CN113061192B

CN113061192B - PDL1 fusion protein with high affinity to PD-1 receptor and application thereof as T cell inhibitor

Info

Publication number: CN113061192B
Application number: CN202110390981.5A
Authority: CN
Inventors: 赵晓庆; 朱晓东; 党春桂; 姜河
Original assignee: Best Nest Shanghai Biotechnology Co ltd
Current assignee: Best Nest Shanghai Biotechnology Co ltd
Priority date: 2021-04-12
Filing date: 2021-04-12
Publication date: 2023-08-22
Anticipated expiration: 2041-04-12
Also published as: CN113061192A

Abstract

The invention discloses PDL1 fusion proteins with high affinity to PD-1 receptors and application thereof as T cell inhibitors. The high-affinity PDL1 fusion protein can be used as a novel T cell inhibitor, has good biological activity compared with natural PDL1 molecules, has stronger binding capacity to PD-1 receptors, can inhibit T cell activation more effectively, can be applied to the field of disease treatment characterized by T cell overactivation and immunopathogenic injury, and belongs to the technical field of preparation of anti-autoimmune disease drugs.

Description

PDL1 fusion protein with high affinity to PD-1 receptor and application thereof as T cell inhibitor

Technical Field

The invention belongs to the technical field of fusion protein medicine preparation, and relates to PDL1 fusion proteins with high affinity to PD-1 receptors and application of PDL1 fusion proteins as T cell inhibitors.

Background

The physiological functions of the immune system are immune defenses, immune homeostasis and immune surveillance. The premise of the immune system to achieve the functions is to identify the 'own' and 'non-own' components, generate immune response to the 'non-own' components and remove the immune response; does not respond to the 'own' component and maintains tolerance. Under certain pathological conditions, the organism can respond to the autoantigen, namely, autoimmune; the quality and quantity of autoimmune response are abnormal, the self-tolerance mechanism is destroyed, the self-reactive T cells and the self-reactive B cells can be activated to attack and destroy the self-tissue cells, and the pathological changes and corresponding clinical manifestations of the organism are caused, namely autoimmune diseases (autoimmune disease, AID), such as diseases of Rheumatoid Arthritis (RA), systemic Lupus Erythematosus (SLE), multiple Sclerosis (MS) and the like. For most AIDs, T cell overactivation and immunopathogenic damage due to immune imbalance are their primary pathogenesis. The AID treatment common drugs mainly comprise hormone, immunosuppressant and the like, and have great toxic and side effects after long-term use; in recent years, monoclonal drugs and various JAK inhibitors which take cytokines such as TNF-alpha, IL-6 and the like as targets are new progress of AID treatment, but due to the fact that various factors form a complex network in an AID pathogenesis, long-term and stable curative effects are difficult to achieve aiming at single cytokines or signal molecules as targets. Therefore, we need to change the therapeutic thinking, shift from "point" to "face" and try to find new therapeutic approaches to AID targeting T cell activity modulation.

Programmed death receptor-1 (PD-1) is one of the most important immune checkpoint molecules expressed on the surface of activated T cells, and after being combined with ligand PDL1 molecules, the receptor-1 is intracellular to transduce inhibition signals, so that the receptor-1 can inhibit activation and proliferation of the T cells, and is favorable for maintaining the immune self-stability of the organism, thereby preventing the occurrence of excessive immune injury and AID, and the PD-1/PDL1 pathway is one of the most promising AID treatment targets at present.

The previous research results of the applicant subject group show that if the super-activated T cell PD-1 receptor is taken as a target point, the inhibitory signal of the self-reactive T cells of the organism is properly enhanced through the exogenous PDL1 molecule, the super-activated T cell PD-1 receptor is expected to have good treatment effect on AID, and the super-activated T cell PD-1 receptor can be applied to the field of treating various diseases with the super-activated T cells and immunopathogenic injury as main pathogenesis. However, because the lesions of AID are in a continuous and repeated attack process, and the binding capacity of the natural PDL1 molecules and the PD-1 receptor is limited, the use of the natural PDL1 molecules has the defects of large use amount, low patentability and the like, and is not beneficial to the later conversion application.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a novel PDL1 fusion protein with high affinity to a PD-1 receptor and application thereof as a T cell inhibitor, wherein the PDL1 fusion protein has higher affinity to the PD-1 receptor of the T cell, can inhibit T cell activation more effectively, and is expected to be applied to treatment of diseases characterized by T cell overactivation and immunopathogenic injury.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses PDL1 fusion proteins with high affinity to PD-1 receptors, wherein the amino acid sequence of the PDL1 fusion proteins with high affinity to the PD-1 receptors is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6;

the nucleotide sequence of the high-affinity PDL1 fusion protein is shown as SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 or SEQ ID No. 12.

The invention also discloses application of the PDL1 fusion protein with high affinity to the PD-1 receptor as a T cell inhibitor.

Preferably, the PDL1 fusion protein is formed by connecting an extracellular domain of a PDL1 molecule with high affinity to a T cell PD-1 receptor and an Fc segment of an immunoglobulin Ig molecule.

Further preferably, the PDL1 molecule ectodomain of the PDL1 fusion protein comprises an IgV domain and an IgC domain.

Preferably, the PD-1 receptor, PDL1 molecule and Ig molecule Fc fragments are all of human origin.

Preferably, the Ig molecules used include IgG1, igG2, igG3, igG4, igA, igD, or IgM.

Preferably, the high affinity PDL1 fusion protein comprises a native PDL1 molecule or a molecule of the native PDL1 molecule responsible for one or more amino acid mutations in the specific binding site for the PD-1 receptor;

wherein the nucleotide sequence of the unmutated natural PDL1 molecule is shown as SEQ.ID.NO. 1;

in a molecule in which one or more amino acid mutations have occurred, the mutation positions are selected from one or more amino acid mutation sites corresponding to the following positions: 26. 56, 58, 66, 113, 115, 117, and 120.

Further preferred, the high affinity PDL1 fusion protein thereof is an amino acid sequence comprising the corresponding amino acid sequence in seq id No.1, or having at least 95% sequence similarity with the sequence of seq id No. 1.

Further preferably, the one or more amino acid mutations in the high affinity PDL1 fusion protein are selected from one or more of 26D/E, 56Y/F, 58E/V, 66Q/W, 113R/T, 115M/V, 117S/A and 120G/H, or from conservative amino acid mutations corresponding to the respective sites of one or more of 26D/E, 56Y/F, 58E/V, 66Q/W, 113R/T, 115M/V, 117S/A and 120G/H.

The invention also discloses application of the PDL1 fusion protein with high affinity to the PD-1 receptor in preparing a medicament for inhibiting overactivation of human immune cells.

Preferably, the agent is an agent whose target site of action is expressed on the PD-1 receptor on the surface of the activation T.

Preferably, the drug is one whose target is an immune cell expressing the PD-1 receptor.

Preferably, the agent is an agent that inhibits immune cell overactivation via the PD-1/PDL1 signaling pathway.

Preferably, the indications involved in immune cell overactivation include immunopathogenic injury-mediated diseases, autoimmune diseases and organ/tissue graft rejection diseases.

Preferably, the immunopathogenic injury-mediated disease comprises cerebral malaria, immunopathogenic injury mediated by viral infection.

Preferably, autoimmune diseases include psoriasis, rheumatoid arthritis, juvenile arthritis, multiple sclerosis, systemic lupus erythematosus, asthma, crohn's disease, polymyositis, systemic vasculitis, scleroderma, ulcerative colitis, ankylosing spondylitis, sjogren's syndrome, and the like.

Preferably, the organ/tissue graft rejection disease comprises a patient graft versus host disease or a host versus graft disease caused by transplantation of various organs/tissues such as kidney, heart, lung, liver, spleen, thymus, hematopoietic stem cells, and the like.

Compared with the prior art, the invention has the following beneficial effects:

the PD-1/PDL1 channel is one of the most important immunosuppression signals for regulating the activity of T cells, the invention adopts a site-directed combination mutation mode to the natural PDL1 molecule, improves the binding capacity of the natural PDL1 molecule and a PD-1 receptor, has stronger inhibition capacity to activated T cells, and can furthest improve the targeted treatment effect of the PDL1 fusion protein on inhibiting the activation degree of self-reactive T cells. In the process of designing the high-affinity PDL1 fusion protein, a design method based on protein characterization, homology modeling, molecular mechanics calculation and molecular dynamics simulation is adopted, and compared with the traditional method (such as phage/yeast display technology), the method greatly improves the design accuracy and greatly reduces the workload; single-point mutation and combined mutation are carried out on key amino acid sites in a natural PDL1 molecule, wherein the key amino acid sites are responsible for binding with a PD-1 receptor, and 5 PDL1 fusion proteins with high affinity to the PD-1 receptor are screened out; the eukaryotic protein expression system is adopted for expression so as to ensure that the eukaryotic protein expression system can correctly fold, modify and maintain biological activity, and western blot experiments prove that the high-affinity PDL1 fusion protein can be smoothly expressed; the proliferation experiment of the activated T cells is inhibited, so that the high-affinity PDL1 fusion protein has good inhibition capability on the activated T cells; by fluorescence polarization detection technology, compared with the natural PDL1 molecule, the high-affinity PDL1 fusion protein has obviously improved affinity to PD-1 receptor; the high-affinity PDL1 fusion protein is expected to be applied to AID disease treatment taking abnormal activation of T cells and immunopathogenic injury as main pathogenesis.

Drawings

Fig. 1 is a schematic diagram of the innovative design concept of the present invention: the inhibition signal of the PD-1 of the T cells is enhanced through the high-affinity PDL1 fusion protein, so that the activation degree of the T cells is reduced;

FIG. 2 is a theoretical prediction of 8 key amino acid positions in PDL1 molecule responsible for binding to PD-1, namely D26, Y56, E58, Q66, R113, M115, S117, G120;

FIG. 3 is a schematic diagram of the total gene synthesis of human PDL1-IgG4Fc fusion gene (AB) and PDL1 mutant gene (A1-A5);

FIG. 4 shows the verification of the natural PDL1 gene and PDL1 mutant gene by PCR; wherein a is the PCR verification result of the natural PDL1 and the high-affinity PDL1 mutant genes A1-A5, and lanes 1-6 correspond to the result of amplifying PDL1 fragments by the natural PDL1 and the PDL1 mutant genes A1-A5; b is the PCR result of amplifying PDL1-IgG4Fc and IgG4Fc fragments from the native PDL1 fusion gene, lane 7 is the result of amplifying PDL1-IgG4Fc genes from PCR, and lane 8 is the result of amplifying IgG4Fc genes from PCR;

FIG. 5 shows endonuclease verification of PDL1-IgG4Fc fusion gene and PDL1 mutant gene; wherein a is double-restriction identification of target PDL1 genes, lanes 1-6 correspond to Xhol I+EcoRI double-restriction identification of PDL1 genes and high-affinity PDL1 mutant genes A1-A5; b is a natural PDL1-IgG4Fc fusion gene identified by double digestion, lane 7 corresponds to EcoRI+NotI double digestion to identify the IgG4Fc gene, and lane 8 corresponds to Xhol I+NotI double digestion to identify the PDL1-IgG4Fc gene;

FIG. 6 shows detection of PDL1 fusion proteins and high-affinity PDL1 fusion proteins by Western blotting experiments, wherein AB is a natural PDL1 fusion protein, and A1B-A5B are high-affinity PDL1 fusion proteins corresponding to PDL1 mutant genes A1-A5, respectively;

FIG. 7 is a graph showing the validation of the biological activity of a high affinity PDL1 fusion protein; adding a suboptimal amount ConA (1 mug/ml) into each group to stimulate the activation proliferation of human peripheral blood T cells, and then adding natural PDL1 fusion proteins and high-affinity PDL1 fusion proteins with different concentrations respectively, and detecting the proliferation condition of each group of cells by a CCK-8 method;

FIG. 8 shows the measurement of the affinity of a high affinity PDL1 fusion protein (A2B) to the PD-1 receptor by fluorescence polarization detection technique.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the attached drawing figures:

carrying out high-affinity PDL1 fusion protein design based on protein characterization, homology modeling, molecular mechanics calculation and molecular dynamics simulation, carrying out single-point mutation and combined mutation on key amino acid sites in a natural PDL1 molecule responsible for combining with a PD-1 receptor, screening out 5 PDL1 fusion proteins with the highest potential and high affinity to the PD-1, and carrying out eukaryotic protein expression and western blotting verification; proved by cytology experiments, the inhibition effect of the high-affinity PDL1 fusion protein on activated T cells is more obvious; the fluorescence polarization experiment proves that the affinity of the PDL1 fusion protein with high affinity to the PD-1 receptor is indeed obviously improved, so that a novel potential T cell inhibitor is obtained.

Referring to fig. 1, a schematic diagram of the innovative design concept of the present invention is shown: the inhibition signal of the PD-1 of the T cells is enhanced through the high-affinity PDL1 fusion protein, so that the activation degree of the T cells is reduced; the invention proposes the following assumptions based on the above research results: in the treatment of autoimmune diseases characterized by abnormal activation of T cells and immunological pathological damage, a high-affinity PDL1 fusion protein has stronger affinity to a PD-1 receptor aiming at a PD-1/PDL1 signal pathway, and has stronger inhibition capability to activated T cells.

Compared with natural PDL1 molecules, the fusion protein has stronger affinity to the PD-1 receptor expressed by activated T cells, has better effect of inhibiting T cell activation and is expected to be applied to treatment of diseases mediated by abnormal activation of the T cells.

1. High affinity PDL1 fusion protein design based on protein characterization, homology modeling, molecular mechanics calculation and molecular dynamics simulation

1.1 protein binding energy and post-mutation binding energy Change analysis

The PD-1/PDL1 crystal structure data is downloaded from a PDB database, the binding energy of the PD-1/PDL1 binding domain is analyzed by using Discovery Studio 2016 (DS) software, nonsense mutation is carried out on all amino acids on the PDL1 binding domain, the binding energy change of each site is calculated, 8 key amino acid sites responsible for binding to a PD-1 receptor in PDL1 molecules are obtained through screening, and the results are respectively 26D, 56Y, 58E, 66Q, 113R, 115M, 117S and 120G as shown in figure 2 and are used as design references of the next step.

1.2 structural analysis and energy minimization operations

Structural analysis is carried out on a site with more prominent binding energy change and a site nearby the site in the PD-1/PDL1 structure in the Pymol software, and then the modified structural model is subjected to energy minimization operation by using DS software, so that the calculated structural data are derived.

1.3 calculation of binding energy and mutation site combination

And uploading the changed protein structure data to an EMBL-EBI database to calculate the binding energy of the protein structure data, comparing the binding energy with the binding energy of the original structure, and finally synthesizing the binding energy data of all single-point mutations, and screening and optimizing to obtain the optimal mutation combination. The 5 best PDL1 high affinity mutant combinations, designated A1, A2, A3, A4, A5, respectively, were selected and the final results are shown in table 1.

TABLE 1 high affinity PDL1 fusion protein combinations predicted theoretically

The PDL1 mutant protein obtained by the method cannot guarantee good biological activity and higher affinity to PD-1 receptor, and needs to be subjected to the next verification.

2. High-affinity PDL1-IgG4Fc fusion gene synthesis and verification

2.1 design of PDL1-IgG4Fc fusion Gene and high affinity PDL1 mutant Gene

The basic structure of the PDL1 fusion protein is formed by connecting the extracellular domain of a human PDL1 molecule and the Fc segment (range-CH 2-CH 3) of a human immunoglobulin IgG4 molecule, wherein the PDL1 segment is a functional structural domain thereof and is responsible for combining with a PD-1 receptor; the IgG4Fc segment endows the fusion protein with the advantages of convenient purification, strong stability, long half-life and the like; igG4Fc and IgG1Fc are two fusion protein drug structures most commonly used at present, the binding force between the IgG4Fc and immune cell Fc receptor is low, and the mediation ADCC (anti-independent cell-mediated cytotoxicity) and CDC (complex-dependent cytotoxicity) effects are weak, so that the fusion protein is suitable for the requirements of the subject; meanwhile, the cysteine of the hinge region of the IgG4Fc is mutated into serine so as to prevent disulfide bond from being generated and further reduce side effects caused by the Fc segment.

The NCBI database was queried for human PDL1 sequences (GeneBank: AH 005273.2) and human IgG4Fc sequences (GeneBank: AAB 59394.1), and PDL1-IgG4Fc fusion genes (see SEQ ID NO.1 for nucleotide sequences) and mutant PDL1 genes A1-A5 (see SEQ ID NO. 2-6) were designed and sent to Beijing Aokara Biotechnology Co., ltd for total gene sequence synthesis and inserted into the T vector.

2.2 PCR verification products

The following primers were synthesized:

PDL1 F：5-’AGTCTCGAGAGTATGAGGAT-3’

PDL1 R：5-’ACTGAATTCACTCCTTTCAT-3’

IgG4Fc F：5’-AGTGAATTCAGTGAGTCCAA-3’

IgG4Fc R：5’-ACTGCGGCCGCACTTCATTT-3’

referring to the gene diagram of FIG. 3, the genes are verified by using different primer combinations respectively, and the result is shown in FIG. 4, which is the verification of the natural PDL1 gene and PDL1 mutant gene by PCR; wherein a is the PCR verification result of the natural PDL1 and the high-affinity PDL1 mutant genes A1-A5, and lanes 1-6 correspond to the result of amplifying PDL1 fragments by the natural PDL1 and the PDL1 mutant genes A1-A5; b is the PCR result of amplifying PDL1-IgG4Fc and IgG4Fc fragments by the natural PDL1 fusion gene, lane 7 is the result of amplifying PDL1-IgG4Fc genes by PCR, and lane 8 is the result of amplifying IgG4Fc genes by PCR, which indicates that the synthesized target genes are correct.

2.3 enzyme digestion verification

Referring to the gene diagram of FIG. 3, the plasmids are subjected to double digestion with endonucleases respectively, and the result is shown in FIG. 5, wherein a is a PDL1 gene of interest identified by double digestion, lanes 1-6 correspond to Xhol I+EcoRI, PDL1 genes and high affinity PDL1 mutant genes A1-A5 identified by double digestion; b is a natural PDL1-IgG4Fc fusion gene identified by double digestion, lane 7 corresponds to EcoRI+NotI double digestion to identify the IgG4Fc gene, and lane 8 corresponds to Xhol I+NotI double digestion to identify the PDL1-IgG4Fc gene, which indicates that the synthesized target gene is correct.

3. Eukaryotic system expression of high affinity PDL1 fusion proteins

3.1 expression of the protein of interest

Resuscitating 293A cells, starting the experiment after the growth condition of the cells is good, and starting the transfection step (taking 24-well plates as an example) when the cell fusion degree is 80-90%;

(1) Diluting 0.8 μg of natural PDL1 gene T vector and high affinity PDL1 mutant gene T vector with 50 μl serum-free culture medium, slightly blowing and sucking for 3-5 times, mixing, and standing at room temperature for 5min;

(2) Gently inverting and mixing the transfection reagent, diluting the transfection reagent with 2.0 mu l Lipofectamine TM2000 by 50 mu l of serum-free culture medium, gently blowing and sucking for 3-5 times, mixing, and standing for 5min at room temperature;

(3) Mixing the transfection reagent and plasmid diluent, slightly blowing and sucking for 3-5 times, mixing, and standing at room temperature for 20min;

(4) The transfection complex is added into a 24-hole cell plate, 100 μl/hole, and the front and back gently shake cell plates are uniformly mixed;

(5) The cell plates were exposed to 5% CO at 37 ℃ ₂ Culturing in incubator for about 6 hr, changing liquid, changing into common culture medium containing 10% serum, culturing at 37deg.C and 5% CO ₂ Continuously culturing in an incubator;

(6) After 48-72h, collecting cell culture fluid supernatant, and detecting target gene expression by western blotting;

(7) After the target Protein is expressed in large quantity, the target Protein is purified by using Protein A.

3.2 purification of the protein of interest

(1) Preparing a sample; the culture supernatant was combined with binding buffer 1:1 mixing, filtering (to prevent clogging of the purification column);

(2) Equilibrium purification column: purifying the column with 5-10 times the volume of binding buffer;

(3) Loading: loading the prepared cell culture supernatant, taking the volume of the loading amount into consideration according to the binding capacity of the purification column;

(4) Eluting the hybrid protein: washing the purification column with a binding buffer until the binding buffer is free of protein;

(5) Collecting the target protein: the eluate was passed through a purification column while collecting the filtrate (about 3-4 ml/tube) until the filtrate was protein-free. The protein content in each collection tube was measured and the protein tubes were pooled. (Note: about 150ul of 1M PH9.0 Tris-HCl buffer is added in advance to the collection tube to prevent target protein from being inactivated in the presence of peracid);

(6) Regeneration of the purification column: regenerating the purification column with 5-10 times of the volume of regeneration liquid;

(7) And (5) collecting target proteins by PBS dialysis.

3.3 Western blot verification of expression of target protein

Protein quantification by BCA method (specific steps refer to kit instructions), protein concentration was calculated;

(1) Processing a protein sample: adding 5×loading Buffer, heating at 95-100deg.C for 5min, and preserving at-20deg.C.

(2) Preparation of SDS-PAGE gel

(3) Loading electrophoresis, namely, carrying out electrophoresis for 1.5h at a concentration of 20 mug/hole for a protein sample and 100V;

(4) Transferring film, namely transferring PVDF film for 120min under 400mA constant current condition;

(5) Sealing with TBST solution containing 5% skimmed milk for 1 hr;

(6) Antibody incubation: HRP-labeled mouse anti-human IgG4Fc monoclonal antibody was diluted to working concentration and incubated overnight at 4 ℃;

(7) Washing the film: 1 XTBST is washed for 3 times and 5min each time;

(8) ECL color development, image acquisition.

As shown in FIG. 6, lane 1 is the native PDL1 fusion protein (AB), lanes 2-6 are the high affinity PDL1 fusion proteins (A1B-A5B), and the proteins were successfully expressed.

4. High affinity PDL1-IgG4Fc fusion protein affinity detection

4.1 T cell proliferation model

Isolation of T cells in human PBMC, T cells were isolated at 2X 10 ⁵ Density of individuals/wells was seeded into 96-well plates; stimulating T cell proliferation by using ConA with different concentrations, detecting the T cell proliferation condition by using a CCK-8 method, and establishing a T cell proliferation curve activated by the ConA with different concentrations; determining a sub-optimal dose of ConA stimulation conditions (which stimulate both T cell activation proliferation and not too intense to inhibit its activation state); this part of the experiment was completed and,the suboptimal dose for ConA stimulation was determined to be 1. Mu.g/ml.

4.2 high affinity PDL1 fusion proteins inhibit T cell proliferation-activating Capacity

T cells were plated at 2X 10 ⁵ Inoculating the density of each hole into a 96-well plate, dividing the 96-well plate into an intervention-free group, a natural PDL1 fusion protein intervention group and a high-affinity PDL1 fusion protein A1B-A5B intervention group, setting a protein concentration gradient of 0-30 mug/ml, adding ConA (1 mug/ml) with sub-proper dose concentration, and incubating for 4d; the inhibition effect of natural PDL1 fusion proteins and A1B-A5B with different concentrations on T cell proliferation is detected by a CCK-8 method, the in vitro biological activity of each protein is evaluated, and PDL1 mutants with higher inhibition capacity on activated T cells are initially screened.

As shown in the experimental result in FIG. 7, the high-affinity PDL1 fusion protein has good biological activity, can inhibit the activation proliferation of T cells, and has potential as a novel T cell inhibitor.

5. High affinity PDL1-IgG4Fc fusion protein affinity detection

Fluorescence polarization detection technique (fluorescence polarization immune assay, FPIA): when fluorescent molecules are excited by plane polarized light, if the molecules remain stationary for the period of excitation (for about 4 nanoseconds for fluorescein), the emitted light will lie in the same plane of polarization. If the molecules rotate or flip away from this plane during the excitation period, the emitted light will lie in a different plane of polarization than the excitation light. If the fluorescein is excited with vertically polarized light, the intensity of the emitted light can be detected in both the vertical and horizontal planes of polarization (the degree to which the emitted light deviates from the vertical plane to the horizontal plane is related to the mobility of the fluorescein-labeled molecules). If the molecules are large, the motion that occurs upon excitation is minimal and the degree of polarization of the emitted light is high. If the molecules are small, the molecules rotate or flip at a fast rate, and the emitted light will be depolarized with respect to the excitation light plane. The method comprises the following steps:

(1) The PDL1 fusion protein influences the signal value of the PDL1 fusion protein by combining with the tracer substrate, a 96-hole black flat bottom half-zone plate is selected, and the total reaction volume is 40 mu l;

(2) Determining the lowest concentration of the tracer polypeptide by measuring signal values of the tracer polypeptides with different concentration gradients, titrating the tracer polypeptide with PDL1 fusion proteins with different concentrations, incubating for 20min at room temperature, and detecting fluorescence polarization values;

(3) And (3) fitting the curve through Origin software to obtain affinity Kd values of different FITC-labeled PD-1 and PDL1 fusion proteins, selecting the smallest Kd value as the trace polypeptide of the high-throughput screening system, and determining the lowest concentration as the concentration used by the screening system.

As a result, referring to fig. 8, taking A2B as an example, fluorescence polarization experiments demonstrated that the affinity of the high affinity PDL1 fusion protein (A2B) to the PD-1 receptor was improved by more than 4 times as compared to the native PDL1 fusion protein.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence listing

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Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

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

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Ser Glu

225 230 235 240

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

245 250 255

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Leu Ser Leu Ser Leu Gly Lys

465 470

<210> 3

<211> 471

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Val Val Glu Tyr

20 25 30

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

35 40 45

Asp Leu Ala Ala Leu Ile Val Phe Trp Val Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Thr Cys Met Ile Ala Tyr Gly His Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

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

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Ser Glu

225 230 235 240

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

245 250 255

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Leu Ser Leu Ser Leu Gly Lys

465 470

<210> 4

<211> 471

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

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

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Trp Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Thr Cys Met Ile Ala Tyr Gly His Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

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

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Ser Glu

225 230 235 240

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

245 250 255

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Leu Ser Leu Ser Leu Gly Lys

465 470

<210> 5

<211> 471

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Val Val Glu Tyr

20 25 30

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

35 40 45

Asp Leu Ala Ala Leu Ile Val Phe Trp Val Met Glu Asp Lys Asn Ile

50 55 60

Ile Trp Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Thr Cys Met Ile Ala Tyr Gly His Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

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

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Ser Glu

225 230 235 240

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

245 250 255

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Leu Ser Leu Ser Leu Gly Lys

465 470

<210> 6

<211> 471

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Val Val Glu Tyr

20 25 30

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

35 40 45

Asp Leu Ala Ala Leu Ile Val Phe Trp Val Met Glu Asp Lys Asn Ile

50 55 60

Ile Trp Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Thr Cys Val Ile Ala Tyr Gly His Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

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

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Ser Glu

225 230 235 240

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

245 250 255

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Leu Ser Leu Ser Leu Gly Lys

465 470

<210> 7

<211> 1442

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

agtctcgaga gtatgaggat atttgctgtc tttatattca tgacctactg gcatttgctg 60

aacgcattta ctgtcacggt tcccaaggac ctatatgtgg tagagtatgg tagcaatatg 120

acaattgaat gcaaattccc agtagaaaaa caattagacc tggctgcact aattgtctat 180

tgggaaatgg aggataagaa cattattcaa tttgtgcatg gagaggaaga cctgaaggtt 240

cagcatagta gctacagaca gagggcccgg ctgttgaagg accagctctc cctgggaaat 300

gctgcacttc agatcacaga tgtgaaattg caggatgcag gggtgtaccg ctgcatgatc 360

agctatggtg gtgccgacta caagcgaatt actgtgaaag tcaatgcccc atacaacaaa 420

atcaaccaaa gaattttggt tgtggatcca gtcacctctg aacatgaact gacatgtcag 480

gctgagggct accccaaggc cgaagtcatc tggacaagca gtgaccatca agtcctgagt 540

ggtaagacca ccaccaccaa ttccaagaga gaggagaagc ttttcaatgt gaccagcaca 600

ctgagaatca acacaacaac taatgagatt ttctactgca cttttaggag attagatcct 660

gaggaaaacc atacagctga attggtcatc ccagaactac ctctggcaca tcctccaaat 720

gaaaggagtg aattcagtga gtccaaatat ggtcccccat gcccaccgtg cccagcacct 780

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 840

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 900

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 960

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1080

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1140

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1380

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatgaag tgcggccgca 1440

gt 1442

<210> 8

<211> 1442

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

agtctcgaga gtatgaggat atttgctgtc tttatattca tgacctactg gcatttgctg 60

aacgcattta ctgtcacggt tcccaaggag ctatatgtgg tagagtatgg tagcaatatg 120

acaattgaat gcaaattccc agtagaaaaa caattagacc tggctgcact aattgtcttc 180

tgggttatgg aggataagaa cattatttga tttgtgcatg gagaggaaga cctgaaggtt 240

cagcatagta gctacagaca gagggcccgg ctgttgaagg accagctctc cctgggaaat 300

gctgcacttc agatcacaga tgtgaaattg caggatgcag gggtgtacac atgcatgatc 360

agctatggtc acgccgacta caagcgaatt actgtgaaag tcaatgcccc atacaacaaa 420

atcaaccaaa gaattttggt tgtggatcca gtcacctctg aacatgaact gacatgtcag 480

gctgagggct accccaaggc cgaagtcatc tggacaagca gtgaccatca agtcctgagt 540

ggtaagacca ccaccaccaa ttccaagaga gaggagaagc ttttcaatgt gaccagcaca 600

ctgagaatca acacaacaac taatgagatt ttctactgca cttttaggag attagatcct 660

gaggaaaacc atacagctga attggtcatc ccagaactac ctctggcaca tcctccaaat 720

gaaaggagtg aattcagtga gtccaaatat ggtcccccat gcccaccgtg cccagcacct 780

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 840

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 900

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 960

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1080

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1140

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1380

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatgaag tgcggccgca 1440

gt 1442

<210> 9

<211> 1442

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

agtctcgaga gtatgaggat atttgctgtc tttatattca tgacctactg gcatttgctg 60

aacgcattta ctgtcacggt tcccaaggag ctatatgtgg tagagtatgg tagcaatatg 120

acaattgaat gcaaattccc agtagaaaaa caattagacc tggctgcact aattgtcttc 180

tgggttatgg aggataagaa cattattcaa tttgtgcatg gagaggaaga cctgaaggtt 240

cagcatagta gctacagaca gagggcccgg ctgttgaagg accagctctc cctgggaaat 300

gctgcacttc agatcacaga tgtgaaattg caggatgcag gggtgtacac atgcatgatc 360

gcatatggtc acgccgacta caagcgaatt actgtgaaag tcaatgcccc atacaacaaa 420

atcaaccaaa gaattttggt tgtggatcca gtcacctctg aacatgaact gacatgtcag 480

gctgagggct accccaaggc cgaagtcatc tggacaagca gtgaccatca agtcctgagt 540

ggtaagacca ccaccaccaa ttccaagaga gaggagaagc ttttcaatgt gaccagcaca 600

ctgagaatca acacaacaac taatgagatt ttctactgca cttttaggag attagatcct 660

gaggaaaacc atacagctga attggtcatc ccagaactac ctctggcaca tcctccaaat 720

gaaaggagtg aattcagtga gtccaaatat ggtcccccat gcccaccgtg cccagcacct 780

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 840

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 900

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 960

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1080

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1140

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1380

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatgaag tgcggccgca 1440

gt 1442

<210> 10

<211> 1442

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

agtctcgaga gtatgaggat atttgctgtc tttatattca tgacctactg gcatttgctg 60

aacgcattta ctgtcacggt tcccaaggac ctatatgtgg tagagtatgg tagcaatatg 120

acaattgaat gcaaattccc agtagaaaaa caattagacc tggctgcact aattgtctat 180

tgggaaatgg aggataagaa cattatttga tttgtgcatg gagaggaaga cctgaaggtt 240

cagcatagta gctacagaca gagggcccgg ctgttgaagg accagctctc cctgggaaat 300

gctgcacttc agatcacaga tgtgaaattg caggatgcag gggtgtacac atgcatgatc 360

gcatatggtc acgccgacta caagcgaatt actgtgaaag tcaatgcccc atacaacaaa 420

atcaaccaaa gaattttggt tgtggatcca gtcacctctg aacatgaact gacatgtcag 480

gctgagggct accccaaggc cgaagtcatc tggacaagca gtgaccatca agtcctgagt 540

ggtaagacca ccaccaccaa ttccaagaga gaggagaagc ttttcaatgt gaccagcaca 600

ctgagaatca acacaacaac taatgagatt ttctactgca cttttaggag attagatcct 660

gaggaaaacc atacagctga attggtcatc ccagaactac ctctggcaca tcctccaaat 720

gaaaggagtg aattcagtga gtccaaatat ggtcccccat gcccaccgtg cccagcacct 780

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 840

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 900

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 960

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1080

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1140

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1380

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatgaag tgcggccgca 1440

gt 1442

<210> 11

<211> 1442

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

agtctcgaga gtatgaggat atttgctgtc tttatattca tgacctactg gcatttgctg 60

aacgcattta ctgtcacggt tcccaaggag ctatatgtgg tagagtatgg tagcaatatg 120

acaattgaat gcaaattccc agtagaaaaa caattagacc tggctgcact aattgtcttc 180

tgggttatgg aggataagaa cattatttga tttgtgcatg gagaggaaga cctgaaggtt 240

cagcatagta gctacagaca gagggcccgg ctgttgaagg accagctctc cctgggaaat 300

gctgcacttc agatcacaga tgtgaaattg caggatgcag gggtgtacac atgcatgatc 360

gcatatggtc acgccgacta caagcgaatt actgtgaaag tcaatgcccc atacaacaaa 420

atcaaccaaa gaattttggt tgtggatcca gtcacctctg aacatgaact gacatgtcag 480

gctgagggct accccaaggc cgaagtcatc tggacaagca gtgaccatca agtcctgagt 540

ggtaagacca ccaccaccaa ttccaagaga gaggagaagc ttttcaatgt gaccagcaca 600

ctgagaatca acacaacaac taatgagatt ttctactgca cttttaggag attagatcct 660

gaggaaaacc atacagctga attggtcatc ccagaactac ctctggcaca tcctccaaat 720

gaaaggagtg aattcagtga gtccaaatat ggtcccccat gcccaccgtg cccagcacct 780

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 840

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 900

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 960

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1080

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1140

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1380

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatgaag tgcggccgca 1440

gt 1442

<210> 12

<211> 1442

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

agtctcgaga gtatgaggat atttgctgtc tttatattca tgacctactg gcatttgctg 60

aacgcattta ctgtcacggt tcccaaggag ctatatgtgg tagagtatgg tagcaatatg 120

acaattgaat gcaaattccc agtagaaaaa caattagacc tggctgcact aattgtcttc 180

tgggttatgg aggataagaa cattatttga tttgtgcatg gagaggaaga cctgaaggtt 240

cagcatagta gctacagaca gagggcccgg ctgttgaagg accagctctc cctgggaaat 300

gctgcacttc agatcacaga tgtgaaattg caggatgcag gggtgtacac atgcgttatc 360

gcatatggtc acgccgacta caagcgaatt actgtgaaag tcaatgcccc atacaacaaa 420

atcaaccaaa gaattttggt tgtggatcca gtcacctctg aacatgaact gacatgtcag 480

gctgagggct accccaaggc cgaagtcatc tggacaagca gtgaccatca agtcctgagt 540

ggtaagacca ccaccaccaa ttccaagaga gaggagaagc ttttcaatgt gaccagcaca 600

ctgagaatca acacaacaac taatgagatt ttctactgca cttttaggag attagatcct 660

gaggaaaacc atacagctga attggtcatc ccagaactac ctctggcaca tcctccaaat 720

gaaaggagtg aattcagtga gtccaaatat ggtcccccat gcccaccgtg cccagcacct 780

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg 840

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag 900

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg 960

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc 1080

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc 1140

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg 1320

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg 1380

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaatgaag tgcggccgca 1440

gt 1442

Claims

1. The PDL1 fusion protein with high affinity to PD-1 receptor is characterized in that the amino acid sequence of the PDL1 fusion protein is shown as SEQ.ID.NO.3 or SEQ.ID.NO. 6.

2. Use of a PDL1 fusion protein having a high affinity for PD-1 receptor according to claim 1, for the manufacture of a medicament for an indication related to immune cell overactivation, wherein said indication related to immune cell overactivation is an immunopathogenic injury-mediated disease, an autoimmune disease and an organ/tissue transplant rejection disease; wherein:

the disease mediated by the immunopathogenic injury is cerebral malaria and immunopathogenic injury mediated by virus infection;

the autoimmune diseases are psoriasis, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, systemic lupus erythematosus, asthma, crohn's disease, polymyositis, systemic vasculitis, scleroderma, ulcerative colitis, ankylosing spondylitis and Sjogren's syndrome;

the organ/tissue graft rejection disease is a patient graft versus host disease or a host versus graft disease caused by kidney, heart, lung, liver, spleen, thymus, hematopoietic stem cell transplantation.