14

<211> 513

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>Encode the nucleotide sequence of PD-1 nano antibodies

<400> 14

atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc 60

atggcccagg tgcagctgca ggagtctgga ggaggctcgg tgcaggctgg agggtctctg 120

agactctcct gtgtagcctc agggttaacc tacctgcgct actccatggg ctggttccgc 180

caggttccag ataaggagcg cgagggggtc gcagctattg gcggtgatgg taggacaagc 240

tacgcagact ccgtaaaggg ccgattcacc atcttcaaag acaacgccaa gaatactctg 300

tatctggaca tgaacagcct gaaccccgag gacactgcca tgtactactg tgcggcagcg 360

gtactcctgg atggtagctt ctcgctcctg gcccctctaa caccatataa gtatgactac 420

tggggccagg ggacccaggt caccgtctcc tcagcggccg catacccgta cgacgttccg 480

gactacggtt cccaccacca tcaccatcac tag 513

Claims (10)

1. a kind of PD-1 nano antibodies, including the Variable domain being made of framework region FR and complementary determining region CDR, feature It is, the Variable domain includes CDR1, CDR2 and CDR3 selected from the following：

(1) CDR1 shown in SEQ ID No.1 or SEQ ID No.2；

(2) CDR2 shown in SEQ ID No.3；

(3) CDR3 shown in SEQ ID No.4 or SEQ ID No.5.

2. PD-1 nano antibodies according to claim 1, which is characterized in that the Variable domain include selected from Under CDR1, CDR2 and CDR3：

(1) CDR3 shown in CDR2 and SEQ ID No.4 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.3；

(2) CDR3 shown in CDR2 and SEQ ID No.5 shown in CDR1 shown in SEQ ID No.2, SEQ ID No.3.

3. PD-1 nano antibodies according to claim 1, which is characterized in that the framework region FR FR1 selected from the following, FR2, FR3 and FR4：

(1) FR1 shown in SEQ ID No.6；

(2) FR2 shown in SEQ ID No.7 or SEQ ID No.8；

(3) FR3 shown in SEQ ID No.9；

(4) FR4 shown in SEQ ID No.10.

4. PD-1 nano antibodies according to claim 1, it is characterised in that：

The amino acid sequence of the PD-1 nano antibodies is as shown in SEQ ID No.11 or SEQ ID No.12.

5. PD-1 nano antibodies according to claim 1 have at least one of following characteristics：

(1) PD-1 and PD-L1 is blocked to combine；

(2) T cell inactivated state caused by PD-L1 is reversed；

(3) inhibit tumour growth.

6. a kind of nucleic acid molecules, it is characterised in that：

Encode Claims 1 to 5 any one of them PD-1 nano antibodies.

7. a kind of recombinant expression carrier, it is characterised in that：

The recombination of nucleotide sequence containing coding Claims 1 to 5 any one of them PD-1 nano antibody amino acid sequences Expression vector.

8. a kind of host cell, it is characterised in that：

It has converted and has incorporated any one of coding Claims 1 to 5 on recombinant expression carrier or genome described in claim 7 The host cell and its progeny cell of the nucleotide sequence of the PD-1 nano antibodies.

9. the cloning expression method of Claims 1 to 5 any one of them PD-1 nano antibodies, which is characterized in that including as follows Step：

The nucleotide sequence of the coding PD-1 nano antibodies is cloned into expression vector, recombinant expression carrier is built；Again The recombinant expression carrier is transferred in expression system and is expressed；Substep purifying is carried out to destination protein, finally obtains institute The PD-1 nano antibodies stated.

10. the application of Claims 1 to 5 any one of them PD-1 nano antibodies, it is characterised in that：

The application is the application in preparing prevention and/or treating cancer drug.