CN107043425A - anti-PD 1 and CD20 bispecific antibodies and uses thereof - Google Patents

Abstract

The present invention relates to an anti-PD 1 and CD20 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 CD20, comprising the heavy chain variable region of an anti-CD 20 specific antibody (anti-CD 20 VH). The bispecific antibody has sufficient targeting property and bioactivity, has a fully human sequence, and effectively avoids HAMA in clinical treatment application, thereby more effectively reducing drug resistance and improving the utilization efficiency and treatment effect of drugs.

Description

Anti- PD1 and CD20 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 CD20 bispecific antibody and its answers With.The invention provides specific recognition and with reference to PD-1 and CD20 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 special The preparation method of heterogenetic antibody or its functional fragment.

Background technology

CD20 molecules are the distinctive marks in B cell surface, and most NHLs are all the B with CD20 Cell, but do not express CD20 molecules in stem cell, ancestral's B cell, normal plasma cells or other normal structures.Therefore, CD20 Molecule is an important drug target with mab treatment NHL.

Anti-CD-20 monoclonal antibody Rituximab (rituximab, Mabthera) is human mouse chimeric antibody, and it is widely used in The treatment of B cell lymphoma.Rituximab is initially applied to NHL treatment, the NHL of 166 relapsed or stubborns of single therapy Patient, total effective rate is 48%, and complete remission rate is 6%, and part remission rate is 42%.The therapeutic monoclonal antibody mesh of the anti-CD20 of the third generation It is preceding not go public also, including ocaratuzumab (AME-133v), PRO131921, obinutuzumab (GA101) etc..At one In the clinical trial of II phase, 44 FL patients for receiving to recur after initial rituximab treatment are treated with ocaratuzumab, 4 times a week, co-injection 375mg/m², general reaction rate is only 36%.

Meanwhile, in tumor patient, the reason such as immunosupress caused by PD-1/PD-L1 paths, body is thin to tumour The immune response of born of the same parents is typically invalid.Block PD-1/PD-L1 signal paths microenvironment can be immunized with reversing tumor, recover T thin The antitumor activity of born of the same parents, so as to strengthen endogenous antineoplastic immune effect.Block the PD-1 and PD-L1 that suppress path antibody can Competitively combined with PD-1 and PD-L1, and then block PD-1/PD-L1 signal paths, strengthened T cell immune response, exercise The function of killing tumor cell.

In recent years, researcher had developed multiple anti-PD-1 and PD-L1 monoclonal antibodies para-immunity checkpoint inhibitor, these Medicine effectively promotes the progress of cancer immunotherapy.Although anti-PD-1 antibody and anti-PD-LI antibody to some cancers Preferable clinical effectiveness has been shown, but regrettably, these antibody are not all effective to all patients.

The content of the invention

The technical problems to be solved by the invention are to design and build a kind of anti-PD-1 and anti-CD20 bispecific antibody, And make it have enough targetings and bioactivity.Importantly, the bispecific antibody has full people's source sequence, effectively Ground avoids the generation of HAMA in clinical treatment application, so as to more effectively reduce the generation of resistance phenomenon, improves The utilization ratio and therapeutic effect of medicine.The new PD-1/CD20 bispecific fusion proteins of present invention offer, which have, to be directed to First specificity of the first purpose antigen and the second specificity for the second purpose antigen, it has for two kinds of not isoacceptors Binding specificity, while the invention further relates to the production method of the bispecific fusion protein, the medicine of the fusion protein Thing purposes and the treatment method using the fusion protein.

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 CD20 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 CD20 domain, it include anti-CD20 specific antibodies weight chain variable district (resist CD20VH)。

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 CD20 domain is combined by the anti-CD20 specific antibodies that are sequentially connected Light chain variable district (anti-CD20VL), weight chain variable district (anti-CD20VH), hinge area and Fc fragments (anti-CD20CH2-CH3) composition, Or by light chain (anti-CD20VL-CL) and heavy chain (the anti-CD20VH-CH1- hinge areas-CH2- of one group of anti-CD20 specific antibody CH3) constitute, or by the light chain (anti-CD20VL-CL) and heavy chain (anti-CD20VH-CH1- hinges of two groups of anti-CD20 specific antibodies Area-CH2-CH3) composition, or it is (anti-by the light chain variable district (anti-CD20VL) and weight chain variable district of anti-CD20 specific antibodies CD20VH) constitute.

According to the present invention a preferred embodiment, the anti-PD-1 and CD20 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 CD20, it is (anti-by the light chain of one group of anti-CD20 specific antibody CD20VL-CL) constituted with heavy chain (anti-CD20VH-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 CD20 passes through one or more disulfide bond, such as one or more disulfide bond positioned at hinge area connect Connect.

According to the present invention a preferred embodiment, the anti-PD-1 and CD20 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 CD20, it is by the light chain for the anti-CD20 specific antibodies being sequentially connected Variable region (anti-CD20VL), weight chain variable district (anti-CD20VH), hinge area and Fc fragments (anti-CD20CH2-CH3) composition.

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 CD20 passes through one or more disulfide bond, such as one or more disulfide bond positioned at hinge area connect Connect.

According to the present invention a preferred embodiment, the anti-PD-1 and CD20 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 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 CD20, it is (anti-by the light chain of one group of anti-CD20 specific antibody TCD20VL-CL) constituted with heavy chain (anti-CD20VH-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 CD20 passes through one or more disulfide bond, such as one or more disulfide bond positioned at hinge area connect Connect.

According to the present invention a preferred embodiment, the anti-PD-1 and CD20 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 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 CD20 domain, its by two groups of anti-CD20 specific antibodies light chain variable district (anti-CD20VL) and weight chain variable district (anti-CD20VH) 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-CD20VH) of each anti-CD20 specific antibodies in CD20 domain is connected by chemical bond.

Preferably, described anti-PD-1 and CD20 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 such as SEQ ID NO of light chain variable district:3rd, shown in 7,11 or 23.

Preferably, the specific recognition and combine CD20 domain in weight chain variable district amino acid sequence such as SEQ ID NO:13rd, shown in 17 or 25, the amino acid sequence such as SEQ ID NO of light chain variable district: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-CD20VH-CH1- hinge areas-CH2-CH3) of the anti-CD20 specific antibodies Acid sequence is SEQ ID No:35, nucleotide coding sequence is SEQ ID No:36；The light chain of the anti-CD20 specific antibodies The amino acid sequence of (anti-CD20VL-CL) is SEQ ID No:37, nucleotide coding sequence is SEQ ID No:38；By connecting successively Light chain variable district (anti-CD20VL), weight chain variable district (anti-CD20VH), hinge area and the Fc pieces of the anti-CD20 specific antibodies connect The amino acid sequence of the specific recognition of section (anti-CD20CH2-CH3) composition and combination CD20 domain is SEQ ID No:39, nucleotide coding sequence is SEQ ID No:40；By anti-CD20 specific antibodies light chain variable district (anti-CD20VL) and The amino acid sequence of the specific recognition of weight chain variable district (anti-CD20VH) composition and combination CD20 domain 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 CD20 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 such as SEQ ID NO of light chain variable district:4th, shown in 8,12 or 24.

Preferably, the specific recognition and combine CD20 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 CD20 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 CD20 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 CD20 bispecific resist Body, mutant and its functional fragment.Bispecific antibody, mutant or its functional fragment of the present invention has following characteristic At least one：

A. it can be combined with PD-1 with high specific, and block 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 CD20 with high specific；

D. do not combined with other B cell differentiation antigens (such as CD19, CD22)；

E. immune cell activated (such as tumor specific T cells), and specific recognition CD20 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 the anti-PD-1/CD20 bispecific antibodies of humanization or its variant or its feature piece Section.The humanized antibody is designed via computer simulation by the mouse source antibody that mouse generation is immunized and combines phage display skill Art and obtain, and according to its binding characteristic with PD-1 the and CD20 ectodomains of different plant species, identify that its is corresponding Combination epitope.On the anti-PD-1/CD20 bispecific antibodies of humanization of the present invention, variant and its functional fragment possess State the advantageous feature of PD-1/CD20 bispecific antibodies and its functional fragment.

Variant and function fragment of the present invention include that by chemical modification or by mixing half can be increased in liposome 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.

Those skilled in the art can will encode the DNA molecular gram of anti-PD-1/CD20 bispecific antibodies of the present invention It is grand into carrier, and then convert host cell.Therefore, present invention also offers the weight by taking bispecific antibody shown in Figure 1A-D as an example Group DNA vector.The variant of PD-1/CD20 bispecific antibodies shown in code pattern 2 and the carrier of function fragment and host cell also exist 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 place Chief cell can be Escherichia coli, hay bacillus, lactic acid bacteria, streptomycete or proteus mirabilis etc..The eukaryotic host cell can Think such as pichia pastoris phaff, saccharomyces cerevisiae, fission yeast, trichoderma fungi, such as meadow mythimna separata insect cell, such as tobacco Deng plant cell, such as bhk cell, Chinese hamster ovary celI, COS cells, myeloma cell's mammalian cell.In some embodiments In, host cell of the present invention is preferably mammalian cell, and more preferably bhk cell, Chinese hamster ovary celI, NSO cells or COS are thin Born of the same parents.

The present invention obtains corresponding bispecific antibody from recombinant nutrient solution, and with cell ELISA and fluidic cell Art detects its binding activity to PD-1 and CD20.Test result indicates that, bispecific antibody of the invention can be combined Cell positive CD20, such as Raji cells can also combine T lymphocytes, therefore the bispecific antibody constructed by the present invention Can be with efficient targeting CD20 positive lymphs oncocyte and T lymphocytes.

In view of the shortcoming of above PD-1 antibody and CD20 monoclonal antibodies, if T lymphocyte functions can be strengthened PD-1 antibody molecules are merged with targetting the molecule CD20 antibody molecules of B lymphoma cells, and this makes it possible to effectively in Fei Huoqi T lymphocytes are enriched with around golden lymphoma cell, being capable of more effectively target killing tumour on the basis of defence immunologic escape Cell, so as to substantially increase the effect of killing tumor cell.

Therefore, the present invention by PD-1 antibody with CD20 antibody designs into PD-1/CD20 bispecific antibodies, make cytotoxicity T lymphocytes are targeted to tumour cell, immunologic escape, and the accurate killing tumor cell of energy are both avoided, than utilizing PD-1 merely The killing efficiency of antibody or CD20 antibody more fully, more accurately, substantially increases Antybody therapy patients with non Hodgkin lymphoma Clinical effectiveness, there is more preferable clinical value.

Present invention application purification process is obtained after the bispecific antibody of high-purity, detects it to T lymphocytes in vitro Activation, also detected using In vitro cell model and internal animal model its to B cell lymphoma grow suppression make With.In testing in vitro, the bispecific antibody for detecting the present invention with methods such as MTT expresses PD-1 positive T lymphocytes Proliferation function, or detect with methods such as MTT its growth inhibition for expressing CD20 positive lymphoma cell such as Raji cells Effect.In testing in vivo, tumour cell positive above-mentioned CD20 can be for building mouse tumor model, and tumor model can lead to The methods such as abdomen dorsal subcutaneous, abdominal cavity, tail vein structure is crossed, moves real for observing the antitumor or anti-rotation of bispecific antibody Test.

The people source bispecific antibody albumen comprising PD-1 antibody and CD20 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 CD20, and people's T lymphs can be activated Cell, target killing NHL cell.

The beneficial effects of the present invention are the PD-1/CD20 bispecific antibodies there is provided a kind of humanization, it can be tied The positive cells of PD-1 and CD20 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 and CD20 monoclonal antibodies of more current clinical practice, with more powerful killing ability, There is 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 anti-PD-1/CD20 bispecific antibodies of the present invention.

Fig. 2 is the functional fragment of anti-PD-1/CD20 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, CD20 ectodomain.

Fig. 4 is anti-PD-1 monoclonal antibodies and the ELISA testing results of anti-CD-20 monoclonal antibody.

Fig. 5 shows the combination of anti-PD-1 monoclonals 3,21,45 and 77 and CD28 family proteins respectively.

Fig. 6 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. 7 shows the specific binding of anti-CD-20 monoclonal 8,65 and CD20 albumen respectively.

The recombinant plasmid collection of illustrative plates of the bispecific antibody of Fig. 8 code displayings present invention.

Fig. 9 shows Flow cytometry bispecific antibody to the result of the combination effect of Raji cells, left figure：It is anti- PD1/ anti-CD 20s；Right figure：Control antibodies.

Figure 10 shows the affinity capacity experimental result of bispecific antibody moderate resistance PD-1 antibody moieties；

Specific bond of the PD-1/CD20 bispecific antibodies of Figure 11 display present invention to PD-1 and CD20；

Figure 12 shows the tumor cell in vitro growth inhibitory activity of bispecific antibody；

Figure 13 shows that bispecific antibody suppresses the experimental result of the growth of transplantable tumor in Mice Body, and wherein ordinate is swollen Knurl volume.

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

The structure design of the anti-PD-1/CD20 bispecific antibodies of embodiment 1

PD-1 and the bispecific antibody of CD20 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-CD20 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 CD20 light chain variable districts (anti-CD20VL), weight chain variable district (anti-CD20VH), hinge area and The bispecific binding protein of Fc fragments (anti-CD20CH2-CH3) fusion protein composition；(C) anti-PD-1 light chain variable districts, heavy chain The bispecific knot that the light-heavy chain pair of variable region, hinge area and Fc fragment fusion proteins and anti-CD20 specific antibodies is constituted Hop protein；(D) heavy chain of the light chain of anti-PD-1 specific antibodies and anti-PD-1 specific antibodies, anti-CD20 light chain variable districts are (anti- CD20VL) and weight chain variable district (anti-CD20VH) fusion protein composition bispecific binding protein.

The variant of anti-PD-1/CD20 bispecific antibodies and the embodiment of functional fragment are as shown in Fig. 2 in Fig. 2 E-H The formation of bispecific antibody variant and functional fragment based on known to art technology person fusogenic peptide technology (with polypeptide chain or Protein connects different structure territory, and then realizes amalgamation and expression), the formation of heterodimer is based on art technology in Fig. 2 I 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-CD20 The fusion protein of weight chain variable district (anti-CD20VH)；(F) the scFv groups of anti-PD-1 single chain variable fragment (scFv) and anti-CD20 Into fusion protein；(G) non-immunoglobulin that anti-PD-1 single chain variable fragment (scFv) and anti-CD20 scFv are constituted is melted Hop protein；(H) anti-PD-1 light chain variable districts, weight chain variable district, hinge area, Fc fragments and anti-CD20 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-CD20 light chain variable districts, weight chain variable district, hinge area and Fc fragment fusion proteins (note：Specific recognition PD-1 and CD20 function fragment position are interchangeable in figure).

The preparation of the anti-PD-1/CD20 bispecific antibodies antigen of embodiment 2 (people PD-1 and CD20 extracellular can solubilization domain)

The coding gene sequence of people source PD-1 and CD20 ectodomain is obtained in UniProt databases, passes through full genome The mode of synthesis obtains encoding gene PD-1ED-CDS and CD20ED-CDS, and synthetic product is through BamH I, EcoR I (PD- 1ED-CDS) and BamH I, Xba I (CD20ED-CDS) double digestion after, be cloned into pcDNA3.1 carriers, by the use of Chinese hamster ovary celI as Host cell carries out protein expression, and destination protein is purified using purification techniques such as affinity chromatography, ion-exchange chromatographies.

Fig. 3 shows people source PD-1 and CD20 ectodomains protein purification and carries out the SDS-PAGE after deglycosylation processing Testing result figure.

The anti-human PD-1 of embodiment 3 and CD20 antibody acquisition

1. immune animal

The purified people PD-1 of 1mg/ml or the μ l of CD20 ectodomains protein 10 0 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, at room temperature 2h is stood, 25 DEG C, 6000-8000rpm centrifuges 10-15min, takes supernatant, pass through ELISA experiment detection potency.If bioactivity Whole blood can be taken with i.e. eye, 2h be stood at room temperature, 25 DEG C, 6000-8000rpm centrifuges 10min, take supernatant, -80 DEG C of preservations.If Potency is unavailable, continues 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

The egg with reference to PD-1 or CD20 ectodomains can be secreted by being reacted using ELISA in screening monoclonal hybridoma Bai Kangti clone, is to filter out the preferred clone with reference to PD-1 (clone 3,21,45,77) and CD20 (clone 8,65) respectively, Clone can the corresponding antigen protein of specific recognition.

The combination identification of the anti-PD-1 antibody of embodiment 4 and other CD28 family proteins

The specificity of the PD-1 antibody gone out for evaluation and screening, by member protein PD-1, CD28, CTLA-4 of CD28 families and ICOS (R＆D Systerm) stands coating 2h in ELISA Plate in 37 DEG C of incubators；4 DEG C of closings are stayed overnight；100 μ l are added after washing Candidate antibody (1 μ g/ml), 37 DEG C of incubation 1h；The HRP mark goat anti-mouse antibodies (Abcam) of dilution are added after washing 5 times, It is incubated at room temperature 1h；The 2M H that 50 μ l/ holes are added after the μ l/ holes of OPD substrate solutions 100, colour developing 5min are added after washing 5 times₂SO₄Eventually Only react.The light absorption value in each hole at 490nm is determined with ELIASA.Experimental result such as Fig. 5, the antibody of candidate clone secretion is not combined CD28 family member's albumen in addition to PD-1.

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), 60min is incubated at 4 DEG C, concussion in every 10 minutes is once；Rinsed and diluted with PBS, cell suspension is injected Be filled with the special test tube of stainless steel wool, be put into separator and isolated and purified, under high-intensity magnetic field T cell can not by separator, Purified.

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

Human PD-L 1 ectodomain coding gene sequence is obtained in UniProt databases, by way of full genome is synthesized Encoding gene is obtained, synthetic product is cloned into pEGFP-N1 carriers after EcoR I, Xho I double digestions, thin using CHO-S Born of the same parents carry out protein expression as host cell, and destination protein is entered using purification techniques such as affinity chromatography, ion-exchange chromatographies Row purifying, people source PD-L1 ectodomain the albumen rh-PD-L1-GFP, Fig. 6 (A) for obtaining green fluorescent protein amalgamation and expression shows Show the expression and purification of fusion protein and carry 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.

By Fig. 6 (B-F), clone 3,21 and 77 can hinder the combination of the PD-1 acceptors on PD-L1 and human T-cell, clone 45 Barrier effect it is poor.

The anti-CD 20 antibodies of embodiment 6 and CD19, CD33 and CD138 combination identification

CD20, CD19, 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. 7, the antibody of candidate clone secretion does not combine CD19, CD33 and CD138 albumen.

The acquisition of the anti-PD-1 and CD20 candidate antibodies variable region sequences of embodiment 7

Expand the anti-PD-1 of culture respectively and clone 3,21,77 and anti-CD20 clones 8,65, culture to cell concentration is 10⁹It is individual, from The heart collects cell, removes cell culture fluid, gently cell is washed twice with the PBS (6-8mL) of precooling；Tried with Trizol Agent box (TAKARA) extracts total serum IgE, with UV spectrophotometer measuring RNA purity and concentration；Using total serum IgE as template, reverse Record 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 chain and chain variable region amino acid sequence are shown in SEQ No:1 and 3,5 and 7,9 and 11；Encode the core of corresponding amino acid Nucleotide sequence is shown in SEQ No:2 and 4,6 and 8,10 and 12.The anti-CD20 obtained clones 8,65 heavy chain and light chain variable district amino Acid sequence is 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 Hes 20。

The anti-PD-1 and CD20 antibody of embodiment 8 it is humanization modified

Using the anti-PD-1 and CD20 antibody of acquisition variable region amino acid sequence (sequence that embodiment 7 is obtained), utilize IMGT/V-Quest line servers analyze antibody subtype, the CDR region of tri- kinds of antibody of comprehensive Kabat, Chothia and IMGT Numbering schemes determine six CDR region sequences of antibody light chain and heavy chain；Mouse parental antibody Fab is carried out using DS softwares And its chimeric humanized antibody Fab space structure, obtain chimeric antibody molecular model after, to parent Fab and chimeric antibody Fab initial models are comprehensively optimized using molecular dynamics simulation, extract stable average conformation；Examined using epitope scanning The humanized antibody linear epitope that may be present and comformational epitope of design are tested, it is determined that the Framework Region amino acid site that must retain Afterwards, different amino acid mutation sites are designed and obtains humanized antibody.

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 34, and the heavy chain and light-chain amino acid sequence of clone 81 are respectively SEQ ID No:21 and 23, The nucleotide sequence for encoding corresponding amino acid is respectively SEQ ID No:22 and 24；The heavy chain and light-chain amino acid sequence of clone 34 Respectively 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/CD20 bispecific antibodies expression vector of embodiment 9

Anti- PD-1's (clone 81, preparation method is shown in embodiment 8) in the anti-PD-1/CD20 bispecific antibodies of the present invention Variable region coding nucleotide PD-1-VH and PD-1-VL, the variable region coding of anti-CD20 (clone 34, preparation method is shown in embodiment 8) Nucleotides CD20-VH and CD20-VL, IgG1 heavy chain constant region CH1, hinge area, Fc and Fc-knob and Fc-hole encoding nucleosides Acid, and constant region of light chain CL kappa coding nucleotide, and link scFv polypeptide (GGGGS)₃Encoding gene is derived from In Life Technologies Inc. (Carlsbad, CA).

Meanwhile, it in order to which anti-PD-1/CD20 antibody can be expressed in Chinese hamster ovary celI, and can be secreted into culture medium, have selected point The leader peptide sequences of TNF-α are secreted as secreting signal peptide.Bispecific antibody PD-1/CD20 N-terminal adds secreting signal peptide And the labels of His 6, it is not only able to ensure that it is secreted into outside mammalian cell, but also affinitive layer purification, and energy can be utilized It is enough further to remove label and only retentive activity bispecific antibody albumen using digestion.

Each different chain shown in Fig. 1 in the bispecific antibody of 4 kinds of structures, VL and CL, VH and CH1, CH1 and Fc (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 is as expression vector to prepare bispecific antibody.

Expression and purifying of the bispecific antibody of the present invention of embodiment 10 in cell

The bispecific antibody of the present invention is expressed and is secreted into nutrient solution in CHO-S cells, and utilizes nickel post The method purifying of affinity chromatography is obtained.

1. transient expression of the anti-PD-1/CD20 bispecific antibodies in CHO-S cells

After the recombinant plasmid for obtaining high-purity encoding bispecific antibody PD-1/CD20, Lipofectamine is utilized 2000 plasmid transfection kits (Invitrogen companies) are by Transfected Recombinant Plasmid Chinese hamster ovary celI (ATCC), in serum free medium Middle culture collects Chinese hamster ovary celI supernatant after three days, can use the expression of immune-blotting method bispecific antibody.The method can use In rapidly obtaining a small amount of bispecific antibody albumen, its concentration can quantitatively detect that primary antibody used can be with ELISA method The anti-antibody of His 6.

2. stable expression of the anti-PD-1/CD20 bispecific antibodies in CHO-S cells

The recombinant plasmid of encoding bispecific antibody is transfected into CHO with the plasmid transfection kits of Lipofectamine 2000 Cell, cultivates in serum free medium and adds hygromycin two days later, carries out cell clone culture using limiting dilution assay, about The cell clone of picking hygromycin resistance carries out the expansion culture of cell after 14 days, and chooses cell in good condition in liquid nitrogen Freezen protective.CHO-S cells after stable transfection further expand culture to produce substantial amounts of bispecific antibody albumen, this side Method can be used for obtaining substantial amounts of bispecific antibody PD-1/CD20, and its concentration can quantitatively be detected with ELISA method, primary antibody used Can be the anti-antibody of His 6.

3. bispecific antibody PD-1/CD20 purifying

Cell culture fluid comprising bispecific antibody can be purified using the method for affinity chromatography.Nickel dam is analysed After post is balanced with buffer solution, the concentrated CHO-S cell culture supernatant liquid sample introductions of ultrafilter are supervised with A280 (nm) Survey, uncombined albumen is washed till with cleaning fluid and is all eluted, elution associated proteins are then used.After purification double special Property antibody ELISA method detection concentration.

The vitro binding assay of the bispecific antibody of the present invention of embodiment 11 and cell

The bispecific antibody (sequence is referring to embodiment 8) of the present invention can be enriched with T lymphocytes to corresponding in vitro Target cell.The present invention cell positive using Raji cells as CD20, and detect that its is thin with the bispecific antibody of the present invention Born of the same parents' binding activity.

1. the binding activity of bispecific antibody and Raji cells

Control antibodies or the μ l of bispecific antibody 20 and 1 × 10⁶Individual Raji mixing with cells, is incubated 30 minutes at 37 DEG C.PBS Buffer solution is washed 2 times, is then incubated 30 minutes on ice with the antibody of the anti-His of mouse 6.PBS is washed, the rabbit marked with PE Anti-mouse antibody is incubated 30 minutes in dye solution on ice.PBS is washed 2 times, and flow cytometer is analyzed. As a result show, bispecific antibody of the invention is compared with CD20 monoclonal antibodies, with the positive Raji of stronger combination CD20 The ability of cell, and difference is more significantly (see Fig. 9).

2. the affinity identification of bispecific antibody moderate resistance PD-1 antibody

PD-1-His recombinant proteins are coated with into ELISA Plate by 2 μ g/ml, stood overnight at 4 DEG C.Washed with PBST 3 times.With 0.1% BSA room temperatures are closed 1 hour.Washed with PBST 3 times, the PD-1/CD20 antibody that embodiment 2 is prepared is configured to 200 μ g/ml concentration, and gradient dilution is carried out, 30 μ l are added per hole, are stored at room temperature 30 minutes.Washed with PBST 3 times, add coupling HRP Anti-human Fc ELIAS secondary antibody, be stored at room temperature 1 hour.Washed with PBST 4 times, add TMB colour developings, and with 2M H₂SO₄Terminate anti- Should, ELIASA reading.Can be seen that from result, screening obtained antibody has preferable affinity to PD-1, and its ability with Positive control Nivolumab close (Figure 10).

The specific binding identification of the anti-PD-1/CD20 bispecific antibodies of embodiment 12

PD-1, CTLA-4, CD20 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；4 kinds of antibody (, referring to embodiment 8, structure is referring to Figure 1A-D for sequence) (100 μ g/ of purifying are added after washing Ml gradient dilution 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 of incubation 1h；The HRP mark goat anti-mouse antibodies (Abcam) of dilution are added after washing 5 times, 1h is incubated at room temperature； The 2M H that 50 μ l/ holes are added after the μ l/ holes of OPD substrate solutions 100, colour developing 5min are added after washing 5 times₂SO₄Terminating reaction.Use enzyme Mark the light absorption value that instrument determines each hole at 490nm.Experimental result such as Figure 11,4 kinds of bispecific antibodies can be while specific recognition PD- 1 and CD20.

The tumor cell in vitro growth inhibitory activity detection of the bispecific antibody of the present invention of embodiment 13

The present invention detects growth in vitro of the bispecific antibody to Raji cells by the use of Raji cells as cell model Inhibitory action.

Raji cells are inoculated with 96 porocyte culture plates, according to T lymphocytes and Raji cells 10：1 ratio drenches T Bar cell is added in culture plate, while the bispecific antibody of various concentrations is added into cell culture well.Continue to cultivate thin After born of the same parents, add 20 μ l MTT per hole.Continue after being incubated 4 hours, inhale the supernatant abandoned in cell culture well, add 150 μ l diformazans per hole Base sulfoxide (DMSO), the absorption value of each hole 490nm wavelength is determined with enzyme-linked immunosorbent assay instrument, draws cell growth curve.As a result It has been shown that, compared with PD-1 antibodyomes and CD20 monoclonal antibody groups, (sequence is shown in embodiment 8 to bispecific antibody of the invention, and structure is shown in Fig. 1 D) growth of Raji cells can be significantly inhibited, there were significant differences (see Figure 12) with control group.

The detection that the bispecific antibody PD1/CD20 of the present invention of embodiment 14 suppresses to growth of transplanted human in Mice Body

The present invention is inoculated with SCID mice using Raji cells to detect internal life of the bispecific antibody to Raji cells Long inhibitory action.

4-5 week old SCID mice (body weight is 18-20g/) is divided into 4 groups.Mouse oxter inoculation Raji cells l × 10⁷/ Ml, 0.2ml/ only, set up lymthoma nude mice model.One group of Raji receives vehicle (PBS), another group of μ g bispecific of receiving 5 Antibody (PD-1/CD20BsAb) processing, remaining two groups are positive controls：(prepared by embodiment 8 for anti-PD-1 antibody controls group And anti-CD-20 monoclonal antibody control group (embodiment 8 prepare clone 34) 81), processing scheme is tail vein injection every other day to clone 5 times.After 15 days, tumor size is measured, 1 is the results are shown in Table.

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

Group	Gross tumor volume (cm3)
		PBS control group	2.76±0.5
PD-1 antibody control groups	1.65±0.2*
		CD20 monoclonal antibody groups	1.62±0.08*
Dual anti-group	1.20±0.1**#&

Note：Compared * * P with PBS control group<0.01, * P<0.05；Compared with PD-1 antibody control groups^#P<0.05；With CD20 Monoclonal antibody group compares^&P<0.05。

Figure 13 is shown, is compared than PBS control group, anti-PD-1 antibody controls group, anti-CD-20 monoclonal antibody control group and sheet The bispecific antibody group (, referring to embodiment 8, structure is referring to Fig. 1 D for sequence) of invention can substantially suppress the growth (p of transplantable tumor< 0.01)；Compared with anti-PD-1 antibody controls group and anti-CD-20 monoclonal antibody control group, bispecific antibody of the invention can The growth of more efficient suppression Raji transplantable tumors, and difference has conspicuousness (p<0.05).

Variant (5 kinds of variants shown in Fig. 2) in-vivo tumour killing experiments of the anti-PD-1/CD20 bispecific antibodies of embodiment 15

Plantation knurl mouse is divided into 6 groups, every group 4, (schemed with the variant of 5 kinds of purifying anti-PD-1/CD20 bispecific antibodies Dual anti-variant shown in 2E-I, sequence is referring to embodiment 8) tail vein injection is into knurl Mice Body, and consumption is respectively A groups： PBS, B group：30mg/kg variants 1 (Fig. 2 E), C groups：30mg/kg variants 2 (Fig. 2 F), D groups：30mg/kg variants 3 (Fig. 2 G), E groups： 30mg/kg variants 4 (Fig. 2 H), and F groups：30mg/kg variants 5 (Fig. 2 I).Inject 1 time week about, continuous to inject 4 times, record Mouse interior tumor size variation.The variant of 5 kinds of anti-PD-1/TEM-8 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 2 to tumour

Group	Tumor size
		Control group	1495±33mg
Variant 1 (Fig. 2 E) group	218±41mg**
		Variant 2 (Fig. 2 F) group	129±19mg**
Variant 3 (Fig. 2 G) group	86±23mg**
		Variant 4 (Fig. 2 H) group	87±15mg**
Variant 5 (Fig. 2 I) group	82±14mg**

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

Above-described embodiment shows, the anti-PD-1/CD20 bispecific antibodies in people source of the invention can in Chinese hamster ovary celI table Reach, can further pass through affinitive layer purification.Resulting bispecific antibody can combine the positive Raji cells of CD20, And it can suppress the apoptosis of T lymphocytes, the effect of inducer T lymphocyte target killing NHL, and compared with Anti- PD-1 antibody or anti-CD 20 antibodies, which is used alone, has more significant killing efficiency.

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

<120>Anti- PD1 and CD20 bispecific antibodies and its application

<130> SHUNHOBiMab-PD-1XCD20

<160> 42

<170> PatentIn version 3.3

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

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

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

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

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

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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

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Gly Thr Thr Val Thr Val Ser Ser

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cagggccagc tggtgcagag cggcggcgag gtgaagaagc ccggcgccag cctgaggctg 60

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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

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

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

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

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

100 105 110

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gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccagc 60

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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

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

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

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

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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

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

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Ser Ser

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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

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

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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

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

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

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Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

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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

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ggcaccaagc tggagatcaa g 321

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

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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

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

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Val Ser Ser

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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

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

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

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

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

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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

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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

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

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

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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

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

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Tyr Trp Gly Gln Gly Thr Thr Val Thr

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ccggcgcctc tgtaaagatg tcctgtaaag catcaggata tacttttacc gcgtacaata 60

tgcattgggt gaagcaaaca ccagggcgtg gtcttgagtg gattggcgct ctctatccgg 120

gaaacgggga tacgtcgtac aatcagaaat tcaagggtaa agccactcta accgcagaca 180

agagtacaag cacggcgtat atgcaactgt cttccttaac ttcacacgat tcggctgact 240

actattgcgc ccgcagtaac tactatggca ccagctactg gtttttcgat gtttggggag 300

cagggacaac ggtcactgta tctgcg 326

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

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

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Thr Asn Leu Val Thr 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 Arg Arg Ile Asp Ala Ala Thr Tyr His Cys Gln Arg Ser

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Arg Arg Ser

115

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cacttggtca gcgtgcaacc atctcgtgta aagcgacaca agctgtagac tatgatggcg 60

acgcctacct caattggtat cagcaaatac ccggacagcc accgaagcta ctgatttacg 120

atgcaacgaa cttagtgact gggatccctc cccgctttag tggtagcggc tctggaaccg 180

acttcacatt gaatatacat ccagttgaac gacggattga tgcggctacg tatcactgcc 240

aaagatccac taaagacccg tggacctttg ggggtggcac aaagcttgag atcaaaaggc 300

gttcataata gtgatat 317

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

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

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

35 40 45

Gly Ala Leu Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Ala

50 55 60

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

65 70 75 80

Met Gln Leu Thr Thr Leu Thr Ser His Asp Ser Ala Asp Tyr Tyr Cys

85 90 95

Ala Arg Ser Asn Tyr Tyr Gly Thr Ser Tyr Trp Phe Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

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<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 65 weight chain variable districts

<400> 18

ccggcgcctc tgtaaaaatg tcctgtaagg catcaggata tacttttacc gcgtacaata 60

tgcattgggt gaaacaaaca ccagggcgcg gtcttgagtg gattggcgct ctctatccgg 120

gaaacgggga tacgtcgtac aatcagaagg ccaaaggtaa ggcaactcta accgcggaca 180

aaagtacaag cacggcttat atgcaactga ctaccttaac atctcacgat tccgccgact 240

actattgcgc acgatcaaac tactatggca cgtcgtactg gttctttgat gtttggggag 300

cggggactac cgtcacagta agtgct 326

<210> 19

<211> 115

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 65 light chain variable districts

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Arg Arg Ile Asp Ala Ala Thr Tyr His Cys Gln Arg Ser

85 90 95

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

100 105 110

Arg Arg Ser

115

<210> 20

<211> 307

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 65 light chain variable districts

<400> 20

gacccttggt cagcgtgcaa caatctcatg tcgcgcgacg caagctgtag actatgatgg 60

cgacgcctac ctcaattggt atcagcaaat acccggacag ccaccgaaac tactgattta 120

cgatgcaact aacttagtga ccgggatccc tccccgagcg tcgggtagtg gcagcggaac 180

agacttgacg cttaatatac atccagttga acggagaatt gatgctgcca cttatcactg 240

ccaaaggtct accaaggacc cgtggacatt tgggggtggc acgaaactcg agatcaagcg 300

tcgctcc 307

<210> 21

<211> 116

<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> 109

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 34 weight chain variable districts

<400> 25

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 Ala Gln Ser 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 Gln Val Ala Ser Arg Phe Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser 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 Ile His Leu

85 90 95

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

100 105

<210> 26

<211> 363

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 34 weight chain variable districts

<400> 26

caaggtgttc ctgatcgttt ttctggttcc ggcactgact tcaccttaaa aatttcacgc 60

gtcgaagctg aggatttggg agtatatttt tgtttccaag ggatccatct tccctacaca 120

tttggtcagg gcacgaagct cgaaataaaa caccatcacc atcaccatga cgtggttatg 180

actcaatcgc cactaagtct gccggtcacc ttacagcctg ccagcatttc ttgcaagtcc 240

gcacaatcat tgcttcactc ggatggaaaa acatatctct actggtatct acagaagccc 300

gggcaaagtc caaaactgtt aatctaccag gtagcgagcc gattcgctgg ttaatagtga 360

taa 363

<210> 27

<211> 115

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 34 light chain variable districts

<400> 27

Asp Ile 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 Thr Gln Ala Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Thr Asn Leu Val Thr 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 Arg Arg Ile Asp Ala Ala Thr Tyr His Cys Gln Arg Ser

85 90 95

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

100 105 110

Arg Arg Ser

115

<210> 28

<211> 354

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 34 light chain variable districts

<400> 28

gatattgttt taactcaatc tcctgcttcc ttggccgtct cacttggtca gcgtgcaacc 60

atctcgtgta aagcgacaca agctgtagac tatgatggcg acgcctacct caattggtat 120

cagcaaatac ccggacagcc accgaagcta ctgatttacg atgcaacgaa cttagtgact 180

gggatccctc cccgctttag tggtagcggc tctggaaccg acttcacatt gaatatacat 240

ccagttgaac gacggattga tgcggctacg tatcactgcc aaagatccac taaagacccg 300

tggacctttg ggggtggcac aaagcttgag atcaaaaggc gttcataata gtga 354

<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 heavy chain nucleotide coding sequences 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>The amino acid sequence of anti-PD-1 scFv-Fc fusion proteins

<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>The nucleotide sequence of anti-PD-1 scFv-Fc fusion proteins

<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> 436

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of the heavy chain of anti-CD20 specific antibodies

<400> 35

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 Ala Gln Ser 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 Gln Val Ala Ser Arg Phe Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser 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 Ile His Leu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Ser Leu Gly Lys

435

<210> 36

<211> 2031

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide coding sequence of the heavy chain of anti-CD20 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

gacgtggtga tgacccagag ccccctgagc ctgcccgtga ccctgcagcc cgccagcatc 780

agctgcaaga gcgcccagag cctgctgcac agcgacggca agacctacct gtactggtac 840

ctgcagaagc ccggccagag ccccaagctg ctgatctacc aggtggccag ccgcttcgcc 900

ggcgtgcccg accgcttcag cggcagcggc accgacttca ccctgaagat cagccgcgtg 960

gaggccgagg acctgggcgt gtacttctgc ttccagggca tccacctgcc ctacaccttc 1020

ggccagggca ccaagctgga gatcaagagc accaagggcc ccagcgtgtt ccccctggcc 1080

ccctgcagca ggagcaccag cgagagcacc gccgccctgg gctgcctggt gaaggactac 1140

ttccccgagc ccgtgaccgt gagctggaac agcggcgccc tgaccagcgg cgtgcacacc 1200

ttccccgccg tgctgcagag cagcggcctg tacagcctga gcagcgtggt gaccgtgccc 1260

agcagcagcc tgggcaccaa gacctacacc tgcaacgtgg accacaagcc cagcaacacc 1320

aaggtggaca agagggtgga gagcaagtac ggccccccct gccccccctg ccccgccccc 1380

gagttcctgg gcggccccag cgtgttcctg ttccccccca agcccaagga caccctgatg 1440

atcagcagga cccccgaggt gacctgcgtg gtggtggacg tgagccagga ggaccccgag 1500

gtgcagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagcccagg 1560

gaggagcagt tcaacagcac ctacagggtg gtgagcgtgc tgaccgtgct gcaccaggac 1620

tggctgaacg gcaaggagta caagtgcaag gtgagcaaca agggcctgcc cagcagcatc 1680

gagaagacca tcagcaaggc caagggccag cccagggagc cccaggtgta caccctgccc 1740

cccagccagg aggagatgac caagaaccag gtgagcctga cctgcctggt gaagggcttc 1800

taccccagcg acatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag 1860

accacccccc ccgtgctgga cagcgacggc agcttcttcc tgtacagcag gctgaccgtg 1920

gacaagagca ggtggcagga gggcaacgtg ttcagctgca gcgtgatgca cgaggccctg 1980

cacaaccact acacccagaa gagcctgagc ctgagcctgg gcaagtgata a 2031

<210> 37

<211> 221

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of the light chain of anti-CD20 specific antibodies

<400> 37

Asp Ile 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 Thr Gln Ala Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Thr Asn Leu Val Thr 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 Arg Arg Ile Asp Ala Ala Thr Tyr His Cys Gln Arg Ser

85 90 95

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

100 105 110

Arg Arg Ser Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

115 120 125

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

130 135 140

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

145 150 155 160

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

165 170 175

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

180 185 190

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

195 200 205

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 38

<211> 714

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide coding sequence of the light chain of CD20 specific antibodies

<400> 38

gacatcgtgc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccaccca ggccgtggac tacgacggcg acgcctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccaccaa cctggtgacc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggagc gccgcatcga cgccgccacc taccactgcc agcgcagcac caaggacccc 300

tggaccttcg gcggcggcac caagctggag atcaagcgcc gcagcaccgt ggccgccccc 360

agcgtgttca tcttcccccc cagcgacgag cagctgaaga gcggcaccgc cagcgtggtg 420

tgcctgctga acaacttcta cccccgcgag gccaaggtgc agtggaaggt ggacaacgcc 480

ctgcagagcg gcaacagcca ggagagcgtg accgagcagg acagcaagga cagcacctac 540

agcctgagca gcaccctgac cctgagcaag gccgactacg agaagcacaa ggtgtacgcc 600

tgcgaggtga cccaccaggg cctgagcagc cccgtgacca agagcttcaa ccgcggcgag 660

tgctgataaa gcagccccgt gaccaagagc ttcaaccgcg gcgagtgctg ataa 714

<210> 39

<211> 468

<212> PRT

<213>Artificial sequence

<220>

<223>Anti-CD 20 scFv-Fc fusion protein amino acid sequences

<400> 39

Asp Ile 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 Thr Gln Ala Val Asp Tyr Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Thr Asn Leu Val Thr 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 Arg Arg Ile Asp Ala Ala Thr Tyr His Cys Gln Arg Ser

85 90 95

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

100 105 110

Arg Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

His Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 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> 40

<211> 1422

<212> DNA

<213>Artificial sequence

<220>

<223>Anti-CD 20 scFv-Fc fusion protein nucleotide sequences

<400> 40

gacatcgtgc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccaccca ggccgtggac tacgacggcg acgcctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccaccaa cctggtgacc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggagc gccgcatcga cgccgccacc taccactgcc agcgcagcac caaggacccc 300

tggaccttcg gcggcggcac caagctggag atcaagcgcc gcagcggcgg cggcggcagc 360

ggcggcggcg gcagcggcgg cggcggcagc gacgtggtga tgacccagag ccccctgagc 420

ctgcccgtga ccctgcagcc cgccagcatc agctgcaaga gcgcccagag cctgctgcac 480

agcgacggca agacctacct gtactggtac ctgcagaagc ccggccagag ccccaagctg 540

ctgatctacc aggtggccag ccgcttcgcc ggcgtgcccg accgcttcag cggcagcggc 600

accgacttca ccctgaagat cagccgcgtg gaggccgagg acctgggcgt gtacttctgc 660

ttccagggca tccacctgcc ctacaccttc ggccagggca ccaagctgga gatcaaggag 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 ggcaagtgat aa 1422

<210> 41

<211> 688

<212> PRT

<213>Artificial sequence

<220>

<223>Anti- PD-1 heavy chains-CD20 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 Asp Ile Val Leu Thr

435 440 445

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

450 455 460

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

465 470 475 480

Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

485 490 495

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

500 505 510

Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Arg Arg

515 520 525

Ile Asp Ala Ala Thr Tyr His Cys Gln Arg Ser Thr Lys Asp Pro Trp

530 535 540

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

545 550 555 560

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val

565 570 575

Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gln Pro Ala Ser

580 585 590

Ile Ser Cys Lys Ser Ala Gln Ser Leu Leu His Ser Asp Gly Lys Thr

595 600 605

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

610 615 620

Ile Tyr Gln Val Ala Ser Arg Phe Ala Gly Val Pro Asp Arg Phe Ser

625 630 635 640

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

645 650 655

Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly Ile His Leu Pro Tyr Thr

660 665 670

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

675 680 685

<210> 42

<211> 2052

<212> DNA

<213>Artificial sequence

<220>

<223>Anti- PD-1 heavy chains-CD20 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 acatcgtgct gacccagagc cccgccagcc tggccgtgag cctgggccag 1380

cgcgccacca tcagctgcaa ggccacccag gccgtggact acgacggcga cgcctacctg 1440

aactggtacc agcagatccc cggccagccc cccaagctgc tgatctacga cgccaccaac 1500

ctggtgaccg gcatcccccc ccgcttcagc ggcagcggca gcggcaccga cttcaccctg 1560

aacatccacc ccgtggagcg ccgcatcgac gccgccacct accactgcca gcgcagcacc 1620

aaggacccct ggaccttcgg cggcggcacc aagctggaga tcaagcgccg cagcggcggc 1680

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acgtggtgat gacccagagc 1740

cccctgagcc tgcccgtgac cctgcagccc gccagcatca gctgcaagag cgcccagagc 1800

ctgctgcaca gcgacggcaa gacctacctg tactggtacc tgcagaagcc cggccagagc 1860

cccaagctgc tgatctacca ggtggccagc cgcttcgccg gcgtgcccga ccgcttcagc 1920

ggcagcggca ccgacttcac cctgaagatc agccgcgtgg aggccgagga cctgggcgtg 1980

tacttctgct tccagggcat ccacctgccc tacaccttcg gccagggcac caagctggag 2040

atcaagtgat aa 2052

Claims (10)

1. a kind of anti-PD-1 and CD20 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

B. specific recognition and combine CD20 domain, it include anti-CD20 specific antibodies weight chain variable district (resist CD20VH)。

2. anti-PD-1 and CD20 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 CD20 domain are by the light chain for the anti-CD20 specific antibodies being sequentially connected Variable region (anti-CD20VL), weight chain variable district (anti-CD20VH), hinge area and Fc fragments (anti-CD20CH2-CH3) composition, or by Light chain (anti-CD20VL-CL) and heavy chain (anti-CD20VH-CH1- hinge areas-CH2-CH3) group of one group of anti-CD20 specific antibody Into, or by light chain (anti-CD20VL-CL) and heavy chain (the anti-CD20VH-CH1- hinge areas-CH2- of two groups of anti-CD20 specific antibodies CH3) constitute, or light chain variable district (anti-CD20VL) and weight chain variable district (anti-CD20VH) group by anti-CD20 specific antibodies Into.

3. anti-PD-1 and CD20 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 CD20 domain, its by one group of anti-CD20 specific antibody light chain (anti-CD20VL- CL) constituted with heavy chain (anti-CD20VH-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 CD20 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 CD20, it is by the light chain variable for the anti-CD20 specific antibodies being sequentially connected Area (anti-CD20VL), weight chain variable district (anti-CD20VH), hinge area and Fc fragments (anti-CD20CH2-CH3) composition；

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 CD20 domain；Or Person

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 the domain for combining CD20, it is (anti-by the light chain of one group of anti-CD20 specific antibody TCD20VL-CL) constituted with heavy chain (anti-CD20VH-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 CD20 domain；Or Person

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 CD20, it is (anti-by the light chain variable district of two groups of anti-CD20 specific antibodies CD20VL) constituted with weight chain variable district (anti-CD20VH)；

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 CD20 Domain in the weight chain variable districts (anti-CD20VH) of each anti-CD20 specific antibodies connected by chemical bond.

4. anti-PD-1 and CD20 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 CD20 bispecific antibodies according to any one of claim 1 to 3 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 such as SEQ ID NO of light chain variable district: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 CD20 domain NO:13rd, shown in 17 or 25, the amino acid sequence such as SEQ ID NO of light chain variable district: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-CD20VH-CH1- hinge areas-CH2-CH3) of the anti-CD20 specific antibodies It is classified as SEQ ID No:35, nucleotide coding sequence is SEQ ID No:36；The light chain of the anti-CD20 specific antibodies is (anti- CD20VL-CL amino acid sequence) is SEQ ID No:37, nucleotide coding sequence is SEQ ID No:38；By being sequentially connected Anti- CD20 specific antibodies light chain variable district (anti-CD20VL), weight chain variable district (anti-CD20VH), hinge area and Fc fragments The amino acid sequence of the specific recognition of (anti-CD20CH2-CH3) composition and combination CD20 domain is SEQ ID No: 39, nucleotide coding sequence is SEQ ID No:40；By the light chain variable district (anti-CD20VL) and again of anti-CD20 specific antibodies The amino acid sequence of the specific recognition of chain variable region (anti-CD20VH) composition and combination CD20 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 such as SEQ ID NO of light chain variable district: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 CD20 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 CD20 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 CD20 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 CD20 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 CD20 in the medicine for the treatment of tumour (such as NHL).