CN113061192A

CN113061192A - PDL1 fusion protein with high affinity to PD-1 receptor and application thereof as T cell inhibitor

Info

Publication number: CN113061192A
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: 2021-07-02
Anticipated expiration: 2041-04-12
Also published as: CN113061192B

Abstract

The invention discloses PDL1 fusion proteins with high affinity to PD-1 receptors and application thereof as a T cell inhibitor. The high-affinity PDL1 fusion protein can be used as a novel T cell inhibitor, compared with a natural PDL1 molecule, the high-affinity PDL1 fusion protein has good biological activity, has stronger binding capacity to a PD-1 receptor, can effectively inhibit T cell activation, can be applied to the field of disease treatment characterized by T cell over-activation and immunopathological damage, and belongs to the technical field of preparation of anti-autoimmune 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 preparation of fusion protein medicines, and relates to PDL1 fusion proteins with high affinity to PD-1 receptors and application thereof as a T cell inhibitor.

Background

The physiological functions of the immune system are immune defense, immune homeostasis and immune surveillance. The premise of the immune system realizing the functions is to recognize the self component and the non-self component, generate immune response to the non-self component and eliminate the non-self component; does not respond to the 'self' component and maintains tolerance. Under certain pathological conditions, the organism can respond to autoantigens, namely autoimmunity; the quality and quantity of autoimmune response are abnormal, and self-tolerance mechanism is destroyed, self-reactive T cells and self-reactive B cells can be activated, attack and destroy self tissue cells, so that the organism has pathological changes and corresponding clinical manifestations, namely autoimmune diseases (AID), such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS) and other diseases. For most AIDs, T cell over-activation and immunopathological damage due to immune imbalance are their major pathogenesis. The common drugs for AID treatment are mainly hormones, immunosuppressants and the like, and have large toxic and side effects after long-term use; in recent years, monoclonal antibody medicines and various JAK inhibitors which take TNF-alpha, IL-6 and other cytokines as targets are new advances of AID treatment, but because various factors in the pathogenesis of AID form a complex network, long-term and stable curative effect is difficult to achieve by taking a single cytokine or signal molecule as a target. Therefore, we need to change the therapeutic thinking, switch from "point" to "face", in an attempt to find new methods of AID therapy that target modulation of T cell activity.

Programmed death receptor-1 (PD-1) is one of the most important immune check point molecules expressed on the surface of activated T cells, and after being combined with a ligand PDL1 molecule, an inhibitory signal is conducted in cells, so that the activation and proliferation of the T cells can be inhibited, the body immunity self-stabilization can be favorably maintained, the excessive immune injury and the occurrence of AID can be prevented, and the PD-1/PDL1 pathway is one of the most promising AID treatment targets at present.

The previous research results of the subject groups of the applicant show that if the over-activated T cell PD-1 receptor is taken as a target, the inhibitory signal of the autoreactive T cells of the organism is properly enhanced through the exogenous PDL1 molecule, the compound is expected to have good treatment effect on AID, and can be even applied to the field of treatment of various diseases taking over the over-activation of the T cells and immunopathological damage as main pathogenesis. However, since AID lesions are in a continuous and repeated outbreak process, and natural PDL1 molecules have limited binding capacity with PD-1 receptors, the use of natural PDL1 molecules has the disadvantages of large use amount, low patent drug property and the like, and is not favorable for later transformation application.

Disclosure of Invention

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

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses PDL1 fusion proteins with high affinity for PD-1 receptors, wherein the amino acid sequence of PDL1 fusion proteins with high affinity for 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 PDL1 fusion protein with high affinity 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 a fusion protein formed by connecting an extracellular domain of a PDL1 molecule and an Fc segment of an immunoglobulin Ig molecule, wherein the extracellular domain has high affinity for a T cell PD-1 receptor.

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

Preferably, the PD-1 receptor, the PDL1 molecule and the Fc fragment of the Ig molecule are all derived from human.

Preferably, the Ig molecule used comprises IgG1, IgG2, IgG3, IgG4, IgA, IgD or IgM.

Preferably, the high affinity PDL1 fusion protein comprises a molecule of native PDL1 molecule or 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 native PDL1 molecule is shown in SEQ ID No. 1;

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

Further preferably, the high affinity PDL1 fusion protein thereof is an amino acid sequence comprising the corresponding amino acid sequence of seq.id.no.1, or an amino acid sequence having at least 95% sequence similarity to 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 conservative amino acid mutations corresponding to corresponding positions selected from 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 over-activation of human immune cells.

Preferably, the medicament is a medicament with an action target expressed in a PD-1 receptor on the surface of activated T.

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

Preferably, the drug is a drug that inhibits the over-activation of immune cells via the PD-1/PDL1 signaling pathway.

Preferably, the indications involved in the over-activation of immune cells include diseases mediated by immunopathological damage, autoimmune diseases and organ/tissue transplant rejection diseases.

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

Preferably, the autoimmune disease includes 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 transplant rejection disease includes graft-versus-host disease or host-versus-graft disease of patients 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 pathway is one of the most important immune suppression signals for regulating the activity of T cells, the invention adopts a site-directed combination mutation mode to natural PDL1 molecules to improve the binding capacity of the natural PDL1 molecules and PD-1 receptors, has stronger suppression capacity to activated T cells, and can furthest improve the targeted treatment effect of PDL1 fusion protein on inhibiting the activation degree of autoreactive T cells. In the process of designing the high-affinity PDL1 fusion protein, a design method based on protein characterization, homologous modeling, molecular mechanics calculation and molecular dynamics simulation is adopted, and compared with the traditional method (such as a phage/yeast display technology), the method greatly improves the design accuracy and greatly reduces the workload; carrying out single-point mutation and combined mutation on key amino acid sites which are responsible for being combined with a PD-1 receptor in a natural PDL1 molecule, and screening 5 most potential PDL1 fusion proteins with high affinity to the PD-1 receptor; an eukaryotic protein expression system is adopted for expression so as to ensure that the eukaryotic protein can be correctly folded, modified and maintained with biological activity, and a western blot experiment proves that the high-affinity PDL1 fusion protein can be smoothly expressed; the experiments of inhibiting the proliferation of the activated T cells prove that the high-affinity PDL1 fusion protein has good inhibition capacity on the activated T cells; the fluorescence polarization detection technology proves that compared with the natural PDL1 molecule, the affinity of the high-affinity PDL1 fusion protein to the PD-1 receptor is obviously improved; the high-affinity PDL1 fusion protein is expected to be applied to AID disease treatment with abnormal activation of T cells and immunopathological injury as main pathogenesis.

Drawings

FIG. 1 is a schematic diagram of the innovative design concept of the present invention: strengthening a T cell PD-1 inhibition signal through the high-affinity PDL1 fusion protein, and reducing the activation degree of the T cell;

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

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

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

FIG. 5 shows endonuclease validation of PDL1-IgG4Fc fusion gene and PDL1 mutant gene; wherein a is PDL1 gene for double enzyme digestion identification, lanes 1-6 correspond to Xhol I + EcoR I double enzyme digestion identification PDL1 gene and high-affinity PDL1 mutant gene A1-A5; b is a natural PDL1-IgG4Fc fusion gene identified by double enzyme digestion, wherein a Lane 7 corresponds to EcoR I + Not I double enzyme digestion to identify an IgG4Fc gene, and a Lane 8 corresponds to Xhol I + Not I double enzyme digestion to identify a PDL1-IgG4Fc gene;

FIG. 6 shows Western blot analysis to detect PDL1 fusion protein and high affinity PDL1 fusion protein, wherein AB is native PDL1 fusion protein, and A1B-A5B are PDL1 mutant gene A1-A5 corresponding high affinity PDL1 fusion protein;

FIG. 7 shows the confirmation of the biological activity of the high affinity PDL1 fusion protein; adding a suboptimal dose of ConA (1 mu g/ml) to each group to stimulate the T cells of human peripheral blood to activate and proliferate, then respectively adding natural PDL1 fusion protein and high-affinity PDL1 fusion protein with different concentrations, and detecting the proliferation condition of each group of cells by a CCK-8 method;

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

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or 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 accompanying drawings:

the method comprises the following steps of designing high-affinity PDL1 fusion protein based on protein characterization, homologous modeling, molecular mechanics calculation and molecular dynamics simulation, carrying out single-point mutation and combined mutation on key amino acid sites which are responsible for being combined with PD-1 receptors in natural PDL1 molecules, screening 5 most potential PDL1 fusion proteins with high affinity for PD-1, and carrying out eukaryotic protein expression and western blot verification; cytology experiments prove that the high-affinity PDL1 fusion protein has more obvious inhibition effect on activated T cells; fluorescence polarization experiments prove that the affinity of the fusion protein to the high-affinity PDL1 to the PD-1 receptor is obviously improved, so that a potential novel T cell inhibitor is obtained.

Referring to fig. 1, a schematic diagram of the innovative design concept of the present invention is shown: strengthening a T cell PD-1 inhibition signal through the high-affinity PDL1 fusion protein, and reducing the activation degree of the T cell; the present 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 immunopathological damage, aiming at a PD-1/PDL1 signal pathway, the high-affinity PDL1 fusion protein has stronger affinity to a PD-1 receptor and stronger inhibition capability on activated T cells, and the fusion protein serving as a novel T cell inhibitor is expected to be applied to the treatment of the autoimmune diseases mainly characterized by abnormal activation of the T cells.

The invention constructs a novel T cell inhibitor PDL1 fusion protein with high affinity to PD-1 receptor, compared with natural PDL1 molecule, the fusion protein has stronger affinity to PD-1 receptor expressed by activated T cell, has better effect on inhibiting T cell activation, and is expected to be applied to the treatment of diseases mediated by abnormal activation of T cell.

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

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

The PD-1/PDL1 crystal structure data is downloaded from a PDB database, the binding energy of a PD-1/PDL1 binding domain is analyzed by utilizing 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 in PDL1 molecules which are responsible for binding with PD-1 receptors are obtained through screening, and the results are respectively 26D, 56Y, 58E, 66Q, 113R, 115M, 117S and 120G which are used as design references in the next step and are shown in FIG. 2.

1.2 structural analysis and energy minimization operation

And performing structural analysis on the more prominent binding energy change site and the nearby site in the structure of PD-1/PDL1 in Pymol software, and performing energy minimization operation on the modified structural model by using DS software to derive the structural data after operation.

1.3 binding energy calculation and combination of mutation sites

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

Table 1 theoretical prediction of high affinity PDL1 fusion protein combinations

The PDL1 mutant protein obtained by the method cannot ensure that the protein has good biological activity and higher affinity to a PD-1 receptor, and needs to be verified in the next step.

2. Synthesis and verification of high-affinity PDL1-IgG4Fc fusion gene

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

The PDL1 fusion protein is basically formed by connecting a human PDL1 molecular ectodomain and a human immunoglobulin IgG4 molecular Fc segment (hinge-CH2-CH3), wherein a PDL1 segment is a functional domain and is responsible for binding with a PD-1 receptor; the IgG4Fc segment endows the fusion protein with the advantages of convenient purification, strong stability, long half-life period and the like; IgG4Fc and IgG1Fc are two most commonly used fusion protein drug structures at present, the former has low binding force with an Fc receptor of an immune cell, and the two fusion protein drug structures have weak effects of mediating ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complementary-dependent cytotoxicity), and are suitable for the requirements of the subject; and cysteine in the hinge region of IgG4Fc is mutated into serine so as to prevent generation of disulfide bond and further reduce side effect caused by Fc segment.

An NCBI database is queried, a human PDL1 sequence (GeneBank: AH005273.2) and a human IgG4Fc sequence (GeneBank: AAB59394.1) are searched, a PDL1-IgG4Fc fusion gene (nucleotide sequence is shown in SEQ ID NO.1) and a mutant PDL1 gene A1-A5 (nucleotide sequence is shown in SEQ ID NO.2-6) are designed, and the gene sequences are sent to Beijing Okoku Biotechnology Limited liability company for whole gene sequence synthesis and inserted into a T vector.

2.2 PCR validation of the product

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

2.3 enzyme digestion verification

Referring to the gene schematic diagram of FIG. 3, the plasmid is subjected to double digestion with endonuclease, and the results are shown in FIG. 5, where a is PDL1 gene for double digestion identification, lanes 1-6 correspond to Xhol I + EcoR I, and PDL1 gene and high-affinity PDL1 mutant gene A1-A5 are identified; b is the natural PDL1-IgG4Fc fusion gene identified by double enzyme digestion, lane 7 corresponds to EcoR I + Not I and identifies IgG4Fc gene, lane 8 corresponds to Xhol I + Not I and identifies PDL1-IgG4Fc gene, which indicates that the synthesized target gene is correct.

3. Eukaryotic system expression of high affinity PDL1 fusion protein

3.1 expression of the protein of interest

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

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

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

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

(4) adding the transfection compound into a 24-hole cell plate, wherein each hole is 100 mu l, and slightly shaking the cell plate back and forth to mix the transfection compound uniformly;

(5) the cell plates were incubated at 37 ℃ with 5% CO₂Culturing in incubator for about 6 hr, changing culture medium to 10% serum, and culturing at 37 deg.C under 5% CO₂Continuously culturing in the incubator;

(6) collecting cell culture solution supernatant after 48-72h, and detecting target gene expression by a western blot method;

(7) after the target Protein is expressed in a large amount, the Protein of interest is purified using Protein A.

3.2 purification of the protein of interest

(1) Preparing a sample; culture supernatants were mixed with binding buffer 1: 1 mixing, filtering (preventing from blocking the purification column);

(2) and (3) balancing and purifying the column: passing the Protein A purification column with 5-10 volumes of binding buffer;

(3) loading: loading the prepared cell culture supernatant, and considering the volume of the loading amount according to the binding capacity of the purification column;

(4) and (3) eluting the hybrid protein: washing the purification column with binding buffer until the binding solution does not contain protein;

(5) collecting the target protein: the eluent was passed through a purification column while the permeate (about 3-4 ml/tube) was collected until it was free of protein. And (4) determining the protein content in each collection tube, and combining the protein tubes. (Note: about 150ul of 1M Tris-HCl buffer pH9.0 was added to the collection tube to prevent inactivation of the target protein in a peracid environment);

(6) regeneration of a purification column: regenerating the purifying column by using 5-10 times of volume of regeneration liquid;

(7) the target protein was collected by PBS dialysis.

3.3 Western blot to verify the expression of the target protein

Protein quantification by the BCA method (the specific steps refer to the kit instruction), and protein concentration is calculated;

(1) treating the protein sample: adding 5 Xloading Buffer, heating at 95-100 deg.C for 5min, and storing at-20 deg.C.

(2) Preparation of SDS-PAGE gel

(3) Carrying out sample loading electrophoresis, wherein the protein sample is 20 mu g/hole and is subjected to electrophoresis for 1.5h at 100V;

(4) transferring the film, performing electric transfer for 120min under the condition of 400mA constant current;

(5) blocking with TBST solution containing 5% skimmed milk for 1 h;

(6) antibody incubation: diluting an HRP-labeled mouse anti-human IgG4Fc monoclonal antibody to a working concentration, and incubating overnight at 4 ℃;

(7) washing the membrane: washing the membrane with 1 × TBST for 3 times, 5min each time;

(8) ECL color development and image acquisition.

As shown in FIG. 6, lane 1 shows native PDL1 fusion protein (AB), and lanes 2 to 6 show high-affinity PDL1 fusion protein (A1B-A5B), which were successfully expressed.

4. High affinity PDL1-IgG4Fc fusion protein affinity detection

4.1T cell proliferation model

Isolating T cells from human PBMC, and selecting the T cells at 2X 10⁵Seed/well density into 96-well plates; stimulating the proliferation of T cells by ConA with different concentrations, detecting the proliferation condition of the T cells by a CCK-8 method, and establishing T cell proliferation curves activated by the ConA with different concentrations; determining the ConA stimulation condition of suboptimal dose (which can stimulate T cells to activate and proliferate and can not stimulate the T cells too strongly to inhibit the activation state); this part of the experiment was completed and a suboptimal dose of 1. mu.g/ml for ConA stimulation was determined.

4.2 high affinity PDL1 fusion proteins inhibit the ability of T cells to activate proliferation

The T cells were arranged at 2X 10⁵Inoculating the protein/well density to a 96-well plate, dividing the protein/well density into a non-intervention 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 mu g/ml, adding ConA (1 mu g/ml) with a suboptimal dose concentration, and incubating for 4 d; through a CCK-8 method, the inhibitory action of natural PDL1 fusion proteins with different concentrations and A1B-A5B on T cell proliferation is detected, the in vitro biological activity of each protein is evaluated, and a PDL1 mutant with higher inhibitory capacity on activated T cells is preliminarily screened.

The experimental result is shown in fig. 7, and the high-affinity PDL1 fusion protein has good biological activity, can inhibit T cell activation proliferation, and has potential to be used as a novel T cell inhibitor.

5. High affinity PDL1-IgG4Fc fusion protein affinity detection

Fluorescence polarization assay (FPIA): when a fluorescent molecule is excited by plane-polarized light, the emitted light will lie in the same plane of polarization if the molecule remains stationary for the excited period (about 4 nanoseconds for fluorescein). If the molecules rotate or flip out of this plane during the excitation period, the emitted light will be 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 is deflected 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 occurring 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 quickly, and the emitted light will be depolarized relative to the plane of the excitation light. The method comprises the following steps:

(1) FITC marked PD-1 molecules are tracer substrates, PDL1 fusion protein influences the signal value of the tracer substrates through combination with the tracer substrates, a 96-hole black flat-bottom half-area plate is selected, and the total reaction volume is 40 mu l;

(2) determining the minimum concentration of tracer polypeptide by measuring signal values of tracer polypeptide with different concentration gradients, titrating the tracer polypeptide with different concentrations by PDL1 fusion protein, incubating for 20min at room temperature, and detecting a fluorescence polarization value;

(3) obtaining Kd values of the affinity of different FITC marked PD-1 and PDL1 fusion proteins by Origin software fitting, selecting the highest Kd value as the tracer polypeptide of the high-throughput screening system, and using the determined lowest concentration as the concentration used by the screening system.

Results referring to fig. 8, fluorescence polarization experiments using A2B as an example confirmed that the affinity of the high affinity PDL1 fusion protein (A2B) for the PD-1 receptor was improved by more than 4-fold compared to the native PDL1 fusion protein.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of 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. PDL1 fusion protein with high affinity to PD-1 receptor,

the amino acid sequence of the PDL1 fusion protein with high affinity to PD-1 receptor 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;

2. Use of the PDL1 fusion protein of claim 1 having high affinity for the PD-1 receptor as a T-cell inhibitor.

3. The use of claim 2, wherein said PDL1 fusion protein is a fusion protein comprising a PDL1 molecule extracellular domain having high affinity for the T-cell PD-1 receptor linked to an Fc fragment of an immunoglobulin Ig molecule.

4. The use of claim 3, wherein the extracellular domain of the PDL1 molecule of the PDL1 fusion protein comprises an IgV domain and an IgC domain.

5. The use of claim 3, wherein the PD-1 receptor, the PDL1 molecule and the Ig molecule Fc fragment are all of human origin.

6. The use as claimed in claim 3 wherein the Ig molecule used comprises IgG1, IgG2, IgG3, IgG4, IgA, IgD or IgM.

7. The use according to claim 3, wherein the high affinity PDL1 fusion protein comprises a native PDL1 molecule or a molecule of native PDL1 molecule responsible for one or more amino acid mutations in the specific binding site for the PD-1 receptor;

8. The use according to claim 7, wherein the high affinity PDL1 fusion protein is an amino acid sequence comprising the corresponding amino acid sequence of SEQ ID No.1 or an amino acid sequence having at least 95% sequence similarity to the sequence of SEQ ID No. 1.

9. The use of claim 7, wherein the one or more amino acid mutations in the high affinity PDL1 fusion protein is selected from one or more of 26D/E, 56Y/F, 58E/V, 66Q/W, 113R/T, 115M/V, 117S/a, and 120G/H, or a conservative amino acid substitution corresponding to a corresponding site selected from one or more of 26D/E, 56Y/F, 58E/V, 66Q/W, 113R/T, 115M/V, 117S/a, and 120G/H.

10. Use of the PDL1 fusion protein according to claim 1, which has a high affinity for the PD-1 receptor, for the preparation of a medicament for inhibiting the overactivation of human immune cells.

11. The use according to claim 10, wherein the medicament is a medicament whose target of action is expressed in the PD-1 receptor on the surface of activated T.

12. The use of claim 10, wherein the medicament is a medicament whose target of action is an immune cell expressing the PD-1 receptor.

13. The use of claim 10, wherein the medicament is a medicament that inhibits over-activation of immune cells via the PD-1/PDL1 signaling pathway.

14. The use of any one of claims 10 to 13, wherein the indications for which over-activation of immune cells is implicated include diseases mediated by immunopathological impairment, autoimmune diseases and organ/tissue transplant rejection.

15. The use of claim 14, wherein the immunopathological injury-mediated disease comprises cerebral malaria, viral infection-mediated immunopathological injury.

16. The use of claim 14, wherein the autoimmune disease comprises 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.

17. The use according to claim 14, wherein the organ/tissue transplant rejection disease comprises graft-versus-host disease or host-versus-graft disease of patients resulting from organ/tissue transplantation of various organs/tissues such as kidney, heart, lung, liver, spleen, thymus, hematopoietic stem cells, and the like.