KR20220046815A - PD-1 variants with increased binding affinity to PD-L1 - Google Patents

Abstract

The present invention relates to PD-1 variants having minimal mutations for enhancing binding ability to PD-L1. The PD-1 variants of the present invention have fewer mutations than existing PD-1 and PD-1 variants and have significantly increased binding ability to PD-L1 compared to the existing mutants, thereby solving the problem of immunogenicity. In addition, since the variants are very small-sized proteins as compared to existing antibody therapeutic agents, PD-1/PD-L1 binding of tumors and immune cells in a tumor microenvironment can be effectively inhibited, and since the problem of low binding ability of PD-L1 to existing PD-1 is solved, the therapeutic effect thereof as a therapeutic agent can be significantly improved. The variants can also be used as an imaging agent for detecting the expression level of PD-L1.

Description

PD-1 variants with increased binding affinity to PD-L1}

The present invention relates to a PD-1 mutant having improved affinity for PD-L1, and to a PD-1 mutant having a minimum mutation for enhancing avidity with PD-L1.

Pharmaceuticals for cancer treatment are largely divided into low molecular weight medicines and high molecular weight medicines, and high molecular weight medicines with specificity are attracting attention as therapeutic agents compared to low molecular weight medicines with relatively large side effects due to lack of specificity. Cancer cells express on the cell surface an immune checkpoint protein used when normal cells suppress immune cell activation to avoid the killing mechanism by immune cells. Research on checkpoint inhibitory proteins is being actively conducted.

Among immune checkpoint inhibitory proteins, it has been reported in academia that blocking PD-1/PD-L1 binding has a great effect on cancer treatment and has fewer side effects compared to other immune checkpoint inhibitory proteins (J. Naidoo et al. (J. Naidoo et al. (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)/natural killer T (NKT) cells, etc. (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 the interaction of PD-1 with one of its ligands, PD-L1, on the tumor surface is an immune checkpoint that reduces the ability of activated T cells to generate an effective immune response. indicates blocking. High levels of expression of PD-L1 on the tumor cell surface enable escape from anti-tumor responses by inhibiting T cell functions, including cytotoxic activity. PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (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 in normal tissues and peripheral blood T lymphocytes, the majority of tumor infiltrating T lymphocytes predominantly express PD-1, which is an antitumor with impaired upregulation of PD-1 on tumor-reactive T cells. suggest that it may contribute to the immune response (Blood 2009 114(8): 1537). This may be due to the utilization of PD-L1 signaling mediated by PD-L1-expressing tumor cells that interact with PD-1 expressing T cells, resulting in attenuation of T cell activation 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 could enhance CD8+ T cell-mediated killing of tumors. Inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity in the treatment of cancer (eg, tumor immunity), and infection, including both acute and chronic (eg, persistent) infections. . Optimal therapeutic treatment may be combined with substances that block the PD-1 receptor/ligand interaction and directly inhibit tumor growth. There remains a need for optimal therapies for treating, stabilizing, preventing and/or delaying the onset of various cancers.

Therapeutic antibodies targeting PD-1 or PD-L1 block ligand-receptor interactions and restore immune function to the tumor microenvironment. The use of these mAbs has shown exciting clinical responses for many cancer types, and an increasing number of mAbs have entered clinical development. Several therapeutic monoclonal antibodies (mAbs) targeting PD-1 and PD-L1 are commercially available, and more than a dozen traditional and bispecific mAbs are being reviewed by the FDA and EMA. BMS' Opdivo (nivolumab), Merck's Keytruda (Pembrolizumab), Regeneron's Libtayo (Cemiplimab), which are anti-PD1 antibody therapeutics that inhibit PD-1 and PD-L1 binding, and Anti-PD-L1 antibody therapeutics Roche's Tecentriq (Atezolizumab), AstraZeneca's Imfinzi (Durvalumab), and Merck Sereno's Bavencio (Avelumab) have recently received US FDA approval, bringing innovation to the treatment of intractable cancer in clinical practice. The clinical demand for these PD-1/PD-L1 interaction inhibitory antibody therapeutics has exploded. Opdivo's 2018 sales were 7.5 billion US$ and Keytruda's 7.1 billion US$, ranking 4th and 6th in the top ten for prescription drugs The clinical demand for these immune checkpoint inhibitory therapeutic antibodies is expected to expand further in the future.

However, since the antibody is a macromolecular protein with a molecular weight of 150,000, it is difficult to penetrate into the cancer tissue, making it difficult to inhibit the PD-1/PD-L1 binding between tumor cells and immune cells inside the cancer micro-environment. this exists For more effective treatment, the need for a protein therapeutic agent that is much smaller than an antibody and easily penetrates into the cancer tissue has emerged.

However, the PD-1 protein exposed as the extracellular domain of human T cells (ectodomain) is small in size and has the property of binding to the PD-L1 molecule expressed in tumors. For this purpose, PD-1, which is smaller in size than PD-L1 and has excellent cell penetration, is more suitable, but wild-type PD-1 has a problem of binding to PD-L1 with very low affinity (equilibrium dissociation constant = ~8.7 μM). .

It is an object of the present invention to provide a PD-1 mutant having increased binding affinity to PD-L1.

It is also an object of the present invention to provide a binding inhibitor of PD-L1 and PD-1.

Another object of the present invention is to provide a composition for detecting PD-L1.

Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of cancer.

Another object of the present invention is to provide a composition for diagnosing cancer.

It is also an object of the present invention to provide a method for specific detection of PD-L1.

In addition, it is an object of the present invention to provide a method for producing a PD-1 mutant having increased binding affinity to PD-L1.

In order to achieve the above object, the present invention provides a PD-1 mutant with increased binding affinity to PD-L1.

In addition, the present invention provides a binding inhibitor of PD-L1 and PD-1 comprising the PD-1 mutant.

In addition, the present invention provides a composition for detecting PD-L1 comprising the PD-1 mutant.

In addition, the present invention provides a pharmaceutical composition for treating or preventing cancer comprising the PD-1 mutant.

In addition, the present invention provides a composition for diagnosing cancer comprising the PD-1 mutant.

In addition, the present invention provides a method for specific detection of PD-L1.

In addition, the present invention provides a method for producing a PD-1 mutant having increased binding affinity to PD-L1.

The PD-1 mutant of the present invention has fewer mutations compared to conventional PD-1 and PD-1 mutants and has significantly increased binding affinity to PD-L1 than these, so that the immunogenicity problem can be solved, and these variants is a very small protein compared to conventional antibody therapeutics, so it can effectively inhibit the PD-1/PD-L1 binding of tumors and immune cells in the tumor microenvironment, and the conventional PD-1 low binding ability to PD-L1 Since the problem has been solved, the therapeutic effect can be significantly improved as a therapeutic agent, and there is an effect that can be used as an imaging agent for detecting the PD-L1 expression level.

1 is a diagram confirming the critical mutation site for binding to PD-L1 of the JY101 mutant discovered by showing the highest binding affinity to PD-L1 in a previous study.
Figure 2 is a diagram comparing the amino acid sequence of the N-IITV mutant and its aglycosylation mutant Q-IITV having a binding affinity increasing mutation with the wild type.
3 is a SDS-PAGE of a pMaz vector containing dimeric human PD-L1 (PD-L1-Fc) and a purified dimeric PD-L1 protein for exploring the binding affinity of PD-1 variants with PD-L1. It is a diagram showing a picture of a gel.
4 is a diagram confirming the activity of the fluorescently-labeled dimer PD-L1 by binding ability with PD-1 mutants.
Figure 5 shows the results of PD-L1 binding affinity analysis of E. coli cells displaying wild-type PD-1, JY101 mutant, N-IITV mutant, and Q-IITV mutant of the previous study, respectively, as a dimeric human PD-L1 (PD-L1-Fc) is a confirmation of
6 is a diagram comparing the results of analysis of binding affinity to PD-L1 of wild-type PD-1, CKJ 52 of the prior patent, CKJ 52-Y69T prepared in Example 5, and the N_IITV mutant of the present invention.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

Throughout this specification, common one-letter and three-letter codes for naturally occurring amino acids are used as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Amino acids referred to by abbreviation in the present invention are also described according to the IUPAC-IUB nomenclature as follows:

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 and valine: V.

In one aspect, the present invention provides PD-L1 (Programmed death-ligand 1) with increased binding affinity, including amino acid substitutions of F13I, M46I, C69T and G100V in the amino acid of wild type PD-1. 1 (Programmed cell death protein-1) relates to a variant.

In one embodiment, the amino acid of wild-type PD-1 may include the amino acid sequence of SEQ ID NO: 1, and the amino acid position may be based on the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the PD-1 mutant of the present invention may include amino acid substitutions of F13I, M46I, C69T and G100V, whereby N-IITV of SEQ ID NO: 2 with the most significantly increased binding to PD-L1 It may be a variant.

The PD-1 variant of the present invention refers to a wild-type PD-1 protein (or peptide) in which some amino acid sequences are substituted,

As used herein, the term "variant" refers to a corresponding amino acid sequence comprising at least one amino acid difference (substitution, insertion or deletion) compared to a reference substance. In certain embodiments a “variant” has high amino acid sequence homology and/or conservative amino acid substitutions, deletions and/or insertions compared to a reference sequence. In addition, as used herein, the term "PD-1 variant" refers to a PD-1 variant protein mutated in one or more amino acids to modulate its binding activity with PD-L1.

Specifically, the PD-1 variants of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other method in the art. Thus, the peptides according to the present invention can be synthesized in a number of ways, including, for example, those comprising:

(a) a method for synthesizing peptides stepwise or by fragment assembly by means of solid or liquid phase methods, and isolating and purifying the final peptide product; or

(b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or

(c) performing cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; or

A method for obtaining a fragment of a peptide by any combination of (a), (b) and (c), and then ligating the fragments to obtain a peptide, and recovering the peptide.

As a more specific example, through genetic manipulation, a gene encoding the PD-1 mutant of the present invention may be prepared, transformed into a host cell, and then expressed to produce the PD-1 mutant of the present invention.

In one aspect, the present invention relates to a nucleic acid molecule encoding the PD-1 variant of the present invention, a vector comprising the same, and a host cell comprising the vector.

As used herein, the term "nucleic acid molecule" refers to deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form, and includes natural nucleic acid analogs unless otherwise specified (Scheit, Nucleotide Analogs, John Wiley). , New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

As used herein, the term "vector" refers to any nucleic acid comprising a competent nucleotide sequence that is inserted into a host cell and recombined with and inserted into the genome of the host cell, or that replicates spontaneously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like.

As used herein, the term "host cell" refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are introduced, and should be understood to refer not only to a specific target cell, but also to its progeny or potential progeny. In fact, the progeny are not identical to the parent cell as certain modifications may occur in subsequent generations due to mutations or environmental influences, but are still included within the scope of the term as used herein.

In one aspect, the present invention relates to a binding inhibitor of PD-L1 and PD-1 comprising a PD-1 variant of the present invention, a nucleic acid molecule thereof, or a vector comprising the same.

In one aspect, the present invention relates to a composition for detecting PD-L1, comprising the PD-1 variant of the present invention.

In one embodiment, the composition can detect and quantify the protein expression level of PD-L1.

In one embodiment, the PD-1 variant may be labeled with one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material, and the fluorescent material is Cy (cyanine) series, It may be a Rhodamine-based, Alexa-based, BODIPY-based, or ROX-based fluorescent material, such as Nile Red, BODIPY, 4,4-difluoro-4-bora-3a,4a. -diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarin, or Alexa.

Since the PD-1 mutant of the present invention can detect and quantify the expression level of PD-L1, it can be used before administration of conventional immune checkpoint inhibitory antibody therapeutics administered after confirming the presence or absence of PD-L1 overexpression in cancer patients. .

In one aspect, the present invention relates to a composition for bioimaging, comprising the PD-1 variant of the present invention.

In one aspect, the present invention relates to a pharmaceutical composition for treating or preventing cancer, comprising the PD-1 mutant of the present invention, a nucleic acid molecule thereof, or a vector comprising the same.

In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, Small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and It may be any one or more selected from the group consisting of pituitary adenoma.

The pharmaceutical composition of the present invention may be used as a single therapy, but may also be used in combination with other conventional biological therapy, chemotherapy, or radiation therapy, and when such a combination therapy is performed, cancer can be treated more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapeutic agents that can be used together with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, Cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunoru daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transpla Transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate and the like. Radiation therapy that can be used together with the composition of the present invention is X-ray irradiation, γ-ray irradiation, and the like.

In the present invention, the term “prevention” refers to any action that inhibits or delays the occurrence, spread, and recurrence of cancer by administration of the PD-1 mutant according to the present invention or a composition comprising the same.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in 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.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological agents. As used herein, the term “pharmaceutically acceptable” refers to exhibiting non-toxic properties to normal cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and for example, Merck Index, 13th ed., Merck & Co. Inc. The compound described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, external preparations, suppositories, sterile injection solutions and sprays.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In addition, the composition of the present invention may contain one or more active ingredients exhibiting the same or similar function. The composition of the present invention comprises 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the patient's weight, age, sex, health status, The range varies depending on the diet, administration time, administration method, excretion rate, and the severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide and administer once to several times a day.

Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweetening agents, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

In one aspect, the present invention relates to a composition for diagnosing cancer comprising the PD-1 mutant of the present invention.

As used herein, the term “detection” or “measurement” refers to quantifying the concentration of a detected or measured target.

In one aspect, the present invention relates to a cancer diagnosis kit comprising the composition for diagnosing cancer of the present invention.

In one embodiment, the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient, as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA or protein from the sample. there is.

As used herein, the term “cancer diagnosis kit” refers to a kit including the cancer diagnosis composition of the present invention. Therefore, the expression “cancer diagnosis kit” can be used interchangeably or mixed with “cancer diagnosis composition”. As used herein, the term “diagnosis” refers to determining a subject's susceptibility to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, or having a specific disease or disorder. Determining a subject's prognosis (e.g., identifying a pre-metastatic or metastatic cancer state, staging the cancer, or determining the responsiveness of the cancer to treatment), or therametrics (e.g., determining the efficacy of a treatment) monitoring the state of the object to provide information about it).

The PD-1 mutant of the present invention has anticancer activity through cancer cell death by immune cells by specifically binding with PD-L1, which is expressed by cancer cells to evade the killing mechanism by immune cells, with significantly increased binding force. , because it specifically binds to cancer cells, it can be used as a theranostic agent used in the diagnosis of cancer. In addition, instead of scFv, CAR (chimeric antigen receptor)-T cells containing a PD-1 mutant can be produced and used as an anticancer agent, and can also be used as an anticancer adjuvant administered simultaneously, separately or sequentially with the anticancer agent. In addition, since it strongly binds to PD-L1 of cancer cells, it can be used as a drug delivery vehicle targeting cancer cells.

In one aspect, the present invention provides a method comprising: contacting a biological sample isolated from a subject with a PD-1 variant of the present invention; confirming the binding level of the PD-1 variant and PD-L1; And it relates to a method for providing information for cancer diagnosis, comprising the step of comparing the binding level of the PD-1 mutant and PD-L1 in a normal control sample.

In one embodiment, when the binding level of the PD-1 mutant and PD-L1 in the biological sample isolated from the subject is higher than the binding level of the PD-1 mutant and PD-L1 in the normal control sample, the test subject It may further include the step of determining that the cancer is.

As used herein, the term “sample (sample)” refers to a biological sample obtained from a subject or patient. Sources of biological samples may include fresh, frozen and/or preserved organ or tissue samples or solid tissue from biopsies or aspirates; blood or any blood component; The cells may be at any point in the pregnancy or development of the subject.

In one aspect, the present invention relates to a method for specific detection of PD-L1, comprising the steps of contacting a PD-1 mutant with a sample and detecting the binding of the PD-1 mutant to PD-L1.

In one aspect, the present invention provides a method comprising: culturing a host cell comprising a vector containing a nucleic acid molecule encoding a PD-1 variant of the present invention; And it relates to a method for producing a PD-1 mutant having increased binding affinity to PD-L1, comprising the step of recovering the PD-1 mutant expressed by the host cell.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Search for mutations important for increasing the binding affinity of PD-1 mutants

Critical mutation of the JY101 variant (L1S, F13I, L17M, S36P, M46I, C69T, G79R, G100V, L114P and A139L) discovered by the inventor of the present invention by showing the highest binding affinity to PD-L1 in previous studies site), a total of 10 mutations S1, I13, M17, P36, I46, T69, R79, V100, P114 and L139 of JY 101 were substituted one by one with wild-type PD-1 amino acids (S1L, I13F, M17L, P36S). , I46M, T69C, R79G, V100G, P114L, L139A). For this purpose, the genome was amplified by Quikchange PCR using the designed primer and Pfu turbo polymerase (Agilent), and the sequence was confirmed by transforming the amplified gene into Jude1. Specifically, E. coli expressing the genes of JY 101 and 10 variants (S1L, I13F, M17L, P36S, I46M, T69C, R79G, V100G, P114L, L139A) was treated with 2% glucose and 40 μg/ml of chloramphenicol, respectively. It was cultured for 16 hours at 37°C and 250 rpm in the included TB medium. The cultured cells were inoculated into 6 ml of TB medium containing 40 μg/ml of chloramphenicol at a ratio of 1:50. After incubation to OD ₆₀₀ = 0.5, after cooling at 25°C and 250 rpm for 20 minutes, 1 mM IPTG was added to overexpress the protein at 25°C at 250 rpm for 5 hours. E. coli overexpressed with protein was placed in an e-tube by the same amount through OD ₆₀₀ normalization, and centrifuged at 14,000 rpm for 1 minute to recover cells. In order to remove the residual medium, the cells in the e-tube were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0) and the washing process of centrifuging at 13,500 rpm for 1 minute was repeated twice. Cells were resuspended using 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution and the outer membrane was removed by rotation at 37°C for 30 minutes. After collecting E. coli by centrifugation at 13,500 rpm for 1 minute, the supernatant was removed. The centrifuged E. coli was resuspended in 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH6.8] and centrifuged at 13,500 rpm for 1 minute. 1 ml of Solution A and 20 μl of a 50 mg/ml lysozyme solution were added to 1 ml of the centrifuged E. coli to resuspend the centrifuged E. coli, and then rotated at 37° C. for 15 minutes to remove the peptidoglycan layer. After centrifugation, remove the supernatant, resuspend in 1 ml of PBS, take 300 μl, add 700 μl of PBS and 3 nM tetrameric PD-L1-Alexa488 probe, and rotate at room temperature to obtain spheroplast. was labeled with a fluorescent probe. After 1 hour of labeling, centrifugation was performed at 13,500 rpm for 1 minute. The supernatant was discarded and the centrifuged E. coli was washed once with 1 ml of PBS and then centrifuged again at 13,500 rpm for 1 minute. After the centrifuged E. coli was resuspended in 1 ml of PBS, it was analyzed using Guava (Merck Millipore) equipment. As a result, when all four of 13I, 46I, 69T, and 100V were replaced with the amino acids of wild-type PD-1, respectively, the binding force was significantly reduced (Fig. 1). I could see that it was important.

Example 2. Variant (N-IITV) and its aglycosylation mutant (Q-IITV) having a mutation increasing binding affinity

2-1. N-IITV production

A PD-1 mutant N-IITV having four binding affinity increasing mutations (13I, 46I, 69T and 100V) of the JY 101 mutant identified in Example 1 was prepared. Specifically, the genome was amplified by Gene assembly PCR using a total of 8 primers and Vent polymerase, and then the amplified genome was treated with Sfi I enzyme. The Sfi I-treated DNA was ligated into the Sfi I-treated pMopac12-NlpA-FLAG vector to prepare a pMopac12-NlpA-PD1_N-IITV-FLAG vector. After that, a single clone was secured by transformation into E. coli Jude1, and the pMopac-12 vector insertion was confirmed through nucleotide sequence analysis, thereby securing PD-1 mutant N-IITV having 4 major mutations that increase binding to PD-L1. (N-IITV in Fig. 2).

2-2. Q-IITV production

Like the N-IITV mutant, in order to produce a mutant with aglycosylation characteristics while having 4 binding affinity-increasing mutations (13I, 46I, 69T and 100V) of the JY 101 mutant, a total of 4 sites of wild-type PD-1 (25N, New PD-1 mutant Q-IITV (F13I, N25Q, N34Q, M46I, N50Q, C69T, N92Q and G100V) were fabricated. For this, the Q-IITV (F13I, N25Q, N34Q, M46I, N50Q, C69T, N92Q and G100V) genome was synthesized (Genescript), and the synthesized genome was amplified by PCR using primers and Vent polymerase, followed by Sfi The pMopac12-NlpA-PD1_Q-IITV-FLAG vector was prepared by ligation to the Sfi I-treated pMopac12-NlpA-FLAG vector by treatment with I enzyme. After that, it was transformed into E. coli Jude1 to secure a single clone, and it was confirmed that it was successfully inserted into the pMopac-12 vector through sequencing (Q-IITV in FIG. 2).

Example 3. Dimeric human PD-L1 (PD-L1-Fc) construction to explore the binding affinity of PD-1 variants with PD-L1

3-1. PD-L1-Fc Cloning

In order to more efficiently search for the binding ability of mutants discovered by increasing the avidity (Avidity) to PD-L1, in order to more efficiently screen aglycosylated PD-1 mutants by increasing the avidity (Avidity), PD-L1 A dimerization reaction was induced by expressing the Fc domain of the antibody IgG at the C-terminal portion, and a GS linker was inserted between Fc and PD-L1 to ensure the fluidity of each protein. Specifically, PD-L1 and Fc genes were amplified using primers and Vent polymerase (New England Biolab), respectively, and then assembly PCR was performed using Vent polymerase. The produced gene was treated with Bss HII and Xba I (New England Biolab), and the restriction enzyme-treated PD-L1-Fc gene was ligated into the same restriction enzyme-treated pMAZ vector. After transforming the ligated plasmid into Jude1 Escherichia coli, a single clone was obtained and it was confirmed through nucleotide sequence analysis that PD-L1-Fc was successfully inserted into the pMAZ vector.

3-2. Animal Cell Expression, Purification and Labeling of PD-L1-Fc

Expi293F cells were subcultured at a density of 2x10 ⁶ cells/ml in 300 ml, and one day later, the PD-L1-Fc expression vector prepared in Example 3-1 was transfected into Expi293F animal cells using PEI. The transfected cells were cultured for 7 days at 37 °C, 125 rpm, and 8% CO ₂ in a CO ₂ shaker incubator, and then centrifuged to take only the supernatant. Then, it was equilibrated with 25x PBS and filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. After adding 1 ml of Protein A resin to the filtered culture, it was stirred at 4 °C for 16 hours, passed through a column to recover the resin, and then washed with 10 ml of PBS. The washed resin was eluted with 100 mM glycine pH 2.7 buffer and then neutralized with 1M Tris-HCl pH 8.0 to obtain purified PD-L1 dimer protein (FIG. 3). The purified PD-L1 dimer protein was fluorescently labeled using the Alexa-488 labeling kit. To confirm the binding affinity of the fluorescently-labeled dimeric PD-L1, E. coli expressing wild-type PD-1 and JY 101 genes were respectively treated in TB medium containing 2% glucose and 40 μg/ml chloramphenicol at 37°C at 250 rpm. was incubated 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. After incubation to OD ₆₀₀ = 0.5, cooling was performed at 25° C. at 250 rpm for 20 minutes, and 1 mM IPTG was added to overexpress the protein at 25° C. at 250 rpm for 5 hours. E. coli overexpressed with protein was put into an e-tube by the same amount through OD ₆₀₀ normalization, and centrifuged at 14,000 rpm for 1 minute to recover cells. In order to remove the residual medium, the cells in the e-tube were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0) and the washing process of centrifuging at 13,500 rpm for 1 minute was repeated twice. Cells were resuspended using 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution, and the outer membrane was removed through rotation at 37°C for 30 minutes. After collecting E. coli by centrifugation at 13,500 rpm for 1 minute, the supernatant was removed. The centrifuged E. coli was resuspended in 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS pH6.8] and centrifuged at 13,500 rpm for 1 minute. 1 ml of a solution of Solution A and 20 μl of 50 mg/ml lysozyme solution was added to resuspend the centrifuged E. coli, and then rotated at 37° C. for 15 minutes to remove the peptidoglycan layer. After centrifugation, remove the supernatant, resuspend in 1 ml of PBS, take 300 μl, add 700 μl of PBS, 25 nM, 50 nM dimeric PD-L1-Alexa488 probe, rotate at room temperature, and add to the spheroplast Labeled with a fluorescent probe. After 1 hour of labeling, centrifugation was performed at 13,500 rpm for 1 minute. The supernatant was discarded and the centrifuged E. coli was washed once with 1 ml of PBS and then centrifuged again at 13,500 rpm for 1 minute. After the centrifuged E. coli was resuspended in 1 ml of PBS, it was analyzed using Calibur (BD Biosciences) equipment. As a result of the analysis, it was confirmed that the fluorescently labeled dimer PD-L1 was active ( FIG. 4 ).

Example 4. PD-L1 avidity analysis of mutant (N-IITV) and its aglycosylation mutant (Q-IITV) having an affinity enhancing mutation (Q-IITV)

In order to verify the binding affinity of PD-1 mutants 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 each treated with 2% glucose and Incubated in TB medium containing 40 μg/ml of chloramphenicol at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated in 6 ml of TB medium containing 40 μg/ml of chloramphenicol at a ratio of 1:100, incubated to OD ₆₀₀ = 0.5, cooled at 25° C. for 20 minutes at 250 rpm, and 1 mM IPTG was added to overexpress the protein at 25 °C, 250 rpm, for 5 hours. E. coli overexpressed with protein was put into an e-tube in the same amount and centrifuged at 14,000 rpm for 1 minute to recover cells. To remove the residual medium, the cells placed in the e-tube were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0) and washed twice by centrifugation at 13,500 rpm for 1 minute. Cells were resuspended using 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution, and the outer membrane was removed by rotation at 37°C for 30 minutes, followed by 13,500 rpm for 1 minute. After collecting E. coli by centrifugation, the supernatant was removed. The centrifuged E. coli was resuspended in 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH6.8] and centrifuged at 13,500 rpm for 1 minute. 1 ml of Solution A and a mixed solution of 20 μl of 50 mg/ml lysozyme solution were added to 1 ml of centrifuged E. coli and then rotated at 37° C. for 15 minutes to remove the peptidoglycan layer. After centrifugation, remove the supernatant, resuspend in 1 ml of PBS, take 300 μl, put 700 μl of PBS and 50 nM dimeric PD-L1-Fc-Alexa488 probe prepared in Example 3, and rotate at room temperature to Loplast was labeled with a fluorescent probe. After 1 hour of labeling, centrifugation was performed at 13,500 rpm for 1 minute, the supernatant was discarded, and the centrifuged E. coli was washed once with 1 ml of PBS, and then centrifuged again at 13,500 rpm for 1 minute. After the centrifuged E. coli was resuspended in 1 ml of PBS, binding to PD-L1 was analyzed using Calibur (BD Biosciences) equipment, respectively. Also, since the expression level of PD-1 protein displayed by E. coli can affect the fluorescence intensity, 300 μl of the remaining E. coli is taken from the remaining E. coli after resuspending in PBS to check the expression level, and 700 μl of PBS and 0.5 μl of Anti-FLAG-FITC was added and rotated at room temperature to label spheroplasts with a fluorescent probe. After 1 hour of labeling, centrifugation was performed at 13,500 rpm for 1 minute, the supernatant was discarded, and the centrifuged E. coli was washed once with 1 ml of PBS, and then centrifuged again at 13,500 rpm for 1 minute. After centrifuged E. coli was resuspended in 1 ml of PBS, the expression level of PD-1 proteins was indirectly analyzed using Calibur (BD Biosciences) equipment.

As a result, although the expression levels of the N-IITV mutant and Q-IITV mutant discovered in the present invention were lower than that of wild-type PD-1 and the JY 101 mutant discovered in the previous study, the N-IITV mutant and It was shown that the Q-IITV variant had a higher binding affinity to PD-L1 ( FIG. 5 ). Through this, it was confirmed that N-IIITV and Q-IITV bound to PD-L1 significantly stronger than that of JY 101, and in particular, it was confirmed that N-IITV had a very superior binding force to PD-L1 than Q-IITV.

Example 5. Comparison of binding force according to the type of amino acid mutation

In order to confirm the difference in binding strength with PD-L1 according to the type of amino acid mutated at the amino acid mutation position of the N-IITV variant of the present invention, the CKJ 52 mutant (F13) of the present inventor's prior patent (No. 10-2019-0011181) , M46I and C69Y) (SEQ ID NO: 54 of the prior patent) was further prepared in a CKJ 52-Y69T variant in which the C69Y mutation was replaced with Y69T. For this purpose, the genome was amplified by Quikchange PCR using the primer designed for the pMopac12-NlpA-CKJ52-FLAG plasmid prepared for this purpose and Pfu turbo polymerase (Agilent), and then the amplified gene was transformed into Jude1 to confirm the sequence. -Y69T mutant was additionally obtained. Thereafter, wild-type PD-1, CKJ 52, CKJ 52-Y69T and wild-type PD-1, CKJ 52, CKJ 52-Y69T and E. coli each expressing N-IITV was cultured in TB medium containing 2% glucose and 40 μg/ml of chloramphenicol, respectively, at 37° C. and 250 rpm for 16 hours. The cultured cells were inoculated in 6 ml of TB medium containing 40 μg/ml of chloramphenicol at a ratio of 1:100, incubated to OD ₆₀₀ = 0.5, and then cooled at 25°C and 250 rpm for 20 minutes, 1 The protein was overexpressed at 25°C, 250 rpm, for 5 hours by adding mM IPTG. E. coli overexpressed with protein was put into an e-tube in the same amount and centrifuged at 14,000 rpm for 1 minute to recover cells. In order to remove the residual medium, the cells placed in the e-tube were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0) and washed twice by centrifugation at 13,500 rpm for 1 minute. Cells were resuspended using 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution, and the outer membrane was removed by rotation at 37°C for 30 minutes, followed by 13,500 rpm for 1 minute. After collecting E. coli by centrifugation, the supernatant was removed. The centrifuged E. coli was resuspended in 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH6.8] and centrifuged at 13,500 rpm for 1 minute. 1 ml of a solution of Solution A and 20 μl of 50 mg/ml lysozyme solution was added to resuspend the centrifuged E. coli, and then rotated at 37° C. for 15 minutes to remove the peptidoglycan layer. After centrifugation, remove the supernatant, resuspend in 1 ml of PBS, take 300 μl, add 700 μl of PBS and 200 nM PD-L1-Fc-Alexa488 probe, rotate at room temperature, and label the spheroplast with a fluorescent probe. did After 1 hour of labeling, centrifugation was performed at 13,500 rpm for 1 minute, the supernatant was discarded, and the centrifuged E. coli was washed once with 1 ml of PBS, and then centrifuged again at 13,500 rpm for 1 minute. After centrifuged E. coli was resuspended in 1 ml of PBS, binding to PD-L1 was analyzed using Calibur (BD Biosciences) equipment, respectively.

As a result, CKJ 52-Y69T mutants (13I, 46I and 69T) in which amino acid 69 was substituted with T than CKJ 52 showed higher binding affinity to PD-L1, and the present invention having an additional 100V mutation It was found that the N-IITV variants (13I, 46I, 69T and 100V) exhibited significantly higher binding affinity ( FIG. 6 ).

<110> Korea University Research and Business Foundation <120> PD-1 variants with increased binding affinity to PD-L1 <130> DP-2020-0575 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 143 <212> PRT <213> Homo sapiens <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 Gln Phe Gln 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 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 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

Claims (14)

Programmed cell death protein (PD-1) with enhanced binding to PD-L1 (Programmed death-ligand 1), including amino acid substitutions of F13I, M46I, C69T and G100V in the amino acids of wild type PD-1 1) Variant. The PD-1 mutant of claim 1, wherein the amino acid of wild-type PD-1 comprises the amino acid sequence of SEQ ID NO: 1. A nucleic acid molecule encoding the PD-1 variant of claim 1. A vector comprising the nucleic acid molecule of claim 3. A host cell comprising the vector of claim 4. A binding inhibitor of PD-L1 and PD-1 comprising the PD-1 variant of claim 1, the nucleic acid molecule of claim 3, or the vector of claim 4. A composition for detecting PD-L1, comprising the PD-1 variant of claim 1. The composition for detecting PD-L1 according to claim 7, wherein the PD-1 variant is labeled with one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material. A pharmaceutical composition for treating or preventing cancer comprising the PD-1 variant of claim 1, the nucleic acid molecule of claim 3, or the vector of claim 4 as an active ingredient. 10. The method of claim 9, wherein the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer , small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, renal or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma And any one or more selected from the group consisting of pituitary adenoma, a pharmaceutical composition for the treatment or prevention of cancer. A composition for diagnosing cancer comprising the PD-1 mutant of claim 1. a) contacting the biological sample isolated from the subject with the PD-1 variant of claim 1;
b) determining the binding level of the PD-1 variant and PD-L1; and
c) Comprising the step of comparing the binding level of the PD-1 mutant and PD-L1 in a normal control sample, an information providing method for cancer diagnosis. A method for specific detection of PD-L1 comprising the steps of contacting the PD-1 mutant of claim 1 with a sample and detecting the binding of the PD-1 mutant to PD-L1. a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the PD-1 variant of claim 1; and
b) A method for producing a PD-1 mutant with increased binding affinity to PD-L1, comprising the step of recovering the PD-1 mutant expressed by the host cell.

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