CN106939050A

CN106939050A - anti-PD 1 and CD19 bispecific antibodies and uses thereof

Info

Publication number: CN106939050A
Application number: CN201710189120.4A
Authority: CN
Inventors: 王继明; 张云; 赵青; 朱泽; 李镜; 李向臣; 李相国; 钟殿胜; 陈镭; 王立祥; ***; 李忠廉; 刘卫平
Original assignee: Shunho Cell Biotechnology Tianjin Co ltd
Current assignee: Shunho Cell Biotechnology Tianjin Co ltd
Priority date: 2017-03-27
Filing date: 2017-03-27
Publication date: 2017-07-11
Anticipated expiration: 2037-03-27
Also published as: CN106939050B

Abstract

The present invention relates to an anti-PD 1 and CD19 bispecific antibody, or a variant thereof, or a functional fragment thereof, comprising: a domain that specifically recognizes and binds to an immune cell surface antigen PD-1, comprising the heavy chain variable region of an anti-PD-1 specific antibody (anti-PD-1 VH); and a domain that specifically recognizes and binds CD19, comprising the heavy chain variable region of an anti-CD 19 specific antibody (anti-CD 19 VH). The bispecific antibody has a fully human sequence, effectively avoids the occurrence of HAMA in clinical treatment application, can more effectively reduce the occurrence of drug resistance, and improves the utilization efficiency and treatment effect of drugs. The anti-PD-1 and CD19 bispecific antibody provided by the invention can obtain better effect of mediating T cells to efficiently and specifically kill B lymphocyte-derived tumor cells than the existing anti-PD-1 and anti-CD 19 specific antibodies.

Description

Anti- PD1 and CD19 bispecific antibodies and its application

Technical field

The invention belongs to antibody drug exploitation and production field, it is related to anti-PD-1 and CD19 bispecific antibody and its answers With.The invention provides specific recognition and with reference to PD-1 and CD19 bispecific antibody or its functional fragment, also provide The nucleic acid molecules of the described bispecific antibody of coding or its functional fragment, for express the bispecific antibody or its The expression vector and host cell of functional fragment, and their applications in the medicine for preparing treating cancer, and it is described double The preparation method of specific antibody or its functional fragment.

Background technology

PD-1 genes are a kind of 55kDa transmembrane proteins, and its molecule is made up of 4 subunits：δ, ε, γ, ζ, respective molecule Quality is respectively 18.9kDa, 23.1kDa, 20.5kDa, 18.7kDa, and it is a member of Ig gene superfamilies.PD-1 is in activation B cell, T cell and myeloid cell on the inhibition member of CD28 families that expresses.

CD19 is 95kDa membrane receptor, and it breaks up early expression in B cell, and continues expression until B cell is triggered Final differentiation.Ball is immunized in the C2 types that CD19 extracellular domain contains two separated by domains by less potential disulfide bond Albumen (IG) spline structure domain.CD19 cytoplasm domains are unique, but highly conserved between people, mouse and cavy in structure (Fujimoto et al., Semin Immunol. (1998) 10:267).CD19 is the protein found on bone-marrow-derived lymphocyte surface A part for compound.

CD19 has expression in normal and malignant B, and one be considered as in B cell growth course covers rank The longer surface marker the most reliable of section.In normal lymphoid tissue, CD19 is expressed in the B cell and folliculus of centrum germinativum The dendron shape maxicell in T cell area, essentially identical with CD19 and CD22 staining patterns between BMDC, jacket cell, folliculus.But Compared with CD22, CD19 is also expressed in pre B cell.In addition, by flow cyctometry detection method, CD19 is in tissue point From can be detected in obtained thick liquid cell.As a rule, CD19 is expressed in bone-marrow-derived lymphocyte knurl, including bone-marrow-derived lymphocyte Lymthoma, small lymphocyte lymthoma, lymphoma mantle cell, follicular lymphoma, Burkitt lymthomas, marginal zone lymphoma.

Bone-marrow-derived lymphocyte leukaemia and malignant lymphoma are the evils for being primary in medulla hematopoietic system and lymph node and diffusing whole body Property tumour.Although traditional chemicotherapy has certain curative effect, but without selectivity, the damage of normal tissue is very big.In recent years, it is raw Thing treatment method is widely used in oncotherapy, especially monoclonal antibody, and is received because of its specific target tropism, high-affinity Good result.Monoclonal antibody is combined by Fc sections with effector cell's surface-active acceptor PD-L1, so that lethal effect is mediated, but tool There is the T cell that immunologic cytotoxicity is acted on because surface lacks above-mentioned acceptor and can not effectively be mediated, so as to weaken body to swollen The immunological effect of knurl.

Chronic lymphocytic leukemia (CLL) is a kind of tumor disease of lymphocyte clone propagation, lymphocyte In the aggregation of marrow, lymph node, blood, spleen, liver and other organs.More than 95% CLL is the clonal expansion of B cell (i.e. B-CLL), the only case less than 5% are T cell phenotype (i.e. T-CLL).With deeply grinding to PD-1/PD-L paths Study carefully, its effect in BMDC (DC), T lymphocytes, bone-marrow-derived lymphocyte etc. is gradually revealed, and CD19 is that B lymphs are thin Cellular surface specific marker, is that molecular weight is 95 × 10³Wear membrane glycoprotein, contactin member, with B cell Activation is relevant with the transduction of signal, in pre-B lymphocyte, immature B lymphocyte, ripe bone-marrow-derived lymphocyte (cytotoxic T Lymphocyte, CTL) in tumor-killing, it is considered to be most hopeful to remove outside traditional operative treatment, chemicotherapy method Residual tumor cells and small metastatic lesion are so as to effect a radical cure the auxiliary treatment measure of tumour, and many zooperies and clinical test are The verified curative effect of the biological therapy measure.The PD-1 expression quantity on B cell surface is apparently higher than healthy group in patient's body；B is thin Born of the same parents' Lymphoma, malignant cell surface PD-L1 expression quantity also increase extremely, B in chronic lymphocytic leukemia body Often there is acute consumption in lymphocyte, has been reduced using this phenomenon after PD-1 retarding agents.Block in addition after PD-1, B cell Reactivity also strengthened.

Antibody drug is based on the biology of the antibody engineering technology preparation of cell engineering and technique for gene engineering Macromolecular drug, its have the advantages that specific high, property it is homogeneous, can for specific target spot beam system for.Monoclonal antibody exists In terms of being clinically mainly used in three below：Oncotherapy, immunity disease treatment and anti-infective therapy.Wherein, tumour Treatment be field that current monoclonal antibody is most widely used, the stage of clinical test and monoclonal antibody launch is come at present, Product quantity accounting for oncotherapy is about 50%.Mab treatment tumour is a kind of special for sick cell Target spot stimulating immune system kills the immunotherapy of target cell, in order to strengthen the effector function of antibody, particularly kills tumour The effect of cell, people attempt a variety of method engineered antibody molecules, and bispecific antibody is to improve the development of Antybody therapy effect One of direction, the focus as antibody engineering research field.

Bispecific antibody (bispecific antibody, BiAb) is containing two species-specific antigen binding sites Two antigen-binding sites in the antibody molecule of artificial antibody, i.e., one can be respectively in connection with the anti-of two kinds of different epitopes Body, can erect bridge between target cell and functional molecular (cell), produce the effector function of guidance quality.Bispecific antibody One arm combination tumor markers, another arm combination T- cell specific surface antigens, such as PD-1 can overcome above mentioned problem And connect tumour cell and T- cells.BiAb is in biomedicine, particularly with wide in the immunization therapy of tumour Application prospect.The CDCC mediated by BiAb kills the focus that tumour cell is current immunization therapy application study, its Immunological effect can directly be triggered in combination with the target molecule on tumor associated antigen and immune effector cell by being mainly characterized by BiAb The specific killing of cells against tumor cells.

Bispecific antibody for immunization therapy is the artificial antibody containing 2 species-specific antigen binding sites, can be swashed Immune response of the hair with guidance quality, has broad application prospects in the immunization therapy of tumour.Bispecific antibody is logical Cross the chemical coupling of mouse monoclonal antibody, a kind of non-natural prepared by 3 developing stage of double cross knurl and genetic engineering Antibody or antibody hybrid molecule, with being showing improvement or progress day by day for biotechnology, the correlative study of domestic and international bispecific antibody is increasingly deep Enter, it has potential application value in clinical treatment as a kind of new secondary guidance system.

With the fast development of computer science, Computer-Aided Drug Design turns into research and development new drug in recent years Brand-new technology, hence it is evident that accelerate the speed and efficiency of new drug design.The continuous parsing of protein structure, PDB data constantly increase, Make it possible CAD pharmaceutical grade protein.Pharmaceutical grade protein research based on protein steric structure is extremely weighed Depending on.

The content of the invention

The technical problems to be solved by the invention are to provide a kind of new anti-PD-1 and CD19 bispecific antibodies and its should With.The bispecific antibody has full people's source sequence, efficiently avoid the generation of HAMA in clinical treatment application, can more have Effect ground reduces the generation of resistance phenomenon, improves the utilization ratio and therapeutic effect of medicine.The present invention provide new anti-PD-1 and CD19 bispecific fusion proteins have for the first specificity of the first purpose antigen and for the second purpose antigen Second specificity, with the binding specificity to two kinds of not isoacceptors, while the invention further relates to the bispecific fusion The production method of albumen, the medicinal usage of the fusion protein and the treatment method using the fusion protein.Therefore, the present invention is provided Anti- PD-1 and CD19 bispecific antibodies to result in than the more preferable mediation T of more anti-PD-1, anti-CD19 specific antibodies thin The effect of the tumour cell in the efficient specific killing bone-marrow-derived lymphocyte source of born of the same parents.

The technical scheme that the present invention is used to solve above-mentioned technical problem is as follows：

A kind of anti-PD-1 and CD19 bispecific antibodies or its variant or its functional fragment, it includes：

A. specific recognition and immune cell surface antigenic PD-1 domain is combined, it includes anti-PD-1 specific antibodies Weight chain variable district (anti-PD-1VH)；And

B. specific recognition and combine CD19 domain, it include anti-CD19 specific antibodies weight chain variable district (resist CD19VH)。

Preferably, the specific recognition and combination immune cell surface antigenic PD-1 domain are anti-by what is be sequentially connected Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and the Fc fragments of PD-1 specific antibodies (resist PD-1CH2-CH3) constitute, or by the light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH- of one group of anti-PD-1 specific antibody CH1- hinge areas-CH2-CH3) composition, or it is (anti-by the light chain (anti-PD-1VL-CL) and heavy chain of two groups of anti-PD-1 specific antibodies PD-1VH-CH1- hinge areas-CH2-CH3) composition, or light chain variable district (anti-PD-1VL) by anti-PD-1 specific antibodies and again Chain variable region (anti-PD-1VH) is constituted.

Preferably, the specific recognition and CD19 domain is combined by the anti-CD19 specific antibodies that are sequentially connected Light chain variable district (anti-CD19VL), weight chain variable district (anti-CD19VH), hinge area and Fc fragments (anti-CD19CH2-CH3) composition, Or by light chain (anti-CD19VL-CL) and heavy chain (the anti-CD19VH-CH1- hinge areas-CH2- of one group of anti-CD19 specific antibody CH3) constitute, or by the light chain (anti-CD19VL-CL) and heavy chain (anti-CD19VH-CH1- hinges of two groups of anti-CD19 specific antibodies Area-CH2-CH3) composition, or it is (anti-by the light chain variable district (anti-CD19VL) and weight chain variable district of anti-CD19 specific antibodies CD19VH) constitute.

According to the present invention a preferred embodiment, the anti-PD-1 and CD19 bispecific antibodies or its variant, Or its functional fragment includes：

A ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specificity Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition of antibody；And

B ' specific recognitions and the domain for combining CD19, it is (anti-by the light chain of one group of anti-CD19 specific antibody CD19VL-CL) constituted with heavy chain (anti-CD19VH-CH1- hinge areas-CH2-CH3).

Preferably, the specific recognition and the domain with reference to immune cell surface antigenic PD-1 are known with the specificity Domain that is other and combining CD19 passes through one or more disulfide bond, such as one or more disulfide bond positioned at hinge area connect Connect.

A " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specificity Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition of antibody；And

B " specific recognitions and the domain for combining CD19, it is by the light chain for the anti-CD19 specific antibodies being sequentially connected Variable region (anti-CD19VL), weight chain variable district (anti-CD19VH), hinge area and Fc fragments (anti-CD19CH2-CH3) composition.

A " ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by the anti-PD-1 that is sequentially connected Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and Fc fragments (the anti-PD- of specific antibody 1CH2-CH3) constitute；And

B " ' specific recognitions and the domain for combining CD19, it is (anti-by the light chain of one group of anti-CD19 specific antibody CD19VL-CL) constituted with heavy chain (anti-CD19VH-CH1- hinge areas-CH2-CH3).

A " " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by two groups of anti-PD-1 specificity Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition of antibody；And

B " " specific recognitions and combine CD19 domain, its by two groups of anti-CD19 specific antibodies light chain variable district (anti-CD19VL) and weight chain variable district (anti-CD19VH) are constituted.

Preferably, each anti-PD-1 in the specific recognition and combination immune cell surface antigenic PD-1 domain The CH3 of the heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) of specific antibody respectively with the specific recognition and combination The weight chain variable district (anti-CD19VH) of each anti-CD19 specific antibodies in CD19 domain is connected by chemical bond.

Preferably, described anti-PD-1 and CD19 bispecific antibodies or its variant or its functional fragment are inosculating antibodies Body, humanized antibody or human antibody.

Preferably, the specific recognition and combine PD-1 domain in weight chain variable district amino acid sequence such as SEQ No:1st, shown in 5,9 or 21；The amino acid sequence of light chain variable district such as SEQ ID NO:3rd, shown in 7,11 or 23.

Preferably, the specific recognition and combine CD19 domain in weight chain variable district amino acid sequence such as SEQ ID NO:13rd, shown in 17 or 25；The amino acid sequence of light chain variable district such as SEQ ID NO:15th, shown in 19 or 27.

Preferably, the amino of the heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) of the anti-PD-1 specific antibodies Acid sequence is SEQ ID No:29, nucleotide coding sequence is SEQ ID No:30；The light chain of the anti-PD-1 specific antibodies The amino acid sequence of (anti-PD-1VL-CL) is SEQ ID No:31, nucleotide coding sequence is SEQ ID No:32；By connecting successively Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and the Fc pieces of the anti-PD-1 specific antibodies connect The amino of the specific recognition of section (anti-PD-1CH2-CH3) composition and combination immune cell surface antigenic PD-1 domain Coding sequences are SEQ ID No:33, nucleotide coding sequence is SEQ ID No:34.

Preferably, the amino of the heavy chain (anti-CD19VH-CH1- hinge areas-CH2-CH3) of the anti-CD19 specific antibodies Acid sequence is SEQ ID No:35, nucleotide coding sequence is SEQ ID No:36；The light chain of the anti-CD19 specific antibodies The amino acid sequence of (anti-CD19VL-CL) is SEQ ID No:37, nucleotide coding sequence is SEQ ID No:38；By connecting successively Light chain variable district (anti-CD19VL), weight chain variable district (anti-CD19VH), hinge area and the Fc pieces of the anti-CD19 specific antibodies connect The amino acid sequence of the specific recognition of section (anti-CD19CH2-CH3) composition and combination CD19 domain is SEQ ID No:39, nucleotide coding sequence is SEQ ID No:40；By the light chain variable district for the anti-CD19 specific antibodies being sequentially connected The amino of the specific recognition of (anti-CD19VL) and weight chain variable district (anti-CD19VH) composition and combination CD19 domain Acid sequence is SEQ ID No:41, nucleotide coding sequence is SEQ ID No:42.

On the other hand, present invention also offers the anti-PD-1 and CD19 bispecific antibodies described in a kind of coding or its change The nucleic acid molecules of body or its functional fragment, either a kind of expression vector comprising the nucleic acid molecules or one kind include institute State the host cell of expression vector.

Preferably, the specific recognition and combine PD-1 domain in weight chain variable district nucleotide sequence such as SEQ ID NO:2nd, shown in 6,10 or 22；The nucleotide sequence of light chain variable district such as SEQ ID NO:4th, shown in 8,12 or 24.

Preferably, the specific recognition and combine CD19 domain in weight chain variable district nucleotide sequence such as SEQ ID NO:14th, shown in 18,26；The nucleotide sequence of light chain variable district such as SEQ ID NO:16th, shown in 20,28.

Another further aspect, present invention also offers a kind of pharmaceutical composition, it includes described anti-PD-1 and CD19 is double special Property antibody or its variant or its functional fragment, or described nucleic acid molecules, or described expression vector or described host Cell.Preferably, described pharmaceutical composition also includes other one or more antineoplastics；Preferably, the drug regimen Thing also with other one or more antitumour treatments, such as chemotherapy, radiotherapy and/or biotherapy make together With.

Further, present invention also offers described anti-PD-1 and CD19 bispecific antibodies or its variant or its work( Energy property fragment or described nucleic acid molecules or described expression vector or described host cell are preparing the medicine for the treatment of tumour Application in thing.Specifically, the tumour is NHL.

The invention provides can be while the specific bond programmed death factor 1 (PD-1) and CD19 bispecific resist Body or its variant or its functional fragment.The bispecific antibody or its variant or its functional fragment of the present invention have with At least one of lower characteristic：

A. it can be combined with PD-1 with high specific, and be blocked PD-1 and PD-L1 interaction；

B. do not combined with other CD28 family members (such as ICOS, CTLA-4 and CD28)；

C. it can be combined with CD19 with high specific；

D. do not combined with other B cell differentiation antigens (such as CD20, CD22)；

E. immune cell activated (such as tumor specific T cells), and specific recognition CD19 positive tumor cells, promote CD8⁺ T cell enters tumor tissues, greatly increases the level of the immunoeffectors such as IFN-γ, so as to realize that target killing tumour is thin Born of the same parents.

Present invention also offers humanization PD-1/CD19 bispecific antibodies or its variant or its functional fragment.Institute State the mouse source antibody that humanized antibody produces by immune mouse designed via computer simulation and combine display technique of bacteriophage and Obtain, and according to its binding characteristic with PD-1 the and CD19 ectodomains of different plant species, identify its corresponding knot Close epitope.The anti-PD-1/CD19 bispecific antibodies of humanization of the present invention or its variant or its functional fragment possess The advantageous feature of above-mentioned PD-1/CD19 bispecific antibodies or its functional fragment.

Variant and function fragment of the present invention include in liposome can increase by half by chemical modification or by mixing Any fragment of phase in longevity, the fragment after modification retains and its derived antibodies identical binding specificity and affinity, is achieved in that The new protein molecular with double specific binding properties also in the scope of protection of the invention.

On the premise of not substantial effect antibody activity, those skilled in the art can to the present invention sequence replace, Add and/or lack it is one or more (such as 1,2,3,4,5,6,7,8,9 or 10 or more) amino acid, to obtain State the variant of antibody or the sequence of its functional fragment.They are considered as being included in the scope of protection of the invention.

Bispecific antibody of the present invention, mutant and functional fragment can be full length antibody, can only include two antigens The fusion protein of bound fraction, such as Fab, F (ab ') 2, Fv or single-stranded (scFv) or full length antibody and antigen-binding portion thereof composition (such as Fig. 2), as described above, any form combines formed bispecific binding protein molecule in the scope of protection of the invention It is interior.

Those skilled in the art can will encode the cloned dna molecule of PD-1/CD19 bispecific antibodies of the present invention Into carrier, and then convert host cell.Therefore, present invention also offers the restructuring by taking bispecific antibody shown in Fig. 1 (A) as an example The mutant of PD-1/CD19 bispecific antibodies and the carrier of function fragment shown in DNA vector, code pattern 1 (B-C) and Fig. 2 and Host cell is also in the scope of protection of the invention.

Host cell of the present invention can be prokaryotic host cell, eukaryotic host cell or bacteriophage.The protokaryon Host cell can be Escherichia coli, hay bacillus, lactic acid bacteria, streptomycete or proteus mirabilis etc..The eukaryotic host cell Can be such as pichia pastoris phaff, saccharomyces cerevisiae, fission yeast, trichoderma fungi, such as meadow mythimna separata insect cell, such as cigarette The plant cells such as grass, such as bhk cell, Chinese hamster ovary celI, COS cells, myeloma cell's mammalian cell.In some embodiment party In case, host cell of the present invention is preferably mammalian cell, more preferably bhk cell, Chinese hamster ovary celI, NSO cells or COS cells.

The people source bispecific antibody albumen comprising PD-1 antibody and CD19 antibody that the present invention is provided all has in vivo and in vitro There is good bioactivity and stability, can be in combination with cell positive PD-1 and CD19, and people's T lymphs can be activated Cell, target killing NHL cell, killing rate is up to more than 30%.

The beneficial effects of the present invention are the PD-1/CD19 bispecific antibodies there is provided a kind of humanization, it can be tied The positive cells of PD-1 and CD19 are closed, and the function of T lymphocytes can be strengthened, so as to reach the anti-non-Hodgkin's lymph of targeting The effect of oncocyte, the PD-1 antibody of more current clinical practice is compared with CD19 monoclonal antibodies, with more powerful killing Ability, has good application prospect in clinical practice.

Brief description of the drawings

Hereinafter, embodiment of the present invention is described in detail with reference to accompanying drawing, wherein：

Fig. 1 is the structural representation of PD-1/CD19 bispecific antibodies of the present invention.

Fig. 2 is the functional fragment of PD-1/CD19 bispecific antibodies of the present invention or the structural representation of variant.

Fig. 3 is the SDS-PAGE testing results of purifying humanization PD-1, CD19 ectodomain.

Fig. 4 is the ELISA testing results of anti-PD-1 monoclonal antibodies.

Fig. 5 is the ELISA testing results of anti-CD19 monoclonal antibodies.

Fig. 6 shows the combination of anti-PD-1 monoclonals 3,21,45 and 77 and CD28 family proteins respectively.

Fig. 7 shows the envelope that anti-PD-1 monoclonals 3,21,45 are combined with 77 pairs of PD-L1 albumen with the PD-1 of human T-cell respectively The effect of closing.

Fig. 8 shows the specific binding of anti-CD19 monoclonals 16,75 and CD19 albumen respectively.

The recombinant plasmid collection of illustrative plates of the bispecific antibody of Fig. 9 code displayings present invention.

Specific bond of the PD-1/CD19 bispecific antibodies of Figure 10 display present invention to PD-1 and CD19.

Figure 11 shows that bispecific antibody suppresses the experimental result of the growth of transplantable tumor in Mice Body.

Embodiment

Illustrate the present invention referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only For illustrating the present invention, it does not limit the scope of the present invention in any way.

Experimental method in following embodiments, unless otherwise specified, is conventional method.Original used in following embodiments Material, reagent material etc., unless otherwise specified, are commercially available purchase product.

The structure design of embodiment 1PD-1/CD19 bispecific antibodies

PD-1 and the bispecific antibody of CD19 ectodomains can be specifically bound simultaneously the invention provides some Structural model, the structure of this 4 kinds of main embodiment of antibody-like as shown in figure 1, in Fig. 1 (A-C) formation of 3 kinds of heterodimers it is equal Based on CH3 domains mutating technology (" knob and hole mutation ") (Von known to art technology person Kreudenstein et al., MAbs.2013 5 (5):646-54).(wherein, the light chain of (A) anti-PD-1 specific antibodies is (anti- PD-1VL-CL light chain (anti-PD- of the)-heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) pair with anti-CD19 specific antibodies 1VL-CL)-heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) is to the bispecific binding protein of composition；(B) anti-PD-1 is special The light-heavy chain pair of heterogenetic antibody and CD19 light chain variable districts (anti-CD19VL), weight chain variable district (anti-CD19VH), hinge area and The bispecific binding protein of Fc fragments (anti-CD19CH2-CH3) fusion protein composition；(C) anti-PD-1 light chain variable districts, heavy chain The bispecific knot that variable region, hinge area and Fc fragment fusion proteins are constituted with the light-heavy chain pair of anti-CD19 specific antibodies Hop protein；(D) heavy chain of the light chain of anti-PD-1 specific antibodies and anti-PD-1 specific antibodies, anti-CD19 light chain variable districts are (anti- CD19VL) and weight chain variable district (anti-CD19VH) fusion protein composition bispecific binding protein.

The variant of PD-1/CD19 bispecific antibodies and the embodiment of functional fragment are as shown in Fig. 2 double in Fig. 2 E-H The formation of specific antibody variant and functional fragment is based on the fusogenic peptide technology known to art technology person (with polypeptide chain or egg White matter connects different structure territory, and then realizes amalgamation and expression), the formation of the heterodimer in Fig. 2 I is based on art technology The weight chain variable district (anti-PD-1VH) of CH3 domains mutating technology (same to Figure 1A-C) (E) anti-PD-1 known to member and anti-CD19 The fusion protein of weight chain variable district (anti-CD19VH)；(F) the scFv groups of anti-PD-1 single chain variable fragment (scFv) and anti-CD19 Into fusion protein；(G) anti-PD-1 single chain variable fragment (scFv) melts with the anti-CD19 scFv non-immunoglobulins constituted Hop protein；(H) anti-PD-1 light chain variable districts, weight chain variable district, hinge area, Fc fragments and anti-CD19 light chain variable districts, heavy chain can Become district's groups into tetravalent fusion protein；(I) anti-PD-1 light chain variable districts, weight chain variable district, hinge area and Fc fragment fusion proteins The bispecific binding protein constituted with anti-CD19 light chain variable districts, weight chain variable district, hinge area and Fc fragment fusion proteins (note：Specific recognition PD-1 and CD19 function fragment position are interchangeable in figure).

The preparation of embodiment 2PD-1/CD19 bispecific antibodies antigen (people PD-1 and CD19 extracellular can solubilization domain)

The coding gene sequence of people source PD-1 and CD19 ectodomain is obtained in UniProt databases, passes through full genome The mode of synthesis obtains encoding gene PD-1ED-CDS and CD19ED-CDS, and synthetic product is through BamH I, EcoR I (PD- 1ED-CDS) and after BamH I, Xba I (CD19ED-CDS) double digestion, pcDNA3.1 carriers are cloned into, are made using CHO-S cells Protein expression is carried out for host cell, destination protein is carried out using purification techniques such as affinity chromatography, ion-exchange chromatographies pure Change.

Fig. 3 behaviours source PD-1 and CD19 ectodomains protein purification simultaneously carry out the inspections of the SDS-PAGE after deglycosylation processing Survey result figure.

The anti-human PD-1 of embodiment 3 and CD19 antibody acquisition

1. immune animal

The 1mg/ml μ l of people's PD-1 or CD19 ectodomain protein 10 0 purified are helped completely with isometric Freund respectively After agent is uniformly mixed at room temperature, subcutaneous abdomen multi-point injection enters in 6-8 weeks BALB/C mice body.After 7 days, by 1mg/ml albumen After 100 μ l are uniformly mixed at room temperature with isometric incomplete Freund's adjuvant, subcutaneous abdomen multi-point injection enters in Mice Body.Seven days Afterwards, previous step is repeated.After seven days, previous step is repeated.Three days after 4th injection, disconnected rat-tail point takes the μ l of blood 15, and room temperature is quiet 2h is put, 25 DEG C, 6000-8000rpm centrifuges 10-15min, takes supernatant, pass through ELISA experiment detection potency.If bioactivity can Whole blood is taken with i.e. eye, 2h is stored at room temperature, 25 DEG C, 6000-8000rpm centrifuges 10min, takes supernatant, -80 DEG C of preservations.If potency It is unavailable, continue to be immunized once.

2. cell fusion

It is sterile to take mouse boosting cell and cell suspension is made, by 1 × 10⁸Individual splenocyte and 2-5 × 10⁷Individual myeloma (SP2/ 0-Ag14) cell fusion, 50%PEG makees fusion agent, after HAT Selective agar mediums culture 3-10 days, is trained with HT culture mediums Support, condition of culture is 5%CO₂, 37 DEG C.

3. positive colony is screened

PD-1 or CD19 ectodomain albumen is combined using that can be secreted in ELISA reaction screening monoclonal hybridomas The clone of antibody, is to filter out the preferred clone with reference to PD-1 (clone 3,21,45,77) and CD19 (clone 16,75), inspection respectively Survey result as shown in Figures 4 and 5, clone can the corresponding antigen protein of specific recognition.

The identification that the anti-PD-1 antibody of embodiment 4 is combined with other CD28 family proteins

The specificity of the anti-PD-1 antibody gone out for evaluation and screening, by member protein PD-1, CD28, CTLA-4 of CD28 families Coating 2h is stood in 37 DEG C of incubators in ELISA Plate with ICOS (R＆D Systerm)；4 DEG C of closings are stayed overnight；100 are added after washing μ l candidates antibody (1 μ g/ml), 37 DEG C of incubation 1h；The HRP mark goat anti-mouse antibodies of dilution are added after washing 5 times (Abcam), it is incubated at room temperature 1h；The 2M that 50 μ l/ holes are added after the μ l/ holes of OPD substrate solutions 100, colour developing 5min is added after washing 5 times H₂SO₄Terminating reaction.The light absorption value in each hole at 490nm is determined with ELIASA.Experimental result such as Fig. 6, the antibody of candidate clone secretion CD28 family member's albumen in addition to PD-1 is not combined.

The enclosed experiment that the PD-1 acceptors of the anti-PD-1 antibody on human T cell of embodiment 5 are combined with PD-L1

1. the separation of human peripheral T cell

Healthy volunteer peripheral blood 10mL is extracted, is slowly added in the centrifuge tube equipped with 5ml lymphocyte separation mediums, 2500rpm, 20min, are taken out tunica albuginea layer, are washed 3 times with PBS, obtain human peripheral blood mononuclear cell PBMC.Isolated PBMC, adds 100 μ l/ml CD3 antibody (Invitrogen), and featheriness is mixed, and is incubated at room temperature 15min；Add 5 μ l/ml and magnetic is immunized Pearl (Invitrogen), 4 DEG C of incubation 60min, concussion in every 10 minutes is once；Rinsed and diluted with PBS, cell suspension injection is filled out Have in the special test tube of stainless steel wool, be put into separator and isolated and purified, T cell can not be obtained by separator under high-intensity magnetic field To purify.

2. the preparation of human PD-L 1 ectodomain and green fluorescent protein fusion protein

The coding gene sequence of human PD-L 1 ectodomain, the side synthesized by full genome are obtained in UniProt databases Formula obtains encoding gene, and synthetic product is cloned into pEGFP-N1 carriers, utilizes CHO-S after EcoR I, Xho I double digestions Cell carries out protein expression as host cell, using purification techniques such as affinity chromatography, ion-exchange chromatographies to destination protein Purified, obtained people source PD-L1 ectodomain the albumen rh-PD-L1-GFP, Fig. 7 (A) of green fluorescent protein amalgamation and expression Expression and purification for fusion protein simultaneously carries out the SDS-PAGE results after area's glycosylation processing.

3. the enclosed experiment that the PD-1 acceptors of anti-PD-1 antibody on human T cell are combined with PD-L1

4 kinds of antibody (1 μ g/ml) that preferred clone is produced softly are mixed at room temperature with rh-PD-L1-GFP (1 μ g/ml) 30min, then adds mixed protein solution the human peripheral T cell isolated and purified out, clear with PBS after being incubated at room temperature 15 minutes Wash 3 times, analyzed with flow cytometer.

From Fig. 7 (B-F), clone 3,21 and 77 can hinder the combination of the PD-1 acceptors on PD-L1 and human T-cell, gram Grand 45 barrier effect is poor.

The anti-CD 19 antibodies of embodiment 6 and CD20, CD33 and CD138 combination identification

CD19, CD20, CD33 and CD138 (R＆D Systerm) are stood into coating 2h in ELISA Plate in 37 DEG C of incubators； 4 DEG C of closings are stayed overnight；100 μ l candidates antibody (1 μ g/ml), 37 DEG C of incubation 1h are added after washing；The HRP of dilution is added after washing 5 times Goat anti-mouse antibody (Abcam) is marked, 1h is incubated at room temperature；The μ l/ holes of OPD substrate solutions 100, colour developing are added after washing 5 times The 2M H in 50 μ l/ holes are added after 5min₂SO₄Terminating reaction.The light absorption value in each hole at 490nm is determined with ELIASA.Experimental result is such as Fig. 8, the antibody of candidate clone secretion does not combine CD20, CD33 and CD138 albumen.

The acquisition of the anti-PD-1 and CD19 candidate antibodies variable region sequences of embodiment 7

Expand the anti-PD-1 of culture respectively and clone 3,21,45,77 and anti-CD19 clones 16,75, culture to cell concentration is 10⁹ It is individual, cell is collected by centrifugation, removes cell culture fluid, cell is gently washed twice with the PBS (6-8mL) of precooling；With Trizol kits (TAKARA) extract total serum IgE, with UV spectrophotometer measuring RNA purity and concentration；Using total serum IgE as mould Plate, reverse transcription obtains cDNA, the μ l of reverse transcription system 20, wherein 5 × reverse transcription Buffer (4 μ l), dNTP (1 μ l), MgCl₂(2.4 μ l), PD (N6) random primer (1.5 μ l), RNA templates (2 μ g), reverse transcriptase (1 μ l), DEPC water (to 20 μ l).

Using cDNA as template, the DNA sequence dna of variable region in amplified hybridization knurl, amplimer sequence and antibody variable region first Framework region and constant region complementation (Orlandi R, et al.Cloning immunoglobulin variable domains for expression by polymerase chain reaction.Proc.Natl.Acad.Sci.USA,1989,86： 3833), the μ l of amplification system 50, wherein 5 × PCR Buffer (10 μ l), dNTP (4 μ l), primer (2 μ l), cDNA templates (1 μ l), PCR enzymes (0.5 μ l), PCR water (to 50 μ l).

Amplified production is cloned into pMD19 carriers, after being screened through blue hickie, recon sequencing, the anti-PD-1 clones 3 of acquisition, 21st, 77 heavy chains and chain variable region amino acid sequence are shown in SEQ No.1 and 3,5 and 7,9 and 11；Encode the nucleosides of corresponding amino acid Acid sequence is shown in SEQ No.2 and 4,6 and 8,10 and 12.The anti-CD19 obtained clones 8,65 heavy chains and chain variable region amino acid sequence Row are shown in SEQ No.13 and 15,17 and 19；The nucleotide sequence for encoding corresponding amino acid is shown in SEQ No.14 and 16,18 and 20.

The anti-PD-1 and CD19 antibody of embodiment 8 it is humanization modified

Using the anti-PD-1 of acquisition IMGT/V-Quest online services are utilized with the variable region amino acid sequence of CD19 antibody Device analyzes antibody subtype, and the Numbering schemes of the CDR region of comprehensive tri- kinds of antibody of Kabat, Chothia and IMGT determine antibody Six CDR region sequences of light chain and heavy chain；Mouse parental antibody Fab and its chimeric humanized antibody Fab is carried out using DS softwares Space structure, obtain chimeric antibody molecular model after, Molecule Motion is used to parent Fab and chimeric antibody Fab initial models Mechanical simulation is comprehensively optimized, and extracts stable average conformation；Can using the humanized antibody of epitope scanning test design The linear epitope and comformational epitope that can exist, it is determined that behind the Framework Region amino acid site that must retain, different amino acid are designed Mutational site obtains humanized antibody.

On this basis, we have obtained multiple humanized antibodies, wherein the scoring anti-PD-1 of highest and anti-CD 19 antibodies Sequence is respectively clone 81 and clone 22.

On this basis, we have obtained multiple humanized antibodies, wherein the scoring anti-PD-1 of highest and anti-CD 20 antibodies Sequence is respectively clone 81 and clone 22, and heavy chain and light-chain amino acid sequence the difference SEQ ID No.21 and 23 of clone 81 are compiled Code-phase answers the nucleotide sequence of amino acid to distinguish SEQ ID No.22 and 24；Heavy chain and the light-chain amino acid sequence difference of clone 22 SEQ ID No.25 and 27, the nucleotide sequence for encoding corresponding amino acid is respectively SEQ ID No.26 and 28.

The structure of the anti-PD-1/CD19 bispecific antibodies expression vector of embodiment 9

The anti-PD-1/CD19 bispecific antibodies moderate resistance PD-1's of the present invention (clone 81, preparation method is shown in embodiment 8) is variable Area coding nucleotide PD-1-VH and PD-1-VL, the variable region encoding nucleoside of anti-CD19 (clone 22, preparation method is shown in embodiment 8) Sour CD19-VH and CD19-VL, IgG1 heavy chain constant region CH1, hinge area, Fc and Fc-knob and Fc-hole coding nucleotides, with And constant region of light chain CL kappa coding nucleotide, and link scFv the encoding gene of polypeptide (GGGGS) 3 be obtained from Life Technologies Inc. (Carlsbad, CA).

Each different chain, VL and CL, VH and CH1, CH1 and Fc in the bispecific antibody of 4 kinds of structures shown in Fig. 1 (Fc-knob, Fc-hole), ScFv and Fc (Fc-knob, Fc-hole), Fc and ScFv are obtained by way of over-lap PCR. PcDNA3.1 and pIRES as expression vector to prepare bispecific antibody, the restructuring of 4 kinds of bispecific antibodies shown in Fig. 1 Plasmid map is shown in Fig. 9.

The preparation of the anti-PD-1/CD19 bispecific antibodies of embodiment 10

1. utilize 293F host cell expression bispecific antibodies

24 hours before transfection, by 1 × 10⁶Individual 293F cells are inoculated in the 293freestyle culture mediums in flask In 37 DEG C, 8%CO₂, cultivate under the conditions of 130rpm.100 μ l 293fectin are added into 1ml OPtiMEM, stirring condition Under, it is incubated at room temperature 5 minutes.Meanwhile, recombinant plasmid (prepared by embodiment 9) is dissolved in 1ml OPtiMEM, DNA total amounts are 30μg.Above-mentioned DNA and 293fectin is thoroughly mixed, and cumulative volume is 2ml, is incubated 20 minutes at room temperature.Then should Mixture is added into cell culture.Being cultivated at 37 DEG C in cell, and incubator has 5%CO₂, cultivated 5 days with 130rpm.

2. antibody purification

Cell culture is centrifuged based on 2000 × g, supernatant is collected and is filtered with 0.22 μm of filter.The liquid of collection is used 10 times of (being calculated with volume) combination buffer (9.5mM NaH₂PO₄+40.5mM Na₂HPO₄, pH 7.0) and dilution, then pass through Sepharose Fast Flow protein A affinity chromatographic columns (being purchased from GE companies, 5ml volumes), FabAffinity KBPAgarose is affine filler (is purchased from ACROBiosystems companies, 5ml volumes), and SP cation-exchange chromatography posts (are purchased from GE companies, 10ml) purified, operated according to service manual.

The specific binding identification of the anti-PD-1/CD19 bispecific antibodies of embodiment 11

PD-1, CTLA-4, CD19 and CD19 (R＆D Systerm) are stood into coating in ELISA Plate in 37 DEG C of incubators 2h；4 DEG C of closings are stayed overnight；The gradient dilution of 4 kinds of antibody (100 μ g/ml) according to the preparation of embodiment 10 after purification is added after washing Liquid (PBS serial dilutions (1：10², 1：10³, 1：10⁴, 1：10⁵, 1：10⁶, 1：10⁷, 1：10⁸, 1：10⁹), 100 μ l/ holes, 37 DEG C incubate Educate 1h；The HRP mark goat anti-mouse antibodies (Abcam) of dilution are added after washing 5 times, 1h is incubated at room temperature；Added after washing 5 times The 2MH in 50 μ l/ holes is added after the μ l/ holes of OPD substrate solutions 100, colour developing 5min₂SO₄Terminating reaction.Determined with ELIASA at 490nm The light absorption value in each hole.Experimental result such as Figure 10, Fig. 14 kinds of bispecific antibodies can be while specific recognition PD-1 and CD19.

The internal killing experiments of embodiment 12PD-1/CD19 bispecific antibodies

1. different plant knurl model is set up

NHL Raji cells in exponential phase are resuspended in the RPMI1640 nutrient solutions of serum-free, So that cell concentration is 1 × 10⁷Individual cell/ml.Mouse oxter inoculation Raji cells l × 10⁷Individual/ml, 0.2ml/ only, set up and drenched Bar knurl nude mice model.The longest diameter and most short diameter for surveying a lotus knurl in every two days, the volume formula of lotus knurl：Volume (mm³) =long × wide²/2.Test the 30th day, the volume of tumour has reached 500mm³, mouse is put to death, tumour is separated.

2. anti-PD-1/CD19 bispecific antibodies killing experiments

Plantation knurl mouse is divided into 4 groups, it is every group 4, double special with the PD-1/CD19 that purifying is prepared according to embodiment 10 Property antibody (Fig. 1 D) tail vein injection into knurl Mice Body, consumption is respectively A groups：0mg/kg, B group：5mg/kg, C group： 30mg/kg and D groups：100mg/kg.Inject 1 time week about, it is continuous to inject 4 times, record mouse interior tumor size variation.It is anti- PD-1/CD19 bispecific antibodies not only suppress the growth of mouse interior tumor, while there is the effect for reducing tumour, with 30mg/kg and 100mg/kg dosage inject 6 weeks after, gross tumor volume be contracted to respectively initial volume 49.7% and 37.8% (see Figure 11 A).

The dual anti-inhibitory action result to tumour of table 1

Group	Tumor size	Gross tumor volume gaining rate
			Control group	1255±43mg	1255.8%
5mg/kg groups	427±37mg**	432.6%
			30mg/kg groups	75±19mg**	49.7%
100mg/kg groups	49±16mg**	37.8%

Note：Compared * * P with control group<0.01.

3. anti-PD-1 monoclonal antibodies, anti-CD19 monoclonal antibodies and anti-PD-1/CD19 bispecific antibodies tumor-killing Efficiency comparison is tested

Plantation knurl mouse is divided into 5 groups, it is every group 4, mono- with the anti-PD-1 monoclonal antibodies (clone 81) of purifying, anti-CD19 Clonal antibody (clone 22) and anti-PD-1/CD19 bispecific antibodies (sequence is referring to clone 81 and 22, Fig. 1 of clone D) tail vein It is injected into knurl Mice Body, consumption is respectively A groups：PBS, B group：The anti-PD-1 monoclonal antibodies of 30mg/kg (clone 81), C groups：30mg/ The anti-CD19 monoclonal antibodies of kg (clone 75), C groups：The anti-PD-1/CD19 bispecific antibodies of 30mg/kg (the dual anti-D in Fig. 1).Every one Week injection 1 time, it is continuous to inject 4 times, record mouse interior tumor size variation.Anti- PD-1/CD19 bispecific antibodies reduce swollen The effect of knurl is substantially better than anti-PD-1 monoclonal antibodies and anti-CD19 monoclonal antibodies, double in anti-PD-1 monoclonal antibodies, anti-CD19 monoclonal antibodies and anti-PD-1/CD19 Gross tumor volume is contracted to 71.5%, 62.8% and 85.3% (see Figure 11 B) of initial volume respectively under anti-effect.

Inhibitory action result of the different antibodies of table 2 to tumour

Group	Tumor size	Gross tumor volume gaining rate
			Control group	1412±38mg	1321.8%
Anti- PD-1 monoclonal antibodies group	537±17mg*	71.5%
			Anti- CD19 monoclonal antibodies group	130±16mg**	62.8%
Anti- dual anti-group of PD-1/CD19	49±15mg**##&	85.3%

Note：Compared * * P with control group<0.01；Compared with PD-1 groups^##P<0.01；Compared with CD19 groups^&P<0.05。

Variant (5 kinds of variants shown in Fig. 2) in-vivo tumour killing experiments of the anti-PD-1/CD19 bispecific antibodies of embodiment 13

Plantation knurl mouse is divided into 6 groups, every group 4, (schemed with the variant of 5 kinds of purifying anti-PD-1/CD19 bispecific antibodies Dual anti-variant shown in 2E-I, 22) tail vein injection is into knurl Mice Body referring to clone 81 and clone for sequence, and consumption is respectively A groups：PBS, B group：20mg/kg variants 1 (Fig. 2 E), C groups：20mg/kg variants 2 (Fig. 2 F), D groups：20mg/kg variants 3 (Fig. 2 G), E groups：20mg/kg variants 4 (Fig. 2 H), and F groups：20mg/kg variants 5 (Fig. 2 I).Inject 1 time week about, it is continuous to inject 4 times, Record mouse interior tumor size variation.The variant of 5 kinds of anti-PD-1/CD19 bispecific antibodies is likewise supplied with suppressing tumour growth Effect.(p ＜ 0.01).

Inhibitory action result of the variant of the different antibodies of table 3 to tumour

Note：Compared * * P with control group<0.01.

<110>Along sky cellular biological technique（Tianjin）Limited company

<120>Anti- PD1 and CD19 bispecific antibodies and its application

<130> SHUNHOBiMab-PD-1XCD19

<160> 42

<170> PatentIn version 3.5

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<223>Clone the amino acid sequence of 3 heavy chains

<400> 1

Gln Gly Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

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Ala Arg Arg Asp Tyr Phe Phe Asp Trp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

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<212> DNA

<213>Artificial sequence

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<400> 2

cagggccagc tggtgcagag cggcggcgag gtgaagaagc ccggcgccag cctgaggctg 60

gactgcaagg ccagcggcta caccttcacc aactactaca tgcactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atctggtacg acggcagcaa gaggtactac 180

aacgagaagt tcaagaacag ggtgaccatc accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc caggagggac 300

tacttcttcg actggtactt cgactactgg ggccagggca ccaccgtgac cgtgagcagc 360

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

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Glu Arg Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser

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Gly Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Asp Ala Ser Tyr Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Ser

85 90 95

Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 4

<211> 333

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gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccagc 60

atcagctgca gggccagcca gagcgtgagc accagcggct acagctacct ggcctggtac 120

cagcagaagc ccggccaggc ccccaggctg ctgatctacg acgccagcta cctggagagc 180

ggcatccccg ccaggttcag cggcagcggc agcggcaccg acttcaccct gaagatcagc 240

agcctggagc ccgaggacgt gggcgtgtac tactgccagc acagcagcaa ctggcccctg 300

accttcggcc agggcaccaa gctggagatc aag 333

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Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Tyr Ile Thr Phe Ser Asp

20 25 30

Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 6

<211> 342

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caggtgcagc tggtggagag cggcgccgag gtggtgcagc ccggcaggag cctgaggctg 60

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gcccccggcc acggcctgga gtggatcggc tacatcaacc ccagcaacgg cggcaccaac 180

ttcaacgaga agttcaaggg caggttcacc atcagcaggg acaacagcaa gaacaccctg 240

ttcctgcaga tgaacagcct gcagttcgac gacaccgccg tgtactactg cgccaccaac 300

gacgactact ggggccaggg caccctggtg accgtgagca gc 342

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

1 5 10 15

Glu Arg Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Ser Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Arg Asp Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

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gacatcgtga tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccagc 60

atcagctgca gggccagcaa gggcgtgagc agctacctgc actggtacca gcagaagccc 120

ggccagagcc ccaggctgct gatctacctg gccagctacc tggagagcgg cgtgcccgac 180

aggttcagcg gcagcggcag cggcaccgac ttcaccctga agatcagcag ggtggaggcc 240

gaggacttcg ccgtgtacta ctgccagcag agcagggacc tgccctacac cttcggccag 300

ggcaccaagc tggagatcaa g 321

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Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr Ser Trp Asn

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Trp Ile Arg Gln Ala Pro Ile His Gly Leu Glu Trp Ile Gly Tyr Ile

35 40 45

Asn Tyr Ala Gly Ser Thr Ser Tyr Asn Pro Ser Leu Glu Ala Val Thr

50 55 60

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

65 70 75 80

Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Phe Gly

85 90 95

Ser Thr Ala Trp Tyr Ile Asp Val Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

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cagggccagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccac ctgcaccgtg 60

accggctaca gcatcaccag cgactacagc tggaactgga tcaggcaggc ccccatccac 120

ggcctggagt ggatcggcta catcaactac gccggcagca ccagctacaa ccccagcctg 180

gaggccgtga ccatcaccgc cgacaagagc accagcaccg cctacatgga gctgagcagc 240

ctgaggagcg aggacaccgc cgtgtactac tgcgccaggt ggttcggcag caccgcctgg 300

tacatcgacg tgtggggcca gggcaccacc gtgaccgtga gcagc 345

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Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gln

1 5 10 15

Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ala Leu Leu His Ser Asp

20 25 30

Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Glu Leu Ser Ser Arg Phe Ser Gly Ile Pro Asp

50 55 60

Arg Ile Thr Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val

65 70 75 80

Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly Val His Ile

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Pro Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 327

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gacgtggtga tgacccagag ccccctgagc ctgcccgtga ccctgcagcc cgccagcatc 60

agctgcaaga gcagccaggc cctgctgcac agcgacggca agacctacct gtactggtac 120

ctgcagaagc ccggccagag ccccaagctg ctgatctacg agctgagcag caggttcagc 180

ggcatccccg acaggatcac cggcagcggc accgacttca ccctgaagat cagcagggtg 240

gaggccgagg acctgggcgt gtacttctgc ttccagggcg tgcacatccc ctacagcttc 300

ggccagggca ccaagctgga gatcaag 327

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Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr

115 120

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<211> 363

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caagttcagt tattggaatc tggtgctgag cttgtccgtc ctggctcctc agtaaaaatt 60

tcgtgtaagg ccagtggata tgcatttagc tcttactgga tgaattgggt gaaacaacgc 120

cccgggcagg gtctcgaatg gatcggccaa atatggccag gagatgggga cactaactat 180

aatggtaagt tcaaaggcaa ggcgacccta acagctgatg agtcctcatc gacggcctac 240

atgcagctga gtagcttacg atctgaagac tccgcagttt attcatgcgc gcggagagag 300

actaccacag tcggaaggta ctattacgct atggattatt gggggcaagg tacgactgta 360

acc 363

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Glu Leu Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Arg Ser

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<211> 342

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gaattagttt tgactcaatc tcctgcttcc cttgccgtct cactcggtca gcgtgcaacc 60

atttcgtgta aagcgagtca aagcgtagat tatgacggcg attcttacct aaattggtat 120

cagcaaatcc ccggacagcc accgaagctg ttaatatacg acgcttccaa cttggtgtca 180

gggattcctc cccgcttttc gggtagtggc agcggaacag atttcacgct taatatccat 240

ccagttgaga aagtcgacgc cgcaacttat cactgccaac agtctaccga agatccgtgg 300

acatttgggg gtggcacgaa gctcgagata aaacgacggt cc 342

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Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Leu Gly Thr Thr Val Thr

115 120

<210> 18

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 75 weight chain variable districts

<400> 18

gaattagttt tgactcaatc tcctgcttcc cttgccgtct cactcggtca gcgtgcaacc 60

atttcgtgta aagcgagtca aagcgtagat tatgacggcg attcttacct aaattggtat 120

cagcaaatcc ccggacagcc accgaagctg ttaatatacg acgcttccaa cttggtgtca 180

gggattcctc cccgcttttc gggtagtggc agcggaacag atttcacgct taatatccat 240

ccagttgaga aagtcgacgc cgcaacttat cactgccaac agtctaccga agatccgtgg 300

acatttgggg gtggcacgaa gctcgagata aaacgacggt cc 342

<210> 19

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 75 light chain variable districts

<400> 19

Glu Leu Val Leu Thr Gln Ser Pro Ala Ala Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Ala Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Leu Leu Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Arg Ser

<210> 20

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 75 light chain variable districts

<400> 20

gaattagttt tgactcaatc tcctgctgcc cttgcagtct ccctcggtca gcgtgcgacc 60

atttcatgtg ctgcctcgca aagtgtagat tatgacggcg atagctacct aaattggtat 120

cagcaaatcc ccggacagcc accgaaactg ttaatatacg acgcatctaa cttggtgtcc 180

gggattcctc cccgcttttc aggttcgggc agtggaacag atttcacgct taatatccat 240

ccagttgaga aggtcgacgc gctcctatat cactgccaac agagcactga agatccgtgg 300

acctttgggg gtggcacaaa actggagata aagcgacggt ct 342

<210> 21

<211> 108

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 81 weight chain variable districts

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asp

20 25 30

Tyr Ser Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr Met Glu

65 70 75 80

Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Arg Asp Tyr Arg Phe Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 22

<211> 348

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 81 weight chain variable districts

<400> 22

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agcgactaca gctactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcac cgcctacaac 180

cccgccctga agagggtgac cctgaccacc gacagcagca ccaccaccgc ctacatggag 240

ctgaagagcc tgcagttcga cgacaccgcc gtgtactact gcgccaggag ggactacagg 300

ttcgacatgg gcgacctggg ccagggcacc accgtgaccg tgagcagc 348

<210> 23

<211> 108

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 81 light chain variable districts

<400> 23

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Asp Gly

20 25 30

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

35 40 45

Leu Leu Ile Tyr Glu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 24

<211> 324

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 81 light chain variable districts

<400> 24

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc 60

ctgagctgca gggccagcaa gggcgtgagc gacggcaaga cctacctgta ctggtaccag 120

cagaagcccg gccaggcccc caggctgctg atctacgagg ccagctacct ggagagcggc 180

gtgcccgcca ggttcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240

cccgaggact tcgccgtgta ctactgccag cacagcaggg acctgcccta caccttcggc 300

ggcggcacca aggtggagat caag 324

<210> 25

<211> 91

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 22 weight chain variable districts

<400> 25

Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Cys Thr Val Thr Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp

20 25 30

Val Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Lys Phe Lys Gly Lys

35 40 45

Ala Thr Leu Thr Ala Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Tyr

50 55 60

Tyr Tyr Ala Met Asp Tyr Trp Gly Leu Gly Thr Thr Val Thr Trp Gly

65 70 75 80

Gln Gly Thr Thr Val Thr Val Ser Ser Leu Lys

85 90

<210> 26

<211> 282

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 22 weight chain variable districts

<400> 26

caaggtcagt tagttcaatc tggcgctgaa gtcaaaaagc ctggagccac ttgtaccgta 60

acagggtatg cattttcctc atactggatg aattgggtgt ggcccggtga tggcgacacg 120

aactataata aattcaaggg aaaagcgact ttgaccgctg ttgggcgtta ctattacgcc 180

atggattatt actattacgc aatggactat tggggtcttg gcacaacggt cacttaatgg 240

ggacagggga ccacagtaac ggtgtcgagt ctcaagtagt ga 282

<210> 27

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 22 light chain variable districts

<400> 27

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gln

1 5 10 15

Pro Ala Ser Ile Ser Cys Lys Tyr Asp Gly Asp Ser Tyr Leu Asn Trp

20 25 30

Tyr Gln Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly

35 40 45

Ser Gly Ser Gly Thr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Arg

50 55 60

Phe Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu

65 70 75 80

Ala Glu Asp Leu Gly Val Thr Ala Asp Glu Ser Ser Ser Thr Ser Tyr

85 90 95

Met Gln Leu Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 28

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 22 light chain variable districts

<400> 28

gatgttgtca tgactcaatc tcctttatcc ttgcccgtaa cccttcagcc agcttcaatt 60

tcgtgtaaat atgacggtga tagttacctc aattggtatc aagccagcaa cctagtgtct 120

ggcatcccgc ctcgtttttc cggatcaggg tcgggtacag acgcaagtaa tctggttagc 180

ggcatacccc gcttctctgg atccgggacg gattttactt taaagatttc acgagtcgaa 240

gcggaggact tgggtgtaac cgctgatgaa tcgagtagca catcttacat gcagcttggc 300

acgaaactcg agatcaagcg g 321

<210> 29

<211> 443

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of the heavy chain of anti-PD-1 specific antibodies

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asp

20 25 30

Tyr Ser Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr Met Glu

65 70 75 80

Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Arg Asp Tyr Arg Phe Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 30

<211> 1335

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide coding sequence of the heavy chain of anti-PD-1 specific antibodies

<400> 30

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agcgactaca gctactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcac cgcctacaac 180

cccgccctga agagggtgac cctgaccacc gacagcagca ccaccaccgc ctacatggag 240

ctgaagagcc tgcagttcga cgacaccgcc gtgtactact gcgccaggag ggactacagg 300

ttcgacatgg gcgacctggg ccagggcacc accgtgaccg tgagcagcgc cagcaccaag 360

ggccccagcg tgttccccct ggccccctgc agcaggagca ccagcgagag caccgccgcc 420

ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc 480

gccctgacca gcggcgtgca caccttcccc gccgtgctgc agagcagcgg cctgtacagc 540

ctgagcagcg tggtgaccgt gcccagcagc agcctgggca ccaagaccta cacctgcaac 600

gtggaccaca agcccagcaa caccaaggtg gacaagaggg tggagagcaa gtacggcccc 660

ccctgccccc cctgccccgc ccccgagttc ctgggcggcc ccagcgtgtt cctgttcccc 720

cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg cgtggtggtg 780

gacgtgagcc aggaggaccc cgaggtgcag ttcaactggt acgtggacgg cgtggaggtg 840

cacaacgcca agaccaagcc cagggaggag cagttcaaca gcacctacag ggtggtgagc 900

gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgagc 960

aacaagggcc tgcccagcag catcgagaag accatcagca aggccaaggg ccagcccagg 1020

gagccccagg tgtacaccct gccccccagc caggaggaga tgaccaagaa ccaggtgagc 1080

ctgacctgcc tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac 1140

ggccagcccg agaacaacta caagaccacc ccccccgtgc tggacagcga cggcagcttc 1200

ttcctgtaca gcaggctgac cgtggacaag agcaggtggc aggagggcaa cgtgttcagc 1260

tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgagc 1320

ctgggcaagt gataa 1335

<210> 31

<211> 215

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of the light chain of anti-PD-1 specific antibodies

<400> 31

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Asp Gly

20 25 30

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

35 40 45

Leu Leu Ile Tyr Glu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

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

180 185 190

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

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 32

<211> 651

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide coding sequence of the light chain of anti-PD-1 specific antibodies

<400> 32

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcaa gggcgtgagc gacggcaaga cctacctgta ctggtaccag 120

cagaagcccg gccaggcccc ccgcctgctg atctacgagg ccagctacct ggagagcggc 180

gtgcccgccc gcttcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240

cccgaggact tcgccgtgta ctactgccag cacagccgcg acctgcccta caccttcggc 300

ggcggcacca aggtggagat caagcgcacc gtggccgccc ccagcgtgtt catcttcccc 360

cccagcgacg agcagctgaa gagcggcacc gccagcgtgg tgtgcctgct gaacaacttc 420

tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc 480

caggagagcg tgaccgagca ggacagcaag gacagcacct acagcctgag cagcaccctg 540

accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag 600

ggcctgagca gccccgtgac caagagcttc aaccgcggcg agtgctgata a 651

<210> 33

<211> 468

<212> PRT

<213>Artificial sequence

<220>

<223>Anti- PD-1 scFv-Fc fusion protein amino acid sequences

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Asp Gly

20 25 30

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

35 40 45

Leu Leu Ile Tyr Glu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

115 120 125

Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser

130 135 140

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asp Tyr Ser Tyr Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Asn Pro

165 170 175

Ser Asn Gly Thr Ala Tyr Asn Pro Ala Leu Lys Arg Val Thr Leu Thr

180 185 190

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

195 200 205

Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Asp Tyr Arg Phe

210 215 220

Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu

225 230 235 240

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

245 250 255

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

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

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

420 425 430

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Leu Gly Lys

465

<210> 34

<211> 1410

<212> DNA

<213>Artificial sequence

<220>

<223>Anti- PD-1 scFv-Fc fusion protein nucleotide sequences

<400> 34

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc 60

ctgagctgca gggccagcaa gggcgtgagc gacggcaaga cctacctgta ctggtaccag 120

cagaagcccg gccaggcccc caggctgctg atctacgagg ccagctacct ggagagcggc 180

gtgcccgcca ggttcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240

cccgaggact tcgccgtgta ctactgccag cacagcaggg acctgcccta caccttcggc 300

ggcggcacca aggtggagat caagggcggc ggcggcagcg gcggcggcgg cagcggcggc 360

ggcggcagcc aggtgcagct ggtgcagagc ggcgtggagg tgaagaagcc cggcgccagc 420

gtgaaggtga gctgcaaggc cagcggctac accttcacca gcgactacag ctactgggtg 480

aggcaggccc ccggccaggg cctggagtgg atgggcggca tcaaccccag caacggcacc 540

gcctacaacc ccgccctgaa gagggtgacc ctgaccaccg acagcagcac caccaccgcc 600

tacatggagc tgaagagcct gcagttcgac gacaccgccg tgtactactg cgccaggagg 660

gactacaggt tcgacatggg cgacctgggc cagggcacca ccgtgaccgt gagcagcgag 720

agcaagtacg gccccccctg ccccccctgc cccgcccccg agttcctggg cggccccagc 780

gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcaggac ccccgaggtg 840

acctgcgtgg tggtggacgt gagccaggag gaccccgagg tgcagttcaa ctggtacgtg 900

gacggcgtgg aggtgcacaa cgccaagacc aagcccaggg aggagcagtt caacagcacc 960

tacagggtgg tgagcgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac 1020

aagtgcaagg tgagcaacaa gggcctgccc agcagcatcg agaagaccat cagcaaggcc 1080

aagggccagc ccagggagcc ccaggtgtac accctgcccc ccagccagga ggagatgacc 1140

aagaaccagg tgagcctgac ctgcctggtg aagggcttct accccagcga catcgccgtg 1200

gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc cgtgctggac 1260

agcgacggca gcttcttcct gtacagcagg ctgaccgtgg acaagagcag gtggcaggag 1320

ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380

agcctgagcc tgagcctggg caagtgataa 1410

<210> 35

<211> 418

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of the heavy chain of anti-CD19 specific antibodies

<400> 35

Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Cys Thr Val Thr Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp

20 25 30

Val Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Lys Phe Lys Gly Lys

35 40 45

Ala Thr Leu Thr Ala Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Tyr

50 55 60

Tyr Tyr Ala Met Asp Tyr Trp Gly Leu Gly Thr Thr Val Thr Trp Gly

65 70 75 80

Gln Gly Thr Thr Val Thr Val Ser Ser Leu Lys Ala Ser Thr Lys Gly

85 90 95

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

100 105 110

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

115 120 125

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

130 135 140

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

145 150 155 160

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

165 170 175

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

180 185 190

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

195 200 205

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

210 215 220

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

225 230 235 240

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

245 250 255

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

260 265 270

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

275 280 285

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

290 295 300

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

305 310 315 320

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

325 330 335

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

340 345 350

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

355 360 365

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

370 375 380

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

385 390 395 400

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

405 410 415

Gly Lys

<210> 36

<211> 1257

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide coding sequence of the heavy chain of anti-CD19 specific antibodies

<400> 36

cagggccagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccac ctgcaccgtg 60

accggctacg ccttcagcag ctactggatg aactgggtgt ggcccggcga cggcgacacc 120

aactacaaca agttcaaggg caaggccacc ctgaccgccg tgggccgcta ctactacgcc 180

atggactact actactacgc catggactac tggggcctgg gcaccaccgt gacctggggc 240

cagggcacca ccgtgaccgt gagcagcctg aagagcacca agggccccag cgtgttcccc 300

ctggccccct gcagcaggag caccagcgag agcaccgccg ccctgggctg cctggtgaag 360

gactacttcc ccgagcccgt gaccgtgagc tggaacagcg gcgccctgac cagcggcgtg 420

cacaccttcc ccgccgtgct gcagagcagc ggcctgtaca gcctgagcag cgtggtgacc 480

gtgcccagca gcagcctggg caccaagacc tacacctgca acgtggacca caagcccagc 540

aacaccaagg tggacaagag ggtggagagc aagtacggcc ccccctgccc cccctgcccc 600

gcccccgagt tcctgggcgg ccccagcgtg ttcctgttcc cccccaagcc caaggacacc 660

ctgatgatca gcaggacccc cgaggtgacc tgcgtggtgg tggacgtgag ccaggaggac 720

cccgaggtgc agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 780

cccagggagg agcagttcaa cagcacctac agggtggtga gcgtgctgac cgtgctgcac 840

caggactggc tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggg cctgcccagc 900

agcatcgaga agaccatcag caaggccaag ggccagccca gggagcccca ggtgtacacc 960

ctgcccccca gccaggagga gatgaccaag aaccaggtga gcctgacctg cctggtgaag 1020

ggcttctacc ccagcgacat cgccgtggag tgggagagca acggccagcc cgagaacaac 1080

tacaagacca ccccccccgt gctggacagc gacggcagct tcttcctgta cagcaggctg 1140

accgtggaca agagcaggtg gcaggagggc aacgtgttca gctgcagcgt gatgcacgag 1200

gccctgcaca accactacac ccagaagagc ctgagcctga gcctgggcaa gtgataa 1257

<210> 37

<211> 213

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of the light chain of anti-CD19 specific antibodies

<400> 37

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gln

1 5 10 15

Pro Ala Ser Ile Ser Cys Lys Tyr Asp Gly Asp Ser Tyr Leu Asn Trp

20 25 30

Tyr Gln Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly

35 40 45

Ser Gly Ser Gly Thr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Arg

50 55 60

Phe Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu

65 70 75 80

Ala Glu Asp Leu Gly Val Thr Ala Asp Glu Ser Ser Ser Thr Ser Tyr

85 90 95

Met Gln Leu Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

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

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 38

<211> 645

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide coding sequence of the light chain of CD19 specific antibodies

<400> 38

gacgtggtga tgacccagag ccccctgagc ctgcccgtga ccctgcagcc cgccagcatc 60

agctgcaagt acgacggcga cagctacctg aactggtacc aggccagcaa cctggtgagc 120

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acgccagcaa cctggtgagc 180

ggcatccccc gcttcagcgg cagcggcacc gacttcaccc tgaagatcag ccgcgtggag 240

gccgaggacc tgggcgtgac cgccgacgag agcagcagca ccagctacat gcagctgggc 300

accaagctgg agatcaagcg caccgtggcc gcccccagcg tgttcatctt cccccccagc 360

gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420

cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480

agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 540

agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 600

agcagccccg tgaccaagag cttcaaccgc ggcgagtgct gataa 645

<210> 39

<211> 442

<212> PRT

<213>Artificial sequence

<220>

<223>Anti- CD19 scFv-Fc fusion protein amino acid sequences

<400> 39

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gln

1 5 10 15

Pro Ala Ser Ile Ser Cys Lys Tyr Asp Gly Asp Ser Tyr Leu Asn Trp

20 25 30

Tyr Gln Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser Gly

35 40 45

Ser Gly Ser Gly Thr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Arg

50 55 60

Phe Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu

65 70 75 80

Ala Glu Asp Leu Gly Val Thr Ala Asp Glu Ser Ser Ser Thr Ser Tyr

85 90 95

Met Gln Leu Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Gly Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Cys Thr Val Thr Gly

130 135 140

Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Trp Pro Gly Asp Gly

145 150 155 160

Asp Thr Asn Tyr Asn Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Val

165 170 175

Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Tyr Tyr Tyr Ala Met Asp Tyr

180 185 190

Trp Gly Leu Gly Thr Thr Val Thr Trp Gly Gln Gly Thr Thr Val Thr

195 200 205

Val Ser Ser Leu Lys Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

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

225 230 235 240

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

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

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

370 375 380

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

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 40

<211> 1332

<212> DNA

<213>Artificial sequence

<220>

<223>Anti- CD19 scFv-Fc fusion protein nucleotide sequences

<400> 40

gacgtggtga tgacccagag ccccctgagc ctgcccgtga ccctgcagcc cgccagcatc 60

agctgcaagt acgacggcga cagctacctg aactggtacc aggccagcaa cctggtgagc 120

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acgccagcaa cctggtgagc 180

ggcatccccc gcttcagcgg cagcggcacc gacttcaccc tgaagatcag ccgcgtggag 240

gccgaggacc tgggcgtgac cgccgacgag agcagcagca ccagctacat gcagctgggc 300

accaagctgg agatcaagcg cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 360

ggcagccagg gccagctggt gcagagcggc gccgaggtga agaagcccgg cgccacctgc 420

accgtgaccg gctacgcctt cagcagctac tggatgaact gggtgtggcc cggcgacggc 480

gacaccaact acaacaagtt caagggcaag gccaccctga ccgccgtggg ccgctactac 540

tacgccatgg actactacta ctacgccatg gactactggg gcctgggcac caccgtgacc 600

tggggccagg gcaccaccgt gaccgtgagc agcctgaagg agagcaagta cggccccccc 660

tgccccccct gccccgcccc cgagttcctg ggcggcccca gcgtgttcct gttccccccc 720

aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt ggtggtggac 780

gtgagccagg aggaccccga ggtgcagttc aactggtacg tggacggcgt ggaggtgcac 840

aacgccaaga ccaagcccag ggaggagcag ttcaacagca cctacagggt ggtgagcgtg 900

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 960

aagggcctgc ccagcagcat cgagaagacc atcagcaagg ccaagggcca gcccagggag 1020

ccccaggtgt acaccctgcc ccccagccag gaggagatga ccaagaacca ggtgagcctg 1080

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 1140

cagcccgaga acaactacaa gaccaccccc cccgtgctgg acagcgacgg cagcttcttc 1200

ctgtacagca ggctgaccgt ggacaagagc aggtggcagg agggcaacgt gttcagctgc 1260

agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgagcctg 1320

ggcaagtgat aa 1332

<210> 41

<211> 666

<212> PRT

<213>Artificial sequence

<220>

<223>Anti- PD-1 heavy chains-CD19 scFv amino acid sequences

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asp

20 25 30

Tyr Ser Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr Met Glu

65 70 75 80

Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Arg Asp Tyr Arg Phe Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Pro Val Thr Leu Gln Pro Ala Ser Ile Ser Cys Lys Tyr Asp Gly Asp

465 470 475 480

Ser Tyr Leu Asn Trp Tyr Gln Ala Ser Asn Leu Val Ser Gly Ile Pro

485 490 495

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

500 505 510

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

515 520 525

Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Thr Ala Asp Glu Ser

530 535 540

Ser Ser Thr Ser Tyr Met Gln Leu Gly Thr Lys Leu Glu Ile Lys Arg

545 550 555 560

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

565 570 575

Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr

580 585 590

Cys Thr Val Thr Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val

595 600 605

Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Lys Phe Lys Gly Lys Ala

610 615 620

Thr Leu Thr Ala Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Tyr Tyr

625 630 635 640

Tyr Ala Met Asp Tyr Trp Gly Leu Gly Thr Thr Val Thr Trp Gly Gln

645 650 655

Gly Thr Thr Val Thr Val Ser Ser Leu Lys

660 665

<210> 42

<211> 1974

<212> DNA

<213>Artificial sequence

<220>

<223>Anti- PD-1 heavy chains-CD19 scFv nucleotide sequences

<400> 42

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agcgactaca gctactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcac cgcctacaac 180

cccgccctga agagggtgac cctgaccacc gacagcagca ccaccaccgc ctacatggag 240

ctgaagagcc tgcagttcga cgacaccgcc gtgtactact gcgccaggag ggactacagg 300

ttcgacatgg gcgacctggg ccagggcacc accgtgaccg tgagcagcgc cagcaccaag 360

ggccccagcg tgttccccct ggccccctgc agcaggagca ccagcgagag caccgccgcc 420

ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc 480

gccctgacca gcggcgtgca caccttcccc gccgtgctgc agagcagcgg cctgtacagc 540

ctgagcagcg tggtgaccgt gcccagcagc agcctgggca ccaagaccta cacctgcaac 600

gtggaccaca agcccagcaa caccaaggtg gacaagaggg tggagagcaa gtacggcccc 660

ccctgccccc cctgccccgc ccccgagttc ctgggcggcc ccagcgtgtt cctgttcccc 720

cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg cgtggtggtg 780

gacgtgagcc aggaggaccc cgaggtgcag ttcaactggt acgtggacgg cgtggaggtg 840

cacaacgcca agaccaagcc cagggaggag cagttcaaca gcacctacag ggtggtgagc 900

gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgagc 960

aacaagggcc tgcccagcag catcgagaag accatcagca aggccaaggg ccagcccagg 1020

gagccccagg tgtacaccct gccccccagc caggaggaga tgaccaagaa ccaggtgagc 1080

ctgacctgcc tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac 1140

ggccagcccg agaacaacta caagaccacc ccccccgtgc tggacagcga cggcagcttc 1200

ttcctgtaca gcaggctgac cgtggacaag agcaggtggc aggagggcaa cgtgttcagc 1260

tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgagc 1320

ctgggcaagg acgtggtgat gacccagagc cccctgagcc tgcccgtgac cctgcagccc 1380

gccagcatca gctgcaagta cgacggcgac agctacctga actggtacca ggccagcaac 1440

ctggtgagcg gcatcccccc ccgcttcagc ggcagcggca gcggcaccga cgccagcaac 1500

ctggtgagcg gcatcccccg cttcagcggc agcggcaccg acttcaccct gaagatcagc 1560

cgcgtggagg ccgaggacct gggcgtgacc gccgacgaga gcagcagcac cagctacatg 1620

cagctgggca ccaagctgga gatcaagcgc ggcggcggcg gcagcggcgg cggcggcagc 1680

ggcggcggcg gcagccaggg ccagctggtg cagagcggcg ccgaggtgaa gaagcccggc 1740

gccacctgca ccgtgaccgg ctacgccttc agcagctact ggatgaactg ggtgtggccc 1800

ggcgacggcg acaccaacta caacaagttc aagggcaagg ccaccctgac cgccgtgggc 1860

cgctactact acgccatgga ctactactac tacgccatgg actactgggg cctgggcacc 1920

accgtgacct ggggccaggg caccaccgtg accgtgagca gcctgaagtg ataa 1974

Claims

1. a kind of anti-PD-1 and CD19 bispecific antibodies or its variant or its functional fragment, it includes：

A. specific recognition and immune cell surface antigenic PD-1 domain is combined, it includes the weight of anti-PD-1 specific antibodies Chain variable region (anti-PD-1VH)；And

2. anti-PD-1 and CD19 bispecific antibodies according to claim 1 or its variant or its functional fragment, its Described in specific recognition and combine immune cell surface antigenic PD-1 domain it is anti-by the anti-PD-1 specificity being sequentially connected Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and Fc fragments (anti-PD-1CH2-CH3) group of body Into, or by light chain (anti-PD-1VL-CL) and heavy chain (the anti-PD-1VH-CH1- hinge areas-CH2- of one group of anti-PD-1 specific antibody CH3) constitute, or by the light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinges of two groups of anti-PD-1 specific antibodies Area-CH2-CH3) composition, or by the light chain variable district (anti-PD-1VL) and weight chain variable district (anti-PD- of anti-PD-1 specific antibodies 1VH) constitute；

Preferably, the specific recognition and combination CD19 domain are by the light chain for the anti-CD19 specific antibodies being sequentially connected Variable region (anti-CD19VL), weight chain variable district (anti-CD19VH), hinge area and Fc fragments (anti-CD19CH2-CH3) composition, or by Light chain (anti-CD19VL-CL) and heavy chain (anti-CD19VH-CH1- hinge areas-CH2-CH3) group of one group of anti-CD19 specific antibody Into, or by light chain (anti-CD19VL-CL) and heavy chain (the anti-CD19VH-CH1- hinge areas-CH2- of two groups of anti-CD19 specific antibodies CH3) constitute, or light chain variable district (anti-CD19VL) and weight chain variable district (anti-CD19VH) group by anti-CD19 specific antibodies Into.

3. anti-PD-1 and CD19 bispecific antibodies according to claim 1 or 2 or its variant or its functional fragment,

It includes：

A ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specific antibody Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition；And

B ' specific recognitions and combine CD19 domain, its by one group of anti-CD19 specific antibody light chain (anti-CD19VL- CL) constituted with heavy chain (anti-CD19VH-CH1- hinge areas-CH2-CH3)；

Preferably, the specific recognition and domain with reference to immune cell surface antigenic PD-1 and the specific recognition and Pass through one or more disulfide bond, such as one or more disulfide bonds positioned at hinge area with reference to CD19 domain；Or Person

It includes：

A " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specific antibody Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition；And

B " specific recognitions and the domain for combining CD19, it is by the light chain variable for the anti-CD19 specific antibodies being sequentially connected Area (anti-CD19VL), weight chain variable district (anti-CD19VH), hinge area and Fc fragments (anti-CD19CH2-CH3) composition；

It includes：

A " ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is special by the anti-PD-1 being sequentially connected Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and Fc fragments (the anti-PD-1CH2- of property antibody CH3) constitute；And

B " ' specific recognitions and combine CD19 domain, its by one group of anti-CD19 specific antibody light chain (anti-CD19VL- CL) constituted with heavy chain (anti-CD19VH-CH1- hinge areas-CH2-CH3)；

It includes：

A " " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by two groups of anti-PD-1 specific antibodies Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition；And

B " " specific recognitions and the domain for combining CD19, it is (anti-by the light chain variable district of two groups of anti-CD19 specific antibodies CD19VL) constituted with weight chain variable district (anti-CD19VH)；

Preferably, each anti-PD-1 in the specific recognition and combination immune cell surface antigenic PD-1 domain is special The CH3 of the heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) of property antibody is respectively with the specific recognition and combining CD19 Domain in the weight chain variable districts (anti-CD19VH) of each anti-CD19 specific antibodies connected by chemical bond.

4. anti-PD-1 and CD19 bispecific antibodies according to any one of claim 1 to 3 or its variant or its work( Energy property fragment, it is chimeric antibody, humanized antibody or human antibody.

5. anti-PD-1 and CD19 bispecific antibodies according to any one of claim 1 to 4 or its variant or its work( Energy property fragment, wherein the amino acid sequence such as SEQ of the weight chain variable district in the specific recognition and combination PD-1 domain No:1st, shown in 5,9 or 21；The amino acid sequence of light chain variable district such as SEQ ID NO:3rd, shown in 7,11 or 23；

Preferably, the amino acid sequence such as SEQ ID of the weight chain variable district in the specific recognition and combination CD19 domain NO:13rd, shown in 17 or 25；The amino acid sequence of light chain variable district such as SEQ ID NO:15th, shown in 19 or 27；

Preferably, the amino acid sequence of the heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) of the anti-PD-1 specific antibodies It is classified as SEQ ID No:29, nucleotide coding sequence is SEQ ID No:30；The light chain of the anti-PD-1 specific antibodies is (anti- PD-1VL-CL amino acid sequence) is SEQ ID No:31, nucleotide coding sequence is SEQ ID No:32；By being sequentially connected Anti- PD-1 specific antibodies light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and Fc fragments The amino acid of the specific recognition of (anti-PD-1CH2-CH3) composition and combination immune cell surface antigenic PD-1 domain Coded sequence is SEQ ID No:33, nucleotide coding sequence is SEQ ID No:34；

Preferably, the amino acid sequence of the heavy chain (anti-CD19VH-CH1- hinge areas-CH2-CH3) of the anti-CD19 specific antibodies It is classified as SEQ ID No:35, nucleotide coding sequence is SEQ ID No:36；The light chain of the anti-CD19 specific antibodies is (anti- CD19VL-CL amino acid sequence) is SEQ ID No:37, nucleotide coding sequence is SEQ ID No:38；By being sequentially connected Anti- CD19 specific antibodies light chain variable district (anti-CD19VL), weight chain variable district (anti-CD19VH), hinge area and Fc fragments The amino acid sequence of the specific recognition of (anti-CD19CH2-CH3) composition and combination CD19 domain is SEQ ID No: 39, nucleotide coding sequence is SEQ ID No:40；By the light chain variable district (anti-CD19VL) and again of anti-CD19 specific antibodies The amino acid sequence of the specific recognition of chain variable region (anti-CD19VH) composition and combination CD19 domain is SEQ ID No:41, nucleotide coding sequence is SEQ ID No:42；

Preferably, the nucleotide sequence such as SEQ ID of the weight chain variable district in the specific recognition and combination PD-1 domain NO:2nd, shown in 6,10 or 22；The nucleotide sequence of light chain variable district such as SEQ ID NO:4th, shown in 8,12 or 24；

Preferably, the nucleotide sequence such as SEQ ID of the weight chain variable district in the specific recognition and combination CD19 domain NO:14th, shown in 18,26；The nucleotide sequence of light chain variable district such as SEQ ID NO:16th, shown in 20,28.

6. one kind coding anti-PD-1 and CD19 bispecific antibodies according to any one of claim 1 to 5 or its change The nucleic acid molecules of body or its functional fragment.

7. a kind of expression vector for including nucleic acid molecules according to claim 6.

8. a kind of host cell for including expression vector according to claim 7.

9. a kind of pharmaceutical composition, it includes anti-PD-1 and CD19 bispecifics according to any one of claim 1 to 5 Antibody or its variant or its functional fragment, or nucleic acid molecules according to claim 6, or according to claim 7 institute The expression vector or host cell according to claim 8 stated.

Preferably, described pharmaceutical composition also includes other one or more antineoplastics；Preferably, described pharmaceutical composition Also with other one or more antitumour treatments, such as chemotherapy, radiotherapy and/or biotherapy are used together.

10. anti-PD-1 and CD19 bispecific antibodies according to any one of claim 1 to 5 or its variant or its work( Can property fragment or nucleic acid molecules according to claim 6 or expression vector according to claim 7 or according to power Profit requires host cell described in 8 and makes to be immunized by suppressing the immunosupress such as PD-1, PD-L1 or PD-L2 signal path Applications of the enhancing joint targeting CD19 in the medicine for the treatment of tumour (such as NHL).