PD-1 variants with increased affinity for pd-l1

Abstract

The present application relates to PD-1 variants with minimal mutations for enhancing the binding force with PD-L1, the PD-1 variants of the present application have significantly increased binding force with PD-L1 while having fewer mutations than the previous PD-1 and PD-1 variants, can solve the immunogenicity problem, since these variants are much smaller proteins than the previous antibody therapeutics, can effectively inhibit the PD-1 / PD-L1 binding of tumor and immune cells in the tumor microenvironment, solve the problem of low binding force of PD-L1 to PD-1 in the past, so that the therapeutic effect of the therapeutic agent can be significantly improved, and can also be used as an imaging agent for detecting the expression amount of PD-L1.

Description

TECHNICAL FIELD

[0001] The present application relates to a PD-1 variant having improved affinity to PD-L1. BACKGROUND

[0002] Pharmaceuticals for treating cancer are broadly classified into low molecular weight pharmaceuticals and high molecular weight pharmaceuticals, and high molecular weight pharmaceuticals, which have specificity, are receiving attention as therapeutic agents compared to low molecular weight pharmaceuticals, which have no specificity and have relatively large side effects. Cancer cells express immune checkpoint proteins on the cell surface in order to avoid the killing mechanism by immune cells, and recently, research on immune checkpoint inhibitory proteins is actively being conducted in order to use them as a method for treating cancer.

[0003] Among immune checkpoint inhibitors, the blockade of PD-1 / PD-L1 binding has shown great efficacy in cancer treatment, with academic reports of lower side effects than other immune checkpoint inhibitors (J. Naidoo et al. (2015) Annals of Oncology, Lucia Gelao et al. (2014) Toxins, Gorge K. Philips et al (2015) International Immunology). The PD-1 receptor is expressed on the surface of activated immune cell types, including T cells, B cells, natural killer (NK) cells / natural killer T (NKT) cells, and the like (Goodman, Patel & Kurzrock, PD-1-PD-L1 immune-checkpoint blockade in B-cell lymphomas, Nature Reviews Clinical Oncology, 14:203-220, 2017.). PD-1 is a negative regulator of T cell activity, and on the surface of tumors, the interaction of PD-1 with PD-L1 as one of its ligands exhibits immune checkpoint blockade that reduces the ability of activated T cells to mount an effective immune response. High levels of PD-L1 expression on the surface of tumor cells suppresses T cell function, including cytotoxic activity, enabling them to escape from anti-tumor responses. PD-L1 is overexpressed in many cancers, associated with a variety of poor prognoses (Okazaki T et al., Intern. Immun. 2007 19(7):813; Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, in contrast to T lymphocytes within normal tissues and T lymphocytes in peripheral blood, the majority of tumor-infiltrating T lymphocytes dominantly express PD-1, suggesting that upregulation of PD-1 on tumor-reactive T cells can favor impaired anti-tumor immune responses (Blood 2009 114(8):1537). This can result from the use of PD-L1 signaling by PD-L1 -expressing tumor cells that cause attenuation of T cell activation through interaction with PD-1 -expressing T cells and evasion of immune surveillance (Sharpe et al., Nat Rev 2002, Keir ME et al., 2008 Annu. Rev. Immunol. 26:677). Thus, inhibition of the PD-L1 / PD-1 interaction can enhance CD8+ T cell-mediated apoptosis of tumors.Thus inhibition of PD-L1 signaling is proposed to improve protocols for cancer treatment (e.g., tumor immunity) and T cell immunity of hepatitis including both acute and chronic (e.g., persistent) hepatitis. Optimal therapeutic treatment can be a combination of blockade of PD-1 receptor / ligand interaction with a substance that directly inhibits tumor growth. Optimal therapeutic methods for treatment, stabilization, prevention, and / or delay of onset of various cancers remain to be developed.

[0004] Therapeutic antibodies targeting PD-1 or PD-L1 block the ligand-receptor interaction, restoring immune function in the tumor microenvironment. The use of such monoclonal antibodies (mAbs) has shown interesting clinical responses in many cancer types, with a growing number of monoclonal antibodies entering clinical development. There are several therapeutic monoclonal antibodies (mAbs) targeting PD-1 and PD-L1 on the market, and more than 12 conventional and bispecific mAbs are under investigation by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Anti-PD1 antibody therapeutics such as BMS's Opdivo (nivolumab), Merck's Keytruda (Pembrolizumab), Regeneron's Libtayo (Cemiplimab), and Anti-PD-L1 antibody therapeutics such as Roche's Tecentriq (Atezolizumab), AstraZeneca's Imfinzi (Durvalumab), and Merck Sereno's Bavencio (Avelumab) have recently been approved by the U.S. Food and Drug Administration (US FDA) and have revolutionized the treatment of refractory cancers in the clinic. The demand for such PD-1 / PD-L1 interaction inhibiting antibody therapeutics in the clinic is growing exponentially, with Opdivo sales of 7.5 billion US$ and Keytruda sales of 7.1 billion US$ in 2018, ranking 4th and 6th, respectively, in the ranking of prescription drugs based on sales. The clinical need for such immune checkpoint inhibiting antibody therapeutics is expected to further expand in the future.

[0005] However, antibodies are large molecular proteins having a molecular weight of 150,000, and thus, it is difficult for the antibodies to enter the inside of cancer tissues, and there is a disadvantage in that the antibodies cannot inhibit the binding of PD-1 / PD-L1 between tumor cells and immune cells in a tumor micro-environment. In order to more effectively treat cancer, the necessity of developing a protein therapeutic agent that is much smaller in size than antibodies and easily enters cancer tissues is increasing.

[0006] However, a PD-1 protein exposed to the ectodomain of a human T cell has a small size and a property of being able to bind to a PD-L1 molecule expressed in a tumor, and in order to treat cancer through effective immune checkpoint inhibition, PD-1, which is smaller in size than PD-L1 and has excellent cell permeability, is more suitable, but wild-type PD-1 has a problem in that it binds to PD-L1 with very low affinity (equilibrium dissociation constant = -8.7 µM). SUMMARY

[0007] TECHNICAL PROBLEM

[0008] The present application aims to provide a PD-1 variant that enhances the binding force to PD-L1.

[0009] Also, the present application aims to provide a PD-L1 and PD-1 binding inhibitor.

[0010] Also, the present application aims to provide a composition for detecting PD-L1.

[0011] Also, the present application aims to provide a pharmaceutical composition for treating or preventing cancer.

[0012] Also, the present application aims to provide a composition for diagnosing cancer.

[0013] Also, the present application aims to provide a specific detection method of PD-L1.

[0014] Also, the present application aims to provide a production method of a PD-1 variant that enhances the binding force to PD-L1.

[0015] TECHNICAL SOLUTION

[0016] To achieve the above object, the present application provides a PD-1 variant that enhances the binding force to PD-L1.

[0017] Also, the present application provides a PD-L1 and PD-1 binding inhibitor comprising the above-described PD-1 variant.

[0018] Also, the present application provides a composition for detecting PD-L1 comprising the above-described PD-1 variant.

[0019] Also, the present application provides a pharmaceutical composition for treating or preventing cancer comprising the above-mentioned PD-1 variant.

[0020] Also, the present application provides a composition for diagnosing cancer comprising the above-mentioned PD-1 variant.

[0021] Also, the present application provides a method for specifically detecting PD-L1.

[0022] Meanwhile, the present application provides a method for producing a PD-1 variant having enhanced binding force with PD-L1.

[0023] Effects of the Invention

[0024] The PD-1 variants of the present application have a significantly increased binding force with PD-L1 while having fewer mutations than the conventional PD-1 and PD-1 variants, can solve the immunogenicity problem, are proteins much smaller in size than the conventional antibody therapeutics, and thus can effectively inhibit the PD-1 / PD-L1 binding of tumor and immune cells in the tumor microenvironment, solve the problem of low binding force of the conventional PD-L1 to PD-1, and thus can significantly improve the therapeutic effect as a therapeutic agent, and can also be used as an imaging agent for detecting the expression amount of PD-L1. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A graph for confirming the mutation site important in the binding force with PD-L1 in JY101, a PD-1 variant, by flow cytometry fluorescence sorting technique (FACS).

[0026] Figure 2 A graph for comparing the amino acid sequences of N-IITV variant having a mutation in the binding force and its non-glycosylated variant Q-IITV with those of wild type.

[0027] Figure 3 A graph showing a photograph of sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis of pMaz vector containing dimeric human PD-L1 (PD-L1-Fc) and purified dimeric PD-L1 protein for exploring the binding force of PD-1 variant with PD-L1.

[0028] Figure 4 A graph for confirming dimeric human PD-L1 (PD-L1-Fc) by the PD-L1 binding force analysis results of E. coli cells expressing wild type PD-1, JY101 variant of existing research, N-IITV variant, and Q-IITV variant, respectively.

[0029] Figure 5 A graph showing the amino acid sequence analysis data of the constructed error prone library.

[0030] Figure 6 A graph showing the results of the library enrichment test by flow cytometry analysis.

[0031] Figure 7 A graph showing the results of the analysis of the PD-L1 binding capacity of the E. coli cells expressing the non-glycosylated PD-1 variant.

[0032] Figure 8 A graph showing the gel photograph of the sodium dodecyl sulfate polyacrylamide gel electrophoresis of the expression vector of the non-glycosylated PD-1 variant, the purified non-glycosylated PD-1 variant protein, and the yield.

[0033] Figure 9 A graph showing the sensorgrams of the different concentrations of each variant.

[0034] Figure 10 A graph showing the K D values of the variants.

[0035] Figure 11 A graph showing the sensorgrams of the different concentrations of the HAC as a glycosylated variant and the non-glycosylated variant Q12 (JY_Q12).

[0036] Figure 12 A graph showing the K D values of the HAC as a glycosylated variant and the non-glycosylated variant Q12 (JY_Q12).

[0037] Figure 13 A graph showing the results of the analysis of the PD-L1 binding capacity of the wild type PD-1, the CKJ 52 of the prior art, the CKJ 52-Y69T prepared in Example 5, and the N_IITV variant of the present application.

[0038] Figure 14 A graph showing the amino acid sequence of the PD-1 JY-Q12 glycosylated variant, wherein the sequence at the top is the PD-1 JY-Q12 variant; the second sequence is the amino acid substitution and glycosylation position of JY_Q12-1 (Q25N); the third sequence is the amino acid substitution and glycosylation position of JY_Q12-2 (Q34N); the fourth sequence is the amino acid substitution and glycosylation position of JY_Q12-3 (Q50N); and the fifth is the amino acid substitution and glycosylation position of JY_Q12-4 (Q92N).

[0039] Figure 15Figure showing a photograph of a sodium dodecyl sulfate polyacrylamide gel electrophoresis of the expression vector of the PD-1 JY-Q12 glycosylation variant and the purified PD-1 JY-Q12 glycosylation variant protein.

[0040] Figure 16 Figure showing a sensorgram of each variant at different concentrations using a bio-layer interferometry assay (BLItz).

[0041] Figure 17 Figure showing a K D value of each variant using a bio-layer interferometry assay. DETAILED DESCRIPTION

[0042] Hereinafter, the present application will be explained in detail by examples of the present application with reference to the accompanying drawings. However, the following examples are presented only to illustrate the present application, and detailed description of the technology or structure which can be obvious to those skilled in the art to which the present application pertains will be omitted when it is judged that the detailed description can unnecessarily obscure the gist of the present application, and the present application is not limited to the following examples. The present application can be variously modified and applied within the equivalent scope recited in the appended claims and interpreted by them.

[0043] Also, the terminology used in the present specification, as the terminology for properly expressing preferred embodiments of the present application, can be different according to the intention of the user, the intention of the user, or the management of the technical field to which the present application pertains, etc. Therefore, the terminology should be defined on the basis of the content of the entire specification. In the entire specification, when a certain part is referred to as "including" a certain structural element, unless otherwise specifically noted, it means that other structural elements can also be included, not excluding other structural elements.

[0044] All technical terms used in the present application, unless specifically defined, are used in the same meaning as commonly understood by those skilled in the art to which the present application pertains. Also, although preferred methods or samples are described in the present specification, similar or equivalent ones are also included in the scope of the present application. The content of all publications recited in the present specification is incorporated in the present application.

[0045] In the entire specification, a naturally occurring amino acid is not only used using a single letter or a three letter abbreviation, but also using a commonly accepted three letter abbreviation of other amino acids such as Aib (alpha-aminoisobutyric acid), Sar (N-methylglycine), etc. Also, the amino acid referred to using an abbreviation in the present application is recited using the following IUPAC-IUB nomenclature.

[0046] Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, Valine: V.

[0047] In an embodiment, the PD-1 variant of the present application can include a PD-1 (Programmed cell death protein-1) variant having an enhanced binding force to PD-L1 (Programmed death-ligand 1) by including amino acid substitutions of F13I, M46I, C69T, and G100V in amino acids of wild type PD-1.

[0048] In an embodiment, the amino acids of wild type PD-1 can include the amino acid sequence of SEQ ID NO: 1, and the amino acid substitution positions can be based on the amino acid sequence of SEQ ID NO: 1.

[0049] In an embodiment, the PD-1 variant of the present application can include amino acid substitutions of F13I, M46I, C69T, and G100V, and can be an N-IITV variant of SEQ ID NO: 2 having an enhanced binding force to PD-L1.

[0050] In an embodiment, the PD-1 variant of the present application can further include one or more of amino acid substitutions of N25Q, N34Q, N50Q, and N92Q, and can have a non-glycosylation property.

[0051] In an embodiment, the PD-1 variant of the present application having a non-glycosylation property can include one selected from the group consisting of the amino acid sequences of SEQ ID NO: 3 to SEQ ID NO: 7.

[0052] Since there is an N-linked glycosylation site in the ectodomain of PD-1, there is a problem of glycan heterogenity caused by cell lines, culture processes, and purification processes, but the PD-1 variant of the present application having the non-glycosylation characteristic through the amino acid substitution has a significantly improved binding force to PD-L1 compared to the existing variant, and since it has the non-glycosylation characteristic through the specific amino acid substitution, it is also easily mass-produced at low cost in bacteria, and there is no problem of glycan heterogenity caused by culture processes and purification processes, so it has a very great advantage in the preparation of biological drugs. Also, although N-glycosylation is very important in the binding force of PD-1 protein to PD-L1 and in the stability of the protein, it is a non-glycosylation variant that improves the binding force and stability.

[0053] In an embodiment, the PD-1 variant of the present application can further include amino acid substitutions of W8L, N9D, E37K, A108V, and G140C, can be a glycosylated variant to which a sugar chain (glycosylation) is attached to the N-linked glycosylation site present in the ectodomain of PD-1, can be a variant in which one or more of N25, N34, N50, and N92 are glycosylated, and can enhance the binding force to PD-L1 due to glycosylation at the above sites.

[0054] In an embodiment, the glycosylated PD-1 variant of the present application can include one or more selected from the group consisting of the amino acid sequences of SEQ ID NO: 8 to SEQ ID NO: 11.

[0055] The term "variant" used in the present application means that a part of the amino acid sequence of the PD-1 protein (or peptide) is substituted, and means a corresponding amino acid sequence including a minimum difference (substitution, insertion, or deletion) of amino acids compared to a reference substance. In a specific embodiment, the "variant" has a high amino acid sequence homology and / or a protective amino acid substitution, deletion, and / or insertion compared to the reference sequence. Also, the term "PD-1 variant" used in the present application means a PD-1 variant protein that is mutated in one or more amino acids to adjust the binding activity to PD-L1.

[0056] In particular, the PD-1 variant of the present application can be prepared by standard synthetic methods, recombinant expression systems, or any other methods in the art to which the present application pertains. Thus, the peptide of the present application can be synthesized by various methods including, for example, method (a), synthesis of a peptide by means of solid phase or liquid phase methods in a stepwise or fragment assembly manner, isolation and purification of the final peptide product; or method (b), recovery of an expression product from a host cell culture after expression of a nucleic acid preparation encoding a peptide within a host cell; or method (c), recovery of an expression product after performing cell-free in vitro expression of a preparation encoding a peptide; or a method of obtaining a peptide by obtaining fragments of a peptide by any combination of method (a), method (b), and method (c), followed by ligation of the fragments, and then recovering the relevant peptide.

[0057] More specifically, the PD-1 variant of the present application can be produced by preparing a gene encoding the PD-1 variant of the present application through genetic manipulation, transforming the same into a host cell, and then expressing the same.

[0058] In an embodiment, the present application relates to a nucleic acid molecule encoding the PD-1 variant of the present application, a vector comprising the same, and a host cell comprising the above-mentioned vector.

[0059] The term "nucleic acid molecule" used in the present application means deoxyribonucleic acid or ribonucleic acid existing in a single strand or double strand form, and includes natural nucleic acid analogs (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)) unless otherwise specified.

[0060] The term "vector" used in the present application includes any nucleic acid that is replicated as an insert or an addition by recombination with the genome of a host cell after being inserted into the host cell. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, ribonucleic acid (RNA) vectors, viral vectors, and the like.

[0061] The term "host cell" used in the present application means a eukaryotic or prokaryotic cell into which one or more deoxyribonucleic acid (DNA) or vectors are introduced, and is understood to mean not only the specific subject cell, but also its progeny or potential progeny. Since the progeny can be deformed in the progeny due to mutation or environmental influences, these progeny are in fact different from the parent cell, but are still included in the scope of the above-mentioned term used in the present application.

[0062] In an embodiment, the present application relates to a PD-L1 and PD-1 binding inhibitor comprising the PD-1 variant of the present application, a nucleic acid molecule thereof, or a vector comprising the same.

[0063] In an embodiment, the present application relates to a composition for detecting PD-L1 comprising the PD-1 variant of the present application.

[0064] In an embodiment, the composition described above can detect and quantify the amount of protein expression of PD-L1.

[0065] In an embodiment, the PD-1 variant can be labeled with one selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromophore, a luminescent substance, and a fluorescent substance, the fluorescent substance can be a fluorescent substance of a cyanine series, a Rhodamine series, an Alexa series, a BODIPY series, or a ROX series, can be Nile Red, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, Rhodamine, coumarine, or Alexa.

[0066] Since the PD-1 variant of the present application can detect and quantify the expression amount of PD-L1, it can be used before administering an existing immune checkpoint inhibitor therapeutic agent after confirming whether or not PD-L1 is overexpressed in a cancer patient.

[0067] In an embodiment, the present application relates to a composition for bioimaging comprising the PD-1 variant of the present application.

[0068] In an embodiment, the present application relates to a pharmaceutical composition for treating or preventing cancer comprising the PD-1 variant of the present application, a nucleic acid molecule thereof, or a vector comprising the same.

[0069] In an embodiment, the cancer can be one or more selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, large intestine cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system cancer, primary central nervous lymphoma, spinal cord cancer, brain stem neuroglioma, and pituitary adenoma.

[0070] The pharmaceutical composition of the present application can be used as a sole therapy or can be used in combination with a conventional biological therapy, chemotherapy or radiation therapy, and in the case of practicing such combination therapy, cancer can be treated more effectively. In the case of using the present application for the prevention and treatment of cancer, the chemotherapeutic agent which can be used in combination with the above composition includes cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, etc. The radiation therapy which can be used in combination with the composition of the present application is X-ray irradiation and γ-ray irradiation.

[0071] In the present application, the term "prevention" means all actions to inhibit or delay the onset, spread and recurrence of cancer by administering the PD-1 variant of the present application or the composition comprising the same.

[0072] The therapeutically effective amount of the composition of the present application can vary depending on various factors such as the method of administration, the target site, the state of the patient, etc. Thus, the amount of administration in humans should be properly determined in consideration of safety and effectiveness. The amount for use in humans can be estimated from the effective amount determined through animal experiments. Matters for determining the effective amount are described in, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E. W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0073] The pharmaceutical composition of the present application is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" used in the present application means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined according to factors well known in the medical field including the health state of the patient, the kind of disease, the severity of disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration, and the metabolic ratio, the period of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The composition of the present application can be administered as a sole therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered once or multiple times. The point is to obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, which can be easily determined by those skilled in the art to which the present application pertains.

[0074] The pharmaceutical composition of the present application can contain a carrier, a diluent, an excipient, or a combination of two or more thereof, which are generally used for biological agents. The term "pharmaceutically acceptable" used in the present application means a property of showing no toxicity to normal cells or humans exposed to the above-mentioned composition. The above-mentioned carrier is not particularly limited as long as it is suitable for delivering the composition into a living body, and for example, a compound gathered in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterilized water, Ringer's solution, buffered physiological saline, a glucose solution, a maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components can be used, and other general additives such as an antioxidant, a buffer, a bacteriostatic agent, and the like can be added as necessary. Also, a diluent, a dispersant, a surfactant, a binding agent, and a lubricant can be added to be formulated into an injection dosage form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule, or a tablet. Further, preferred formulation can be made according to each disease or component by using a suitable method in the technical field to which the present application pertains or a method described in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0075] In one embodiment, the above-mentioned pharmaceutical composition can be one or more dosage forms selected from the group consisting of an oral dosage form, a topical agent, a suppository, a sterilized injection solution, and a spray.

[0076] The pharmaceutical composition of the present application can contain a carrier, a diluent, an excipient, or a combination of two or more thereof, which are generally used for biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition into a living body, and for example, a compound gathered in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterilized water, Ringer's solution, buffered physiological saline, a glucose solution, a maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components can be used, and other general additives such as an antioxidant, a buffer, a bacteriostatic agent, and the like can be added as necessary. Also, a diluent, a dispersant, a surfactant, a binding agent, and a lubricant can be added to be formulated into an injection dosage form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule, or a tablet. Further, preferred formulation can be made according to each disease or component by using a suitable method in the technical field to which the present application pertains or a method described in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0077] The composition of the present application can further include one or more active ingredients exhibiting the same or similar functions. The composition of the present application can include the above-mentioned protein in an amount of 0.001 to 10% by weight, preferably 0.001 to 1% by weight, with respect to the total weight of the composition.

[0078] The pharmaceutical composition of the present application can further include a pharmaceutically acceptable additive, in which case, the pharmaceutically acceptable additive can include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, sugar diluent, acacia, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, palm wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, glucose, sorbitol, and talc, etc. Preferably, the pharmaceutical composition of the present application can include the pharmaceutically acceptable additive in an amount of 0.1 to 90 parts by weight, with respect to the above-mentioned composition, but is not limited thereto.

[0079] The composition of the present application can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally according to the intended method, and the amount of administration can vary depending on the body weight, age, sex, health status, diet, administration time, administration method, metabolic rate, and severity of the disease of the patient, etc. The composition of the present application can be administered in an amount of 0.0001 mg / ml to 10 mg / ml per day, preferably 0.0001 mg / ml to 5 mg / ml per day, more preferably once or divided into several times per day.

[0080] The liquid formulation of the composition of the present application for oral administration includes suspensions, internal solutions, emulsions, syrups, etc., and can include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water, liquid paraffin, etc. which are commonly used as simple diluents. The formulation for parenteral administration includes a sterilized aqueous solution, a non-aqueous solvent, a suspension, an oil agent, a freeze-dried formulation, a suppository, etc.

[0081] In one embodiment, the present application relates to a composition for diagnosing cancer comprising the PD-1 variant of the present application.

[0082] The "detecting" or "measuring" used in the present application is the detection or measurement of the concentration of the object of a specified amount.

[0083] In one embodiment, the present application relates to a cancer diagnosis kit comprising the composition for diagnosing cancer of the present application.

[0084] In one embodiment, the kit described above not only includes tools and / or reagents for collecting a biological sample from a subject or patient, but can also include tools and / or reagents for preparing genomic deoxyribonucleic acid, complementary deoxyribonucleic acid (cDNA), ribonucleic acid, or protein from the sample.

[0085] The term "cancer diagnostic kit" used in the present application refers to a kit comprising the composition for diagnosing cancer of the present application. Thus, the expression "cancer diagnostic kit" described above can be used or mixed with "composition for diagnosing cancer" interchangeably. The term "diagnosis" used in the present specification includes determining the susceptibility of an individual to a particular disease or condition, determining whether an individual is currently suffering from a particular disease or condition, determining the prognosis of an individual suffering from a particular disease or condition (e.g., identification of a pre-metastatic or metastatic cancer state, determination of the stage of cancer, or determination of the responsiveness of cancer to treatment), or therametrics (e.g., detecting the state of an individual in order to provide information on the efficacy of treatment).

[0086] The PD-1 variant of the present application has a cancer cell apoptosis activity by immune cells by specifically binding to PD-L1 expressed to avoid the mechanism of killing cancer cells by immune cells with significantly enhanced binding force, and can be used as a therametrics agent in the diagnosis of cancer because it specifically binds to cancer cells. Also, a chimeric antigen receptor (CAR)-T cell containing the PD-1 variant instead of an scFv can be prepared as an anticancer agent, and can be used as an anticancer adjuvant for simultaneous administration, separate administration, or sequential administration with an anticancer agent. At the same time, it can also be used as a drug delivery to target cancer cells due to strong binding to PD-L1 of cancer cells.

[0087] In one embodiment, the present application relates to an information providing method for diagnosing cancer, comprising the steps of: contacting a biological sample isolated from a subject with the PD-1 variant of the present application; confirming the binding level of the PD-1 variant to PD-L1; and comparing the binding level of the PD-1 variant to PD-L1 in the sample with that in a normal control group.

[0088] In one embodiment, the method can further comprise the step of determining that the subject is cancerous when the binding level of the PD-1 variant to PD-L1 in the biological sample isolated from the subject is higher than that in the normal control group.

[0089] The term "sample" as used herein refers to a biological sample obtained from a subject or patient. The source of the biological sample can be: fresh, frozen and / or preserved organ or tissue sample, solid tissue derived from a biopsy or aspirate; blood or any blood cell fraction; cells at any time point of pregnancy or development of the subject.

[0090] In one embodiment, the present application relates to a method for specifically detecting PD-L1, comprising: a step of contacting a PD-1 variant with a sample; and a step of detecting the binding of the PD-1 variant to PD-L1.

[0091] In one embodiment, the present application relates to a method for producing a PD-1 variant having enhanced binding affinity to PD-L1, comprising: a step of culturing a host cell comprising a vector, the vector comprising a nucleic acid molecule encoding the PD-1 variant of the present application; and a step of recovering the PD-1 variant expressed by the host cell.

[0092] In one embodiment, the present application relates to a method for producing a non-glycosylated PD-1 variant having enhanced binding affinity to PD-L1, comprising: a step of culturing a host cell comprising a vector, the vector comprising a nucleic acid molecule encoding the PD-1 variant of the present application; and a step of recovering the non-glycosylated PD-1 variant expressed by the host cell.

[0093] In one embodiment, the present application relates to a method for producing a glycosylated PD-1 variant having enhanced binding affinity to PD-L1, comprising: a step of culturing a host cell comprising a vector, the vector comprising a nucleic acid molecule encoding the PD-1 variant of the present application; and a step of recovering the glycosylated PD-1 variant expressed by the host cell.

[0094] In one embodiment, the present application relates to a method for treating cancer, comprising a step of administering a pharmaceutically effective amount of the PD-1 variant of the present application to an individual having cancer.

[0095] In one embodiment, the present application relates to the use of the PD-1 variant for the preparation of a pharmaceutical composition for the prevention and treatment of cancer.

[0096] Embodiments of the present invention

[0097] The present application will be described in more detail by the following examples. However, the following examples are set forth to provide illustration of the application and should not be construed as limiting the application thereto.

[0098] Example 1. Exploration of main mutations of PD-1 for enhancing the binding affinity to PD-L1

[0099] To confirm the mutation sites necessary for the JY 101 variant (a variant having mutations substituted with S1, I13, M17, P36, I46, T69, R79, V100, P114, L139 in wild-type PD-1) that exhibits very high binding force to PD-L1 discovered in the gene display system to improve the binding force to PD-L1, the 10 mutations (S1L, I13F, M17L, P36S, I46M, T69C, R79G, V100G, P114L, and L139A) of the amino acids of wild-type PD-1 were substituted one by one. Specifically, the genome was amplified by a QuikChange polymerase chain reaction (PCR) method using primers designed for this purpose and a high-fidelity polymerase (Pfu turbo polymerase) (Agilent). After the amplified gene was transformed into Jude1, the sequence was confirmed. Then, JY 101 and its 10 variants (S1L, I13F, M17L, P36S, I46M, T69C, R79G, V100G, P114L, and L139A) were each cultured in a TB medium containing 2% glucose and 40 μg / ml chloramphenicol at 37°C, 250 rpm for 16 hours. After the cultured cells were inoculated in 6 ml of a TB medium containing 40 μg / ml chloramphenicol at a ratio of 1:50, they were cultured to OD 600After the OD600 value reached 0.5, the cells were cooled for 20 minutes at 25°C and 250 rpm, and then 1 mM of isopropyl-β-D-thiogalactoside (IPTG) was added to overexpress the protein at 25°C and 250 rpm for 5 hours. After the E. coli overexpressing the protein was put into the e-tube in the same amount, the cells were recovered by centrifugation at 14,000 rpm for 1 minute. To remove the remaining medium, the cells put into the e-tube were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0), and then centrifuged at 13,500 rpm for 1 minute, and this process was repeated twice. After the cells were resuspended using 1 ml of STE (0.5 M sucrose, 10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0)) solution, the outer membrane was removed by rotation at 37°C for 30 minutes. After the E. coli was aggregated by centrifugation at 13,500 rpm for 1 minute, the supernatant was removed. After the centrifuged E. coli was resuspended using 1 ml of Solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS, pH 6.8), it was centrifuged at 13,500 rpm for 1 minute. After the centrifuged E. coli was resuspended by adding a solution in which 1 ml of Solution A was mixed with 20 μl of 50 mg / ml lysozyme solution to the E. coli, the peptidoglycan layer was removed by rotation at 37°C for 15 minutes. After the supernatant was removed by centrifugation, the E. coli was resuspended using 1 ml of phosphate buffered saline (PBS), and 300 μl was put into 700 μl of PBS and 3 nM of tetrameric PD-L1-Alexa 488 probe to label the fluorescent probe on the spheroplast by rotation at room temperature. After the labeling process for 1 hour, the supernatant was removed by centrifugation at 13,500 rpm for 1 minute, and the centrifuged E. coli was washed once with 1 ml of PBS and then centrifuged at 13,500 rpm for 1 minute again. After the centrifuged E. coli was resuspended using 1 ml of PBS, the change in the binding force of each variant was analyzed using a Guava (Merck Millipore) device.

[0100] As a result, the mutations of 13I, 46I, 69T, and 100V were significantly reduced in the binding force when they were mutated to the wild type, respectively, and thus it can be confirmed that the mutation sites of 4 places are important in the binding force with PD-L1. Figure 1

[0101] ​Example 2. Preparation of a variant having binding force-raising mutations (N-IITV) and its non-glycosylation variant (Q-IITV)

[0102] 2-1. Preparation of N-IITV

[0103] A PD-1 variant N-IITV having the four (13I, 46I, 69T, and 100V) JY 101 variant binding force-raising mutations identified in the above Example 1 was prepared. Specifically, after amplifying the genome using a total of eight primers and Vent polymerase through gene assembly polymerase chain reaction (Gene assembly PCR), the amplified genome was treated with Sfil enzyme. The deoxyribonucleic acid treated with Sfil was ligated with the pMopac12-NlpA-FLAG vector also treated with Sfil to prepare the pMopac12-NlpA-PD1_N-IITV-FLAG vector. Then, after being transformed into E. coli Jude1 to secure a single colony, the insertion into the pMopac-12 vector was confirmed through base sequence analysis, thereby obtaining the PD-1 variant N-IITV having the four mutations (13I, 46I, 69T, and 100V) having the binding force-raising with PD-L1 (SEQ ID NO: 2) Figure 2 N-IITV).

[0104] 2-2. Preparation of Q-IITV

[0105] To prepare a variant having the non-glycosylation property while having the four mutations (13I, 46I, 69T, and 100V) having the binding force-raising with the same JY 101 variant as the above N-IITV variant, a non-glycosylation PD-1 variant Q-IITV (F13I, N25Q, N34Q, M46I, N50Q, C69T, N92Q, and G100V) having the four (25N, 34N, 50N, 92N) N-glycosylation sites replaced with glutamine (Q) having the most similar properties to asparagine (N) and a similar structure but not glycosylated, while having the four mutations (13I, 46I, 69T, and 100V) having the binding force-raising with the JY 101 variant was prepared (SEQ ID NO: 3). To this end, the Q-IITV (F13I, N25Q, N34Q, M46I, N50Q, C69T, N92Q, and G100V) genome was synthesized (Genescript), and after amplifying the synthesized genome using primers and Vent polymerase through polymerase chain reaction, treatment with Sfil enzyme was used to ligate with the pMopac12-NlpA-FLAG vector treated with Sfil to prepare the pMopac12-NlpA-PD1_Q-IITV-FLAG vector. Then, after being transformed into E. coli Jude1 to secure a single colony, successful insertion into the pMopac-12 vector was confirmed through base sequence analysis (Figure 2 Q-IITV).

[0106] Example 3. Preparation of dimeric human PD-L1 (PD-L1-Fc) for exploring the binding force of the discovered PD-1 variant to PD-L1

[0107] 3-1. Cloning of PD-L1-Fc

[0108] To improve the binding activity (Avidity) to more effectively explore the binding force of the discovered variant to PD-L1, to improve the binding activity to more effectively screen the non-glycosylated PD-1 variant, after inducing the dimerization reaction by expressing the Fc domain of IgG at the C-terminal portion of PD-L1, a GS linker was inserted between Fc and PD-L1 to ensure the mobility of each protein. Specifically, after amplifying the PD-L1 and Fc genes using primers and Vent polymerase (New England Biolab), assembly PCR was performed using Vent polymerase. After treating the prepared genes with BssHII and XbaI (New England Biolab), the PD-L1-Fc gene treated with the restriction enzyme was ligated to the pMAZ vector treated with the same restriction enzyme. After transforming the ligated plasmid into Jude1 E. coli, a monoclonal was ensured to confirm that PD-L1-Fc was successfully inserted into the pMAZ vector by base sequence analysis.

[0109] 3-2. Animal cell expression, purification and labeling of PD-L1-Fc

[0110] Expi293F cells were subcultured at a density of 2x10 6 After subculturing 300 ml of Expi293F cells for one day at a density of 2x10 After culturing the transfected cells in a CO2 shaker incubator at 37℃, 125 rpm, 8% CO2 for 7 days, the supernatant was obtained by centrifugal separation. Then, after equilibrating with 25x phosphate buffer solution, it was filtered through a filter (Merck Millipore) of 0.2 μm using an evaluation filter. After putting 1 ml of Protein A resin into the filtered culture solution and stirring at a temperature of 4℃ for 16 hours, the resin was recovered by passing through a chromatography column, and then washed with 10 ml of phosphate buffer solution. After eluting the washed resin with 100 mM glycine pH 2.7 buffer, neutralization was performed using 1M Tris-HCl (pH 8.0) to obtain a purified PD-L1 dimer proteinFigure 3 The purified PD-L1 dimer protein was fluorescently labeled using the Alexa-488 labeling kit.

[0111] Example 4. Analysis of PD-L1 binding affinity of the variant with an increased binding affinity mutation (N-IITV) and its non-glycosylated variant (Q-IITV).

[0112] To examine the binding affinity of PD-1 variants JY 101, N-IITV, and Q-IITV to PD-L1, *E. coli* expressing wild-type PD-1, JY 101, N-IITV, and Q-IITV were cultured for 16 hours in TB medium containing 2% glucose and 40 μg / ml chloramphenicol at 37°C and 250 rpm. The cultured cells were then seeded at a 1:100 ratio into 6 ml of TB medium containing 40 μg / ml chloramphenicol and cultured until OD500. 600= 0.5, 1 mM of isopropyl-β-D-thiogalactopyranoside was added after a cooling process at a temperature of 25°C and a rotation speed of 250 rpm for 20 minutes, and the protein was overexpressed at 25°C and a rotation speed of 250 rpm for 5 hours. After the E. coli overexpressing the protein was put into the e-tube in the same amount, the cells were recovered by centrifugal separation at a rotation speed of 14,000 rpm for 1 minute. In order to remove the remaining medium, the cells put into the e-tube were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0), and the resuspension was repeated twice by centrifugal separation at a rotation speed of 13,500 rpm for 1 minute. After the cells were resuspended using 1 ml of a STE (0.5 M sucrose, 10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (pH 8.0)) solution, the outer membrane was removed by rotation at 37°C for 30 minutes, and the E. coli was aggregated by centrifugal separation at a rotation speed of 13,500 rpm for 1 minute, and the supernatant was removed. The centrifugally separated E. coli was resuspended using 1 ml of a solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS, pH 6.8), and centrifugal separation was performed at a rotation speed of 13,500 rpm for 1 minute. After 1 ml of a solution in which 1 ml of the solution A was mixed with 20 μl of a 50 mg / ml lysozyme solution was added to resuspend the centrifugally separated E. coli, the peptidoglycan layer was removed by rotation at 37°C for 15 minutes. After the supernatant was removed by centrifugal separation, the resuspension was performed using 1 ml of a phosphate buffer solution, and 300 μl was put into 700 μl of a phosphate buffer solution and 50 nM of the dimeric PD-L1-Fc-Alexa 488 probe prepared in Example 3 above, and the fluorescent probe was labeled on the spheroplast by rotation at room temperature. After the labeling process was performed for 1 hour, the supernatant was removed by centrifugal separation at a rotation speed of 13,500 rpm for 1 minute, and the centrifugally separated E. coli was washed once using 1 ml of a phosphate buffer solution, and was again centrifugally separated at a rotation speed of 13,500 rpm for 1 minute. The centrifugally separated E. coli was resuspended using 1 ml of a phosphate buffer solution, and the binding force to PD-L1 was analyzed using a Calibur (BD Biosciences) device. Also, since the expression amount of the PD-1 protein displayed by the E. coli can affect the fluorescence intensity, in order to confirm the expression amount, 300 μl was taken from the remaining E. coli after resuspension using a phosphate buffer solution, and 700 μl of a phosphate buffer solution and 0.5 μl of an anti-FLAG-FITC were put, and the fluorescent probe was labeled in the spheroplast by rotation at room temperature. After the labeling process was performed for 1 hour, the supernatant was removed by centrifugal separation at a rotation speed of 13,500 rpm for 1 minute, and the centrifugally separated E. coli was washed once using 1 ml of a phosphate buffer solution, and was again centrifugally separated at a rotation speed of 13,500 rpm for 1 minute.After resuspending the centrifuged E. coli using 1 ml of a phosphate buffer solution, the expression amount of the PD-1 protein was indirectly analyzed using a Calibur (BD Biosciences) device.

[0113] As a result, although the expression amount of the N-IITV variant and the Q-IITV variant discovered in the present application was lower than that of the wild-type PD-1 and the JY 101 variant discovered in the existing research, the N-IITV variant and the Q-IITV variant exhibited higher binding force to PD-L1 than the JY 101 variant Figure 4 ). Through this, it was confirmed that the N-IIITV and the Q-IITV significantly strongly bind to PD-L1 compared to the JY 101, and in particular, the N-IITV has more excellent binding force to PD-L1 than the Q-IITV.

[0114] Example 5. Additional screening of non-glycosylated PD-1 variants

[0115] 5-1. Preparation of a mega PD-1 error prone library for use in a ultra-high speed screening method

[0116] In order to rapidly explore a non-glycosylated PD-1 variant exhibiting high binding force to PD-L1, a primer enabling all sites of PD-1_Q_IITV to enter errors was designed to include SfiI sites at both sides based on pMopac12-NlpA-PD-1_Q_IITV-FLAG. The designed primer and Taq polymerase (TAKARA), dNTPs (Invitrogen), MgCl2, MnCl2 (SIGMA), etc. were used to amplify the genome through an error prone PCR method. The amplified genome was treated with SfiI enzyme and inserted into the pMopac12-NlpA-FLAG vector treated with SfiI enzyme to be connected, and then transformed into Jude1 cells. The transformed E. coli was spread in a square plate and cultured at a temperature of 37°C for 16 hours, and then recovered using a TB medium containing 2% glucose to obtain an initial library Figure 5

[0117] 5-2. Screening of non-glycosylated PD-1 variants

[0118] After adding 40 μg / ml of chloramphenicol to 25 ml of a TB medium containing 2% glucose, the library prepared in the above Example 5-1 was inoculated into a 250-ml flask and cultured at 37°C and 250 rpm for 4 hours, and then the cultured E. coli was inoculated into 100 ml of a TB medium containing 40 μg / ml of chloramphenicol at a ratio of 1:100. The culture was performed until the OD 600 ​After the temperature was raised to 42℃, the cells were overexpressed for 5 hours at 42℃, 250 rpm, and 1 mM of isopropyl-β-D-thiogalactopyranoside was added after 0.5 hours. After the temperature was raised to 25℃, the cells were cooled for 20 minutes at 25℃, 250 rpm, and 1 mM of isopropyl-β-D-thiogalactopyranoside was added after 0.5 hours. The cells were recovered by centrifugation at 14,000 rpm for 1 minute after the cells were overexpressed for 5 hours at 25℃, 250 rpm. To remove the remaining medium, the cells were resuspended in 1 ml of 10 mM Tris-HCl (pH 8.0) and centrifuged at 13,500 rpm for 1 minute to wash twice. The cells were resuspended in 1 ml of STE (0.5 M sucrose, 10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (pH 8.0)) solution and rotated for 30 minutes at 37℃ to remove the outer membrane. After centrifugation at 13,500 rpm for 1 minute, the supernatant was removed and the cells were resuspended in 1 ml of solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS, pH 6.8). The cells were centrifuged at 13,500 rpm for 1 minute. After the cells were resuspended in 1 ml of a solution in which 1 ml of solution A was mixed with 20 μl of a 50 mg / ml lysozyme solution, the cells were centrifuged at 13,500 rpm for 1 minute. The cells were resuspended in 1 ml of a solution in which 1 ml of solution A was mixed with 20 μl of a 50 mg / ml lysozyme solution and rotated for 15 minutes at 37℃ to remove the peptidoglycan layer. After centrifugation, the supernatant was removed and the cells were resuspended in 1 ml of a phosphate buffer solution. After 300 μl was placed in 700 μl of a phosphate buffer solution and 25 nM of a dimeric PD-L1-Alexa 488 probe, the cells were rotated at room temperature to label the fluorescent probe on the spheroplast. After 1 hour of the labeling process, the supernatant was removed after centrifugation at 13,500 rpm for 1 minute. The cells were washed once with 1 ml of a phosphate buffer solution and centrifuged at 13,500 rpm for 1 minute. The cells were resuspended in 1 ml of a phosphate buffer solution and recovered using an S3sorter (Bio-Rad) device to recover E. coli having high binding ability to PD-L1. After the genes of the recovered E. coli were amplified by polymerase chain reaction using primers, the obtained genome was treated with sfiI restriction enzyme to be ligated with a pMopac12-NlpA-FLAG vector treated with a restriction enzyme. After each plasmid was transformed into Jude1, the cells were recovered after being spread on a petri dish and cultured at 37℃ for 16 hours to be stored frozen. The above-described screening process was repeated 4 times (rounds).

[0119] 5-3. Amplification of non-glycosylated PD-1 variants with increased PD-L1 affinity

[0120] After adding 40 μg / ml of chloramphenicol to 25 ml of TB medium containing 2% glucose, the initial (Initial) library, 1 round library, 2 round library, 3 round library, 4 round library, and 5 round library of Example 5-1 were put into 250 ml flasks, respectively. After culturing the E. coli at 37°C, 250 rpm for 4 hours, the cultured E. coli was inoculated into 100 ml of TB medium containing 40 μg / ml of chloramphenicol at a ratio of 1:100, respectively. After culturing the E. coli to OD 600 = 0.5, the E. coli was cooled at 25°C, 250 rpm for 20 minutes, and 1 mM of isopropyl-β-D-thiogalactopyranoside was added to overexpress the protein at 25°C, 250 rpm for 5 hours. Also, wild type PD-1 and HAC-V-PD-1 cells were inoculated at a ratio of 1:50 into 6 ml of TB medium containing 40 μg / ml of chloramphenicol, and were used as a control group, which were cultured at 37°C, 250 rpm for 16 hours. The cultured cells were inoculated into 6 ml of TB medium containing 40 μg / ml of chloramphenicol at a ratio of 1:50, and were cultured to OD 600After 0.5, the overexpressed protein was recovered by centrifugation at 14,000 rpm for 1 min. To remove the remaining medium, the cells in the e-tube were resuspended with 1 ml of 10 mM Tris-HCl (pH 8.0) and washed twice by centrifugation at 13,500 rpm for 1 min. The outer membrane was removed by resuspending the cells with 1 ml of STE (0.5 M sucrose, 10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (pH 8.0)) solution and rotating at 37°C for 30 min. After the E. coli was aggregated by centrifugation at 13,500 rpm for 1 min, the supernatant was removed. After the centrifuged E. coli was resuspended with 1 ml of solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS, pH 6.8) and centrifuged at 13,500 rpm for 1 min, the solution in which 1 ml of solution A was mixed with 20 μl of 50 mg / ml lysozyme solution was added to resuspend the centrifuged E. coli, which was rotated at 37°C for 15 min to remove the peptidoglycan layer. After the supernatant was removed by centrifugation, 300 μl was resuspended with 1 ml of phosphate buffer solution and rotated at room temperature after 700 μl of phosphate buffer solution and 3 nM of dimeric PD-L1-Alexa 488 probe were added to label the fluorescent probe on the spheroplast. After 1 hour of labeling, centrifugation was performed at 13,500 rpm for 1 min. After the supernatant was removed, the centrifuged E. coli was washed once with 1 ml of phosphate buffer solution and again centrifuged at 13,500 rpm for 1 min. After the centrifuged E. coli was resuspended with 1 ml of phosphate buffer solution, the binding force to PD-L1 was analyzed by measuring the fluorescence signal value (mean fluorescence intensity (MFI).

[0121] As a result, it was confirmed that the variants having high binding force to PD-L1 were gradually amplified in the library as the screening was performed. Figure 6

[0122] 5-4. Obtaining non-glycosylated PD-1 variants with increased PD-L1 binding

[0123] ​After culturing the last round of the monoclonal in the same manner as in the above-described examples, after recovering the E. coli, removing the peptidoglycan layer, and labeling the fluorescence in the spheroplast, the binding force to PD-L1 was analyzed by measuring the fluorescence signal value using a FACSCalibur device. As a result, Q10 (F13I, N25Q, F32L, N34Q, M46I, N50Q, T52M, C69T, V87D, N92Q, T96S, G100V, A108V) (SEQ ID NO: 4), Q12 (W8L, N9D, F13I, N25Q, N34Q, E37K, M46I, N50Q, C69T, N92Q, G100V, A108V, G140C) (SEQ ID NO: 5), Q18 (F13I, N25Q, S31G, F32L, S33P, N34Q, M46I, S47G, N50Q, T52M, C69T, N92Q, G100V, A108V, P128R, P138S) (SEQ ID NO: 6), and Q33 (F13I, N25Q, N34R, M46I, N50Q, C69T, R88G, R90Q, N92Q, G100V, A108V) (SEQ ID NO: 7) showed high binding force to PD-L1, and thus they were selected Figure 7

[0124] Example 6. Expression and purification of non-glycosylated PD-1 variants with improved PD-L1 binding force

[0125] ​After expressing and purifying the four upstream non-glycosylated PD-1 variants (Q10, Q12, Q18, and Q33) that showed high binding ability to PD-L1 in the above Example 5, to examine their binding ability, cloning was performed. To this end, the genes of wild type Q_PD1 in which the N-glycosylation sites of wild type PD-1 were all changed to Q, AHAC as a control group, the variants Q_IITV, Q10, Q12, Q18, and Q33 additionally discovered in the present application were amplified by polymerase chain reaction using primers and Vent polymerase. After treating the amplified genome with BssHII and XbaI enzymes, it was ligated with pMAZ vector as an animal cell expression vector treated with the same enzymes. After transforming the ligated plasmid into Jude1 E. coli, the sequence was confirmed by single colony analysis. The prepared PD-1 variant expression vectors (pMAZ-PD1 Q_WT-His tag, pMAZ-PD1 AHAC-His tag, pMAZ-PD1 Q_IITV-His tag, pMAZ-PD1 Q10-His tag, pMAZ-PD1 Q12-His tag, pMAZ-PD1 Q18-His tag, and pMAZ-PD1 Q33-His tag) were transfected into Expi293F animal cells using PEI, respectively. Then, after culturing the transfected cells in a CO2 shaker incubator at 37℃, 125 rpm, and 8% CO2 for 7 days, only the supernatant was obtained by centrifugal separation. Then, 25x phosphate buffer solution was used to equilibrate. Filtration was performed using a filter (Merck Millipore) having a pore size of 0.2 μm. After placing 0.5 ml of Ni-NTA resin in the filtered culture solution, it was stirred at a temperature of 4℃ for 16 hours, and then passed through a chromatography column to recover the resin. After washing the recovered resin with a phosphate buffer solution containing 10 mM imidazole (Sigma) for 10 CV (column volume), it was washed once more with a phosphate buffer solution containing 20 mM imidazole for 10 CV. Then, elution was performed using a phosphate buffer solution containing 250 mM imidazole, and the buffer was replaced using centrifugal filter units 3K (Merck Millipore). Then, the non-glycosylated PD-1 variant proteins expressed and purified were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0126] As a result, the yield of wild type Q_PD-1 not expressed and the AHAC variant as a control group was very low, in contrast, the variants discovered in the present application were not glycosylated, but the stability was increased, and it was possible to obtain them at high purity and high yield. Figure 8

[0127] ​Example 7. Assay of binding force of non-glycosylated PD-1 variants

[0128] The binding force of CKJ 49 (F13L / N25D / C69S / N92S / R137K), JY 101 (N1S / F13I / L17M / S36P / M46I / C69T / G79R / G100V / L114P / A139L), and non-glycosylated PD-1 variants (Q10, Q12, Q18, and Q33) discovered in the present application to PD-L1 was determined by a Biolayer interferometry assay (BLItz, Pall Fortebio). Specifically, after first activating an AR2G biosensor (Pall Fortebio) using 20 mM EDC and 10 mM s-NHS for 5 minutes, 20 μg / ml of PD-L1-streptavidin was immobilized for 5 minutes, and then 1 M ethanolamine was used for quenching for 5 minutes. Then, after finding a baseline using 1X kinetic buffer for 30 seconds, 1000 nM, 500 nM, 250 nM, and 125 nM of PD-1 variants were used for association for 1 minute, and 1X kinetic buffer was used for dissociation for 60 seconds. In this way, sensorgrams of different concentrations of each variant were analyzed (Figure 7), and the final Kd values were analyzed. Figure 9 D As a result of analyzing the final Kd values, Q12 showed the highest binding force to PD-L1 (Figure 8). Figure 10

[0129] Example 8. Comparison of binding force of non-glycosylated PD-1 variant (JY_Q12) and existing glycosylated PD-1 variant (HAC) to PD-L1

[0130] ​​For PD-1, glycosylation is crucial for its binding affinity to PD-L1. Unglycosylated, non-glycosylated PD-1 almost completely loses its binding affinity to PD-L1. Therefore, this invention compares and analyzes the binding affinity of non-glycosylated PD-1 variants and glycosylated PD-1 variants to PD-L1. For this purpose, biolayer interferometry (BLItz, Pall Fortebio) was used to determine the binding affinity of HAC, a known glycosylated PD-1 variant in existing studies, and the non-glycosylated variant JY_Q12 (Q12) exhibiting the highest binding affinity in Example 7 to PD-L1. Specifically, following the same procedure as in the examples described above, the AR2G biosensor (Pall Fortebio) was first activated with 20 mM EDC and 10 mM s-NHS for 5 minutes, then immobilized with 20 μg / ml PD-L1-streptavidin for 5 minutes, followed by quenching with 1 M ethanolamine for 5 minutes. Then, after establishing a baseline using 1X kinetic buffer for 30 seconds, PD-1 variants at concentrations of 1000 nM, 500 nM, 250 nM, and 125 nM were applied for binding for 1 minute, followed by decomposition using 1X kinetic buffer for 60 seconds. Sensor maps at different concentrations of each variant were analyzed in this manner. Figure 11 ), analyze the final K D value( Figure 12 )

[0131] As a result, the non-glycosylated PD-1 variant Q12 (JY_Q12) discovered in this invention exhibits PD-L1 binding affinity similar to that of the existing glycosylated PD-1 variant (HAC). Figure 11 and Figure 12 This confirms that the non-glycosylated variant of the present invention, even without glycosylation, which is important for PD-L1 binding, achieves a binding strength similar to that of the glycosylated variant. Because it is a non-glycosylated form, it can be produced in bacteria, and there is no glycosylation heterogeneity problem when produced in animal cells. Therefore, it can solve the non-specific receptor binding problem caused by glycosylation in various purposes such as treatment and diagnosis.

[0132] Example 9. Comparison of binding forces based on amino acid variations

[0133] To confirm the binding force of the N-IITV variant of the present application to PD-L1 varies depending on the type of amino acid variation in the amino acid variation site, the present inventors additionally prepared a CKJ 52-Y69T variant in which the C69Y mutation of the CKJ 52 variant (F13I, M46I, and C69Y) of the existing patent (No. 10-2019-0011181) (sequence 54 of the existing patent) was changed to Y69T. After amplifying the genome of the pMopac12-NlpA-CKJ52-FLAG plasmid prepared for this by a rapid change polymerase chain reaction (Quikchange PCR) method using the designed primer and high-fidelity polymerase (Agilent), the sequence was confirmed by transforming the amplified gene into Jude1, and the CKJ 52-Y69T variant was additionally obtained. Then, to test the binding force of wild-type PD-1, the CKJ 52 of the existing patent, the prepared CKJ 52-Y69T, and the N-IITV variant of the present application to PD-L1, Escherichia coli expressing wild-type PD-1, CKJ 52, CKJ 52-Y69T, and N-IITV, respectively, were cultured in a TB medium containing 2% glucose and 40 μg / ml of chloramphenicol at 37°C, 250 rpm for 16 hours. The cultured cells were inoculated into 6 ml of a TB medium containing 40 μg / ml of chloramphenicol at a ratio of 1:100, and cultured until OD 600= 0.5, after a cooling process for 20 minutes at 25°C, 250 rpm, 1 mM of isopropyl-β-D-thiogalactopyranoside was added, and the protein was overexpressed for 5 hours at 25°C, 250 rpm. The E. coli overexpressing the protein was put into an e-tube in the same amount, and was centrifuged at 14,000 rpm for 1 minute to recover the cells. To remove the remaining medium, the cells put into the e-tube were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0), and were centrifuged at 13,500 rpm for 1 minute to be washed twice. After removing the outer membrane by resuspending the cells using 1 ml of a STE (0.5 M sucrose, 10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (pH 8.0)) solution and rotating for 30 minutes at 37°C, the E. coli was collected by centrifugation at 13,500 rpm for 1 minute, and the supernatant was removed. The E. coli centrifuged was resuspended using 1 ml of Solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS, pH 6.8), and was centrifuged at 13,500 rpm for 1 minute. After adding a solution in which 1 ml of Solution A was mixed with 20 μl of a 50 mg / ml lysozyme solution to resuspend the centrifuged E. coli, the peptidoglycan layer was removed by rotating for 15 minutes at 37°C. After removing the supernatant by centrifugation, the cells were resuspended using 1 ml of a phosphate buffer solution, and 300 μl was put into 700 μl of a phosphate buffer solution and 200 nM of a PD-L1-Fc-Alexa 488 probe to label the fluorescent probe on the spheroplast at room temperature. After a labeling process for 1 hour, the supernatant was removed by centrifugation at 13,500 rpm for 1 minute, the centrifuged E. coli was washed once using 1 ml of a phosphate buffer solution, and was again centrifuged at 13,500 rpm for 1 minute. The centrifuged E. coli was resuspended using 1 ml of a phosphate buffer solution, and the binding force to PD-L1 was analyzed using a Calibur (BD Biosciences) device.

[0134] As a result, the CKJ 52-Y69T variant (13I, 46I, and 69T) in which the 69th amino acid was substituted with T showed a higher binding force to PD-L1 than CKJ 52, and the N-IITV variant (13I, 46I, 69T, and 100V) of the present application in which the 100th amino acid was also mutated showed a much higher binding force to PD-L1 than CKJ 52-Y69T. Figure 13 ).

[0135] Example 10. Preparation of a glycosylated PD-1 variant having improved PD-L1 binding force

[0136] Since whether PD-1 is glycosylated is very important in the binding with PD-L1, in order to explore a variant having more improved binding force with PD-L1, N25Q, N34Q, N50Q, and N92Q residues, which are 4 N-glycosylation sites present in PD-1 ECD and substituted with Q in JY-Q12 variant (Sequence 5) of wild type PD-1 (Sequence 1) including amino acid substitutions of W8L, N9D, F13I, N25Q, N34Q, E37K, M46I, N50Q, C69T, N92Q, G100V, A108V, and G140C, were substituted with N again to induce attachment of a sugar chain, thereby glycosylated. To this end, the genome of the JY-Q12 variant was amplified by a rapid amplification of polymersase chain reaction method using primers therefor and a high fidelity polymerase (Agilent) with respect to pMAZ-PD-1 JY-Q12 plasmid in which the gene of the JY-Q12 variant was inserted into a pMAZ vector. After the amplified genome was transformed into Jude1 E. coli, 4 expression vectors (pMAZ-PD1 JY_Q12-1(Q25N), pMAZ-PD1 JY_Q12-2(Q34N), pMAZ-PD1 JY_Q12-3(Q50N), pMAZ-PD1 JY_Q12-4(Q92N)) respectively containing 4 glycosylated PD-1 variants JY_Q12-1(Q25N) (Sequence 8), JY_Q12-2(Q34N) (Sequence 9), JY_Q12-3(Q50N) (Sequence 10), and JY_Q12-4(Q92N) (Sequence 11) were prepared by confirming the sequence through single colony analysis. Figure 14 ).

[0137] Example 11. Expression and purification of glycosylated PD-1 variants

[0138] The PD-1 variant expression vectors (pMAZ-PD1 JY_Q12-1(Q25N), pMAZ-PD1 JY_Q12-2(Q34N), pMAZ-PD1 JY_Q12-3(Q50N), pMAZ-PD1 JY_Q12-4(Q92N)) prepared in the above examples were each transfected into Expi293F animal cells using PEI. Then, after the transfected cells were cultured in a CO2 shaker incubator at 37°C, 125 rpm, 8% CO2 for 7 days, only the supernatant was separated by centrifugation. Then, 25x phosphate buffer solution was equilibrated. The filtered solution was filtered through a 0.2 μm filter (Merck Millipore) using an evaluation filter, and 0.5 ml of Ni-NTA resin was added to the filtered solution, which was then stirred at 4°C for 16 hours. The resin was recovered by passing the solution through a chromatography column. After the recovered resin was washed with a phosphate buffer solution containing 10 mM imidazole (Sigma) for 10 CV, it was washed once more with a phosphate buffer solution containing 20 mM imidazole. Then, the eluted protein was recovered using a centrifugal filter units 3K (Merck Millipore). The expressed and purified non-glycosylated PD-1 variant proteins were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0139] More than 1 mg of each of the four purified variants was obtained, and it was confirmed that they exhibited different sizes due to different glycosylation patterns Figure 15 ).

[0140] Example 12. Measurement of the binding force of glycosylated PD-1 variants to PD-L1

[0141] The binding affinity of wild-type PD-1, HAC (HAC-V PD-1(N91C), a known glycosylated PD-1 variant in existing studies, Proc Natl Acad Sci US A. 2015 Nov 24; 112(47): E6506-E6514.), JY-Q12, and four glycosylated variants of this invention (JY_Q12-1(Q25N), JY_Q12-2(Q34N), JY_Q12-3(Q50N), and JY_Q12-4(Q92N)) to PD-L1 was determined and compared using biolayer interferometry (Pall Fortebio). Specifically, the AR2G biosensor (Pall Fortebio) was first activated for 5 minutes using 20 mM EDC and 10 mM s-NHS. Then, 20 μg / ml of PD-L1-streptavidin was immobilized for 5 minutes, followed by quenching with 1 M ethanolamine for 5 minutes. Next, after baseline establishment using 1X kinetic buffer for 30 seconds, PD-1 variants ranging from 100 nM to 2000 nM were applied for binding for 1 minute, followed by decomposition using 1X kinetic buffer for 60 seconds. Sensing maps of different concentrations of each variant were analyzed in this manner. Figure 16 ), analyze the final equilibrium dissociation constant (K) D The results showed that, among the four glycosylation variants, all except JY_Q12-4 (Q92N) exhibited significantly higher binding affinity to PD-L1 than existing PD-1 variants. In particular, JY_Q12-2 (Q34N) showed the highest binding affinity to PD-L1. Figure 17 ). SEQUENCE LISTING <110> Korea University Industry-Academia Collaboration Group <120> PD-1 variants with increased PD-L1 affinity <130> P22117754WP <150> KR 10-2020-0053981 <151> 2020-05-06 <150> KR 10-2020-0098405 <151> 2020-08-06 <150> KR 10-2020-0129925 <151> 2020-10-08 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> WT PD-1 <400> 1 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro 35 40 45 Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gin Phe Gin 130 135 140 <210> 2 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> N-IITV <400> 2 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Ile Ser Pro 35 40 45 Ser Asn Gin Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gin 50 55 60 Pro Gly Gin Asp Thr Arg Phe Arg Val Thr Gin Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Ala Gin Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140 <210> 3 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> Q_IITV <400> 3 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Gln Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Gln Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Ile Ser Pro 35 40 45 Ser Gln Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Thr Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Gln Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140 <210> 4 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q10 <400> 4 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Gln Ala Thr Phe Thr Cys Ser Leu 20 25 30 Ser Gln Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Ile Ser Pro 35 40 45 Ser Gln Gln Met Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Thr Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Asp Arg Ala Arg Arg Gin Asp Ser Gly Ser 85 90 95 Tyr Leu Cys Val Ala lie Ser Leu Ala Pro Lys Val Gin lie Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gin Phe Gin 130 135 140 <210> 5 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q12 <400> 5 Leu Asp Ser Pro Asp Arg Pro Leu Asp Pro Pro Thr lie Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Gin Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Gin Thr Ser Lys Ser Phe Val Leu Asn Trp Tyr Arg lie Ser Pro 35 40 45 Ser Gin Gin Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gin 50 55 60 Pro Gly Gin Asp Thr Arg Phe Arg Val Thr Gin Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Gin Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Val Gin Ile Lys Glu 100 105 110 Ser Leu Arg Ala Gin Leu Arg Val Thr Gin Arg Arg Ala Gin Val Pro 115 120 125 Thr Ala His Pro Ser Pro Gin Pro Arg Pro Gin Cys Gin Phe Gin 130 135 140 <210> 6 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q18 <400> 6 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Gin Gly Asp Gin Ala Thr Phe Thr Cys Gly Leu 20 25 30 Pro Gin Thr Ser Gin Ser Phe Val Leu Asn Trp Tyr Gin Ile Gly Pro 35 40 45 Ser Gin Gin Met Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gin 50 55 60 Pro Gly Gin Asp Thr Arg Phe Arg Val Thr Gin Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Gin Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Val Gin Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Arg 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Ser Ala Gly Gin Phe Gin 130 135 140 <210> 7 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q33 <400> 7 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Gin Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Arg Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Ile Ser Pro 35 40 45 Ser Gln Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Thr Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Gly Ala Gln Arg Gln Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Val Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln 130 135 140 <210> 8 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q12‑1 <400> 8 Leu Asp Ser Pro Asp Arg Pro Leu Asp Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Gln Thr Ser Lys Ser Phe Val Leu Asn Trp Tyr Arg Ile Ser Pro 35 40 45 Ser Gln Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Thr Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Gln Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Val Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Cys Gln Phe Gln 130 135 140 <210> 9 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q12‑2 <400> 9 Leu Asp Ser Pro Asp Arg Pro Leu Asp Pro Pro Thr lie Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Gin Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Asn Thr Ser Lys Ser Phe Val Leu Asn Trp Tyr Arg lie Ser Pro 35 40 45 Ser Gin Gin Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gin 50 55 60 Pro Gly Gin Asp Thr Arg Phe Arg Val Thr Gin Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Gin Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala lie Ser Leu Ala Pro Lys Val Gin lie Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Cys Gin Phe Gin 130 135 140 <210> 10 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q12-3 <400> 10 Leu Asp Ser Pro Asp Arg Pro Leu Asp Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Gln Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Gln Thr Ser Lys Ser Phe Val Leu Asn Trp Tyr Arg Ile Ser Pro 35 40 45 Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Thr Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Gln Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Val Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Cys Gln Phe Gln 130 135 140 <210> 11 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q12‑4 <400> 11 Leu Asp Ser Pro Asp Arg Pro Leu Asp Pro Pro Thr Ile Ser Pro Ala 1 5 10 15 Leu Leu Val Val Thr Glu Gly Asp Gln Ala Thr Phe Thr Cys Ser Phe 20 25 30 Ser Gln Thr Ser Lys Ser Phe Val Leu Asn Trp Tyr Arg Ile Ser Pro 35 40 45 Ser Gln Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 50 55 60 Pro Gly Gln Asp Thr Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 65 70 75 80 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr 85 90 95 Tyr Leu Cys Val Ala Ile Ser Leu Ala Pro Lys Val Gln Ile Lys Glu 100 105 110 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Cys Gln Phe Gln 130 135 140

Claims

1. A PD-1 variant with enhanced binding affinity to PD-L1, characterized in that, It is composed of amino acid substitutions of F13I, M46I, C69T, and G100V in the amino acids of wild-type PD-1 in sequence 1. The PD-1 variant is composed of the amino acid sequence of sequence 2.

2. A PD-1 variant with enhanced binding affinity to PD-L1, characterized in that, The PD-1 variant is composed of one of the groups consisting of amino acid sequences selected from sequences 3 to 7.

3. A PD-1 variant with enhanced binding affinity to PD-L1, characterized in that, The PD-1 variant is composed of one of the groups consisting of amino acid sequences selected from sequences 8 to 10.

4. A nucleic acid molecule, characterized in that, Encodes the PD-1 variant as described in any one of claims 1 to 3.

5. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It includes the carrier as described in claim 5.

7. A PD-L1 to PD-1 binding inhibitor, characterized in that, It includes the PD-1 variant as described in any one of claims 1 to 3.

8. A composition for detecting PD-L1, characterized in that, It includes the PD-1 variant as described in any one of claims 1 to 3.

9. The composition for detecting PD-L1 according to claim 8, characterized in that, PD-1 variants are markers selected from a group consisting of chromogenic enzymes, radioactive isotopes, and luminescent substances.

10. The composition for detecting PD-L1 according to claim 9, characterized in that, The luminescent material is a chromophore or a fluorescent material.

11. A pharmaceutical composition for treating or preventing cancer, characterized in that, It contains the PD-1 variant as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, or the vector as described in claim 5 as an active ingredient.

12. A composition for diagnosing cancer, characterized in that, It includes the PD-1 variant as described in any one of claims 1 to 3.

13. A method for producing a PD-1 variant with enhanced binding affinity to PD-L1, characterized in that, include: Step a) Culturing host cells containing a vector, wherein the vector contains a nucleic acid molecule encoding a PD-1 variant as described in any one of claims 1 to 3; as well as Step b), recover the PD-1 variant expressed through the host cells described above.

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