KR102605204B1 - Pd-1 variants with increased binding to pd-l1 - Google Patents

Abstract

The present invention relates to PD-1 variants with increased binding affinity to PD-L1 and non-glycosylated properties. The PD-1 variants of the present invention have significantly increased PD-L1 binding capacity compared to conventional PD-1 variants. Although it is a non-glycosylated variant, it has improved binding and stability, so it can simultaneously overcome the low binding affinity of conventional PD-1 to PD-L1 and the problem of glycan heterogeneity in protein therapeutics, even in bacteria. It is easy to mass-produce inexpensively and has the effect of enabling the manufacture of biological drugs without problems with cell lines, culture processes, and purification processes.

Description

PD-1 variants with increased binding to PD-L1 {PD-1 VARIANTS WITH INCREASED BINDING TO PD-L1}

The present invention relates to PD-1 variants that have increased binding affinity to PD-L1 and have non-glycosylated properties.

Medicines for cancer treatment are largely divided into small molecule medicines and high molecule medicines. Compared to low molecule medicines, which have relatively greater side effects due to lack of specificity, high molecule medicines with specificity are receiving more attention as therapeutic agents. Cancer cells express immune checkpoint proteins on the cell surface, which are used by normal cells to suppress immune cell activation in order to avoid the killing mechanism of immune cells. Recently, immunotherapy has been used as a method to treat cancer. Research on checkpoint inhibitor proteins is actively underway.

It has been reported in the academic world that among immune checkpoint inhibitory proteins, blocking PD-1/PD-L1 binding in particular has a great effect in cancer treatment and has fewer side effects compared to other immune checkpoint inhibitory proteins (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). PD-1 receptors are expressed on the surface of activated immune cell types, including T cells, B cells, and natural killer (NK)/natural killer T (NKT) cells (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 acts as an immune checkpoint that reduces the ability of activated T cells to generate an effective immune response. Indicates blocking. High levels of PD-L1 expression 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, suggesting that upregulation of PD-1 on tumor-reactive T cells is impaired in anti-tumor activity. It suggests that it may contribute to the immune response (Blood 2009 114(8): 1537). This may be due to the exploitation 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). Therefore, inhibition of PD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing of tumors. Inhibition of PD-L1 signaling has been proposed in the treatment of cancer (e.g., tumor immunity) and as a means to enhance T cell immunity against infections, including both acute and chronic (e.g., persistent) infections. . The optimal therapeutic treatment may combine blockade of the PD-1 receptor/ligand interaction with agents that directly inhibit tumor growth. There remains a need for optimal therapies to treat, stabilize, prevent and/or delay the development of various cancers.

Therapeutic antibodies targeting PD-1 or PD-L1 block the ligand-receptor interaction and restore immune function to the tumor microenvironment. The use of these mAbs has resulted in exciting clinical responses against 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 under review by the FDA and EMA. Anti-PD1 antibody treatments that inhibit PD-1 and PD-L1 binding capacity include BMS's Opdivo (nivolumab), Merck's Keytruda (Pembrolizumab), Regeneron's Libtayo (Cemiplimab), and anti-PD-L1 antibody treatments. Roche's Tecentriq (Atezolizumab), AstraZeneca's Imfinzi (Durvalumab), and Merck Sereno's Bavencio (Avelumab) were recently approved by the US FDA and are bringing innovation to the treatment of incurable cancer in clinical settings. Clinical demand for these PD-1/PD-L1 interaction-inhibiting antibody treatments has grown explosively, with 2018 sales of Opdivo reaching 7.5 billion US$ and Keytruda reaching 7.1 billion US$, ranking 4th and 6th among prescription drugs in terms of sales. It is expected that the clinical demand for these immune checkpoint inhibitor therapeutic antibodies will further expand in the future.

However, since antibodies are macromolecular proteins with a molecular weight of 150,000, they have difficulty penetrating into cancer tissue and thus have difficulty inhibiting the PD-1/PD-L1 binding of tumor cells and immune cells within the tumor micro-environment. This exists. For more effective treatment, there is a need for protein treatments that are much smaller than antibodies and can easily penetrate into cancer tissue.

The PD-1 protein exposed as the extracellular domain (ectodomain) of human T cells is small and has the property of binding to PD-L1 molecules expressed on tumors, so it is necessary to treat cancer through effective immune checkpoint inhibition. PD-1, which is smaller than PD-L1 and has excellent cell penetration, is more suitable, but wild-type PD-1 has the problem of binding to PD-L1 with very low affinity (equilibrium dissociation constant = ~8.7 μM).

Meanwhile, immune checkpoint inhibitor antibody treatments such as Keytruda and Opdivo are administered after confirming the presence or absence of PD-L1 overexpression in cancer patients, so a method that can effectively diagnose the presence or absence of PD-L1 overexpression among cancer patients is needed.

The purpose of the present invention is to provide PD-1 variants with increased binding affinity to PD-L1.

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

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

Additionally, an object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer.

Additionally, an object of the present invention is to provide a composition for diagnosing cancer.

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

In addition, the purpose of the present invention is to provide a method for producing unglycosylated PD-1 with increased binding affinity to PD-L1.

To achieve the above object, the present invention provides PD-1 variants with increased binding affinity to PD-L1.

Additionally, the present invention provides a binding inhibitor of PD-L1 and PD-1, including the PD-1 variant.

Additionally, the present invention provides a composition for detecting PD-L1 containing the PD-1 variant.

Additionally, the present invention provides a pharmaceutical composition for treating or preventing cancer containing the PD-1 variant.

Additionally, the present invention provides a composition for diagnosing cancer containing the PD-1 variant.

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

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

The PD-1 variant of the present invention has a significantly increased binding ability to PD-L1 compared to the conventional PD-1 variant, and although it is a non-glycosylated variant, the binding strength and stability are rather improved, so it binds to PD-L1 of the conventional PD-1. It can simultaneously overcome the problem of low binding affinity to protein therapeutics and the problem of glycan heterogeneity of protein therapeutics, making it easy to mass-produce inexpensively even in bacteria, and manufacturing biological drugs without problems due to cell lines, culture processes, and purification processes. There is.

Figure 1 is a diagram showing the mutation site important for binding to PD-L1 in PD-1 variant JY 101 confirmed by FACS.
Figure 2 is a diagram confirming the binding affinity of the non-glycosylated PD-1 variant Q_IITV with PD-L1.
Figure 3 is a diagram showing an SDS-PAGE gel photograph of the pMaz vector containing PD-L1-Fc and purified PD-L1 dimer protein.
Figure 4 is a diagram confirming the activity of fluorescently labeled dimeric PD-L1 by binding affinity to PD-1 variants.
Figure 5 is a diagram showing amino acid sequence analysis data of the constructed error prone library.
Figure 6 is a diagram showing the results of a library enrichment test using a flow cytometer.
Figure 7 is a diagram showing the results of PD-L1 binding affinity analysis of E. coli cells displaying non-glycosylated PD-1 variants.
Figure 8 is a diagram showing expression vectors for non-glycosylated PD-1 variants, SDS-PAGE gel photographs and yields of purified non-glycosylated PD-1 variant proteins.
Figure 9 is a diagram showing sensorgrams for each concentration of each variant.
Figure 10 is a diagram showing the K _D value of each variant.
Figure 11 is a diagram showing sensorgrams by concentration of the glycosylated variant HAC and the non-glycosylated variant Q12 (JY_Q12).
Figure 12 is a diagram showing the K _D values of the glycosylated variant HAC and the non-glycosylated variant Q12 (JY_Q12).

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are hereby incorporated by reference.

Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used, as well as generally acceptable codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Additionally, amino acids referred to by abbreviations in the present invention are 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 relates to the 8th, 9th, 13th, 25th, 31st, 32nd, 33rd, 34th, 37th, 46th, and 47th amino acid sequence of wild type PD-1. At least one selected from the group consisting of the 50th, 52nd, 69th, 87th, 88th, 90th, 92nd, 96th, 100th, 108th, 128th, 138th and 140th amino acids It relates to PD-1 (Programmed cell death protein-1) variants with increased binding to PD-L1 (Programmed death-ligand 1) in which amino acids are substituted with a sequence different from that of the wild type.

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 variant of the invention is W8L, N9D, F13I, S31G, F32L, S33P, N34R, E37K, M46I, S47G, T52M, C69T, V87D, R88G, R90Q, T96S, G100V, A108V, P128R. , P138S, and G140C.

In one embodiment, the PD-1 variant of the invention is W8L, N9D, F13I, S31G, F32L, S33P, N34R, E37K, M46I, S47G, T52M, C69T, V87D, R88G, R90Q, T96S, G100V, A108V, P128R. , P138S, and G140C, and may contain one or more amino acid substitutions selected from the group consisting of P138S and G140C, which may increase binding affinity to PD-L1.

In one embodiment, the PD-1 variant of the invention may include amino acid substitutions F13I, M46I, C69T and G100V, including W8L, N9D, N25Q, S31G, F32L, S33P, N34Q, N34R, E37K, S47G, It may further include one or more amino acid substitutions selected from the group consisting of N50Q, T52M, V87D, R88G, R90Q, N92Q, T96S, A108V, P128R, P138S and G140C.

In one embodiment, the PD-1 variant of the present invention may further include amino acid substitutions N25Q, N34Q, N50Q and N92Q, thereby allowing it to have non-glycosylated properties.

In one embodiment, the PD-1 variant of the invention may include amino acid substitutions F13I, N25Q, N34Q, M46I, N50Q, C69T, N92Q, and G100V.

In one embodiment, the PD-1 variant of the invention may include amino acid substitutions F13I, N25Q, F32L, N34Q, M46I, N50Q, T52M, C69T, V87D, N92Q, T96S, G100V, and A108V.

In one embodiment, the PD-1 variant of the invention may include amino acid substitutions W8L, N9D, F13I, N25Q, N34Q, E37K, M46I, N50Q, C69T, N92Q, G100V, A108V, and G140C.

In one embodiment, the PD-1 variant of the invention may include amino acid substitutions F13I, N25Q, S31G, F32L, S33P, N34Q, M46I, S47G, N50Q, T52M, C69T, N92Q, G100V, A108V, P128R, and P138S. there is.

In one embodiment, the PD-1 variant of the invention may include amino acid substitutions F13I, N25Q, N34R, M46I, N50Q, C69T, R88G, R90Q, N92Q, G100V, and A108V.

Because there is an N-linked glycosylation site in the ectodomain of PD-1, there has been a problem of glycan heterogeneity, which is a difference in glycosylation patterns depending on the cell line, culture process, and purification process. The PD-1 variant of the present invention has significantly improved binding ability to PD-L1 compared to existing variants due to the above amino acid substitution, and has non-glycosylated properties due to the specific amino acid substitution, making it easy to mass-produce inexpensively even in bacteria. And, there is no problem of heterogeneity of glycosylation due to cell line, culture process, and purification process, so it has a great advantage in manufacturing biological drugs. In addition, although N-glycosylation is very important for the binding affinity of PD-1 protein to PD-L1 and protein stability, it is an unglycosylated variant with improved binding affinity and stability.

The PD-1 variant of the present invention refers to a partial amino acid sequence substitution in the wild-type PD-1 protein (or peptide),

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

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

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

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

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

A method of obtaining fragments of a peptide using any combination of (a), (b), and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

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

The term “nucleic acid molecule” used in the present invention refers to deoxyribonucleotides or ribonucleotides that exist in single-stranded or double-stranded form, and includes natural nucleic acid analogues unless specifically stated otherwise (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 containing a competent nucleotide sequence that is inserted into a host cell and recombines with and integrates into the host cell genome, or replicates spontaneously as an episome. These vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, etc.

The term “host cell” used in the present invention 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 descendants or potential descendants. In fact, the progeny are not identical to the parent cell since 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 the PD-1 variant of the present invention, its nucleic acid molecule, or a vector containing it.

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 chromophore, a luminescent substance, and a fluorescent substance, and the fluorescent substance is a Cy (cyanine) series, It may be a Rhodamine series, Alexa series, BODIPY series or ROX series fluorescent substance, such as Nile Red, BODIPY, 4,4-difluoro-4-bora-3a,4a -diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine, or Alexa.

Since the PD-1 variant of the present invention can detect and quantify the expression level of PD-L1, it can be used before the administration of conventional immune checkpoint inhibitory antibody treatments 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 biological imaging, comprising the PD-1 variant of the present invention.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer comprising the PD-1 variant of the present invention, its nucleic acid molecule, or a vector containing it.

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, colon cancer, Small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal or ureteral 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 can be used as a stand-alone therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy. When such combination therapy is performed, cancer can be treated more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapy agents that can be used 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 Includes transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate. Radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and γ-ray irradiation.

In the present invention, the term “prevention” refers to all actions that inhibit or delay the occurrence, spread, and recurrence of cancer by administering the PD-1 variant or a composition containing the same according to the present invention.

The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as administration method, target site, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, 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.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the field of medicine. It can be decided depending on 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. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. As used in the present invention, the term “pharmaceutically acceptable” means that the composition exhibits 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, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient 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 including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays.

The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more commonly used in biological products. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar functions. The composition of the present invention contains 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 pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, 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, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained 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 can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on the desired method, and the dosage is determined by the patient's weight, age, gender, health condition, The range varies depending on diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage 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 administer it once or several times a day.

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

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

As used herein, the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.

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

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

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

In one aspect, the present invention includes contacting a biological sample isolated from a subject with the PD-1 variant of the present invention; Confirming the binding level of the PD-1 variant and PD-L1; and comparing the binding level of the PD-1 variant and PD-L1 in a normal control sample.

In one embodiment, when the binding level of the PD-1 variant and PD-L1 in the biological sample isolated from the subject is higher than the binding level of the PD-1 variant and PD-L1 in the normal control sample, the test subject An additional step of determining that this may be cancer may be included.

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

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

In one aspect, the present invention includes culturing a host cell containing a vector containing a nucleic acid molecule encoding the PD-1 variant of the present invention; and a method for producing aglycosylated PD-1 with increased binding affinity to PD-L1, comprising recovering the PD-1 variant expressed by the host cell.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Search for major mutations in PD-1 that improve binding affinity to PD-L1

The JY 101 variant that showed the highest binding affinity to PD-L1 discovered on the display system (a variant with mutations substituted from wild-type PD-1 to S1, I13, M17, P36, I46, T69, R79, V100, P114, and L139) ), its 10 mutations were replaced one by one with the amino acids of wild-type PD-1 (S1L, I13F, M17L, P36S, I46M, T69C, R79G, V100G, P114L and L139A). Specifically, the genome was amplified using the QuikChange PCR technique using primers designed for this purpose and Pfu turbo polymerase (Agilent). The amplified gene was transformed into Jude1 and the sequence was confirmed. Afterwards, JY 101 and its 10 variants (S1L, I13F, M17L, P36S, I46M, T69C, R79G, V100G, P114L and L139A) were grown in TB medium containing 2% glucose and 40 μg/ml chloramphenicol, respectively, for 37 days. Cultured for 16 hours at ℃ and 250 rpm. The cultured cells were inoculated at a ratio of 1:50 in 6 ml of TB medium containing 40 μg/ml chloramphenicol, cultured until OD ₆₀₀ = 0.5, cooled at 25°C and 250 rpm for 20 minutes, and 1 mM IPTG was added. The protein was overexpressed at 25°C, 250 rpm, for 5 hours. Equal amounts of protein-overexpressed E. coli were placed in e-tubes and centrifuged at 14,000 rpm for 1 minute to recover cells. To remove the remaining 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 centrifugation at 13,500 rpm for 1 minute was repeated twice. The cells were resuspended using 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution and rotated at 37°C for 30 minutes to remove the outer cell membrane. E. coli were collected by centrifugation at 13,500 rpm for 1 minute, and the supernatant was removed. The centrifuged E. coli was resuspended with 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS pH6.8] and then centrifuged at 13,500 rpm for 1 minute. 1 ml of a mixture of 1 ml 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, the supernatant was removed, resuspended in 1 ml of PBS, 300 μl was taken, 700 μl of PBS and 3 nM tetrameric PD-L1-Alexa488 probe were added, and the mixture was rotated at room temperature to form spheroplasts. ) was labeled with a fluorescent probe. After the labeling process for 1 hour, 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 centrifuged again at 13,500 rpm for 1 minute. After resuspending the centrifuged E. coli with 1 ml of PBS, the change in binding capacity of each variant was analyzed using Guava (Merck Millipore) equipment.

As a result, when the mutations 13I, 46I, 69T, and 100V were each mutated to the wild type, the binding affinity was found to be significantly reduced, confirming that these four mutation sites are important for binding affinity to PD-L1 (Figure One).

Example 2. Production of non-glycosylated variants

In order to produce a PD-1 variant in which a mutation critical for improving binding affinity to PD-L1 was introduced and the N-glycosylation site was removed, four binding affinity enhancing mutations (13I, 46I) of the JY 101 variant identified in the above example were used. , 69T and 100V), and has a total of four N-glycosylation sites (25N, 34N, 50N, 92N) to glutamine (Q), which has the most similar properties and similar structure to asparagine (N), but is not glycosylated. ) A new non-glycosylated variant Q_IITV (SEQ ID NO: 2) was produced. Specifically, the genome of Q_IITV, a PD-1 variant with mutations of F13I, N25Q, N34Q, M46I, N50Q, C69T, N92Q, and G100V, was synthesized (Genescript), and the synthesized genome was subjected to PCR using primers and Vent polymerase. After amplification, the amplified genome was treated with Sfi I enzyme. To use the NlpA signal peptide, which is a signal peptide that secretes a protein into the E. coli periplasmic region and immobilizes it on the cell inner membrane, the Sfi I-treated DNA was ligated into the Sfi I-treated pMopac12-NlpA-FLAG vector to create pMopac12- The NlpA-PD1_Q_IITV-FLAG vector was prepared. Afterwards, a single clone was obtained by transformation into E. coli Jude1, and the insertion of the pMopac-12 vector was confirmed through base sequence analysis to produce a non-glycosylated PD-1 variant Q_IITV with improved binding to PD-L1. To verify the binding ability of the constructed Q_IITV mutant to PD-L1, E. coli expressing wild-type PD-1, JY 101, and Q_IITV were cultured in TB medium containing 2% glucose and 40 μg/ml of chloramphenicol, respectively, at 37°C and 250°C. Cultured for 16 hours at rpm. The cultured cells were inoculated at a 1:50 ratio in 6 ml of TB medium containing 40 μg/ml chloramphenicol, cultured until OD ₆₀₀ = 0.5, cooled at 25°C and 250 rpm for 20 minutes, and added with 1 mM IPTG. was added to overexpress the protein at 25°C, 250 rpm, for 5 hours. An equal amount of E. coli overexpressing the protein was placed in an e-tube and centrifuged at 14,000 rpm for 1 minute to recover the cells. To remove 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, rotated for 30 minutes at 37°C to remove the outer cell membrane, and then rotated at 13,500 rpm for 1 minute. After collecting E. coli by centrifugation, the supernatant was removed. The centrifuged E. coli was resuspended with 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS pH6.8] and then centrifuged at 13,500 rpm for 1 minute. 1 ml of a mixture of 1 ml of Solution A and 20 μl of 50 mg/ml lysozyme solution was added to resuspend the centrifuged E. coli and rotated at 37°C for 15 minutes to remove the peptidoglycan layer. After centrifugation, the supernatant was removed, resuspended in 1 ml of PBS, 300 μl was taken, 700 μl of PBS and 4 nM tetrameric PD-L1-Alexa488 probe were added, and the spheroplasts were labeled with a fluorescent probe by rotating at room temperature. did. After the labeling process for 1 hour, 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 centrifuged again at 13,500 rpm for 1 minute. After resuspending the centrifuged E. coli with 1 ml of PBS, their binding ability to PD-L1 was analyzed using FACSCalibur (BD Biosciences) equipment.

As a result, it was confirmed that the Q_IITV variant had a similar level of binding affinity to PD-L1 as JY 101 (Figure 2). Accordingly, an aglycosylated variant with improved binding to PD-L1 was engineered using the Q_IITV variant as a template.

Example 3. Production of dimeric human PD-L1 (PD-L1-Fc) for exploration of aglycosylated PD-1 variants

3-1. PD-L1-Fc cloning

In order to screen non-glycosylated PD-1 variants more efficiently by increasing the binding activity (avidity), the Fc domain of antibody IgG is expressed in the C-terminal part of PD-L1 to induce dimerization, and the Fc and PD A GS linker was added between -L1 to ensure the fluidity of each protein. Specifically, the PD-L1 and Fc genes were each amplified using primers and Vent polymerase (New England Biolab), and then assembly PCR was performed using Vent polymerase. Each constructed gene was treated with Bss HII and Xba I (New England Biolab). The restriction enzyme-treated PD-L1-Fc gene was ligated into the pMAZ vector treated with the same restriction enzyme. After transforming the ligated plasmid into Jude1 E. coli, a single clone was obtained, and it was confirmed through base sequence analysis that PD-L1-Fc was successfully inserted into the pMAZ vector.

3-2. 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 in a CO ₂ shaking incubator at 37°C, 125 rpm, and 8% CO ₂ for 7 days, then centrifuged, and only the supernatant was collected. Afterwards, the mixture was equilibrated using 25x PBS and filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. The filtered culture medium was added with 1 ml of Protein A resin, stirred at 4°C for 16 hours, passed through a column to recover the resin, and washed with 10 ml of PBS. The washed resin was eluted with 100mM glycine pH 2.7 buffer and then neutralized using 1M Tris-HCl pH 8.0 to obtain purified PD-L1 dimeric protein (Figure 3). The purified PD-L1 dimer protein was fluorescently labeled using the Alexa-488 labeling kit. To confirm the binding affinity of fluorescently labeled dimeric PD-L1, samples of wild-type PD-1, JY 101, and Q_IITV were analyzed in the same manner as in Example 2 above.

As a result, the signals of wild-type PD-1 and JY 101 and Q_IITV mutants showed a similar pattern to that when tetrameric PD-L1 was used (Figure 2) (Figure 4), indicating that fluorescently labeled dimeric PD-L1 was active. was able to confirm.

Example 4. Construction of a large PD-1 error prone library for using ultra-fast screening techniques

In order to quickly search for non-glycosylated PD-1 variants that show high binding affinity to PD-L1, Sfi I sites on both sides were used to allow errors to enter all regions of PD-1_Q_IITV based on pMopac12-NlpA-PD-1_Q_IITV-FLAG. A primer containing was designed. The genome was amplified using the Error Prone PCR technique using the designed primers, Taq Polymerase (TAKARA), dNTPs (Invitrogen), MgCl2, and MnCl2 (SIGMA). The amplified genome was transferred to Sfi I Enzyme treatment and Sfi I It was inserted into the enzyme-treated pMopac12-NlpA-FLAG vector, ligated, and then transformed into Jude1 cells. Transformed E. coli were spread on a square plate and cultured at 37°C for 16 hours, and then E. coli were recovered with TB containing 2% glucose to obtain an initial library (FIG. 5).

Example 5. Screening of non-glycosylated PD-1 variants

After adding 40 μg/ml of chloramphenicol to 25 ml of TB medium containing 2% glucose, the library prepared in Example 4 was inoculated into a 250 mL flask and incubated at 37°C and 250 rpm for 4 hours, then 40 μg E. coli cultured in 100 ml of TB medium containing chloramphenicol at a ratio of 1:100 was inoculated. After culturing it until OD ₆₀₀ = 0.5, cooling it at 25°C and 250 rpm for 20 minutes, overexpressing the protein by adding 1 mM IPTG for 5 hours at 25°C, 250 rpm, and centrifuging at 14000 rpm for 1 minute, the cells were centrifuged. recovered. To remove 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. The cells were resuspended in 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution and rotated at 37°C for 30 minutes to remove the cell outer membrane. E. coli was obtained by centrifugation at 13,500 rpm for 1 minute, and the supernatant was removed. The centrifuged E. coli was resuspended with 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl ₂ , 10 mM MOPS pH6.8] and then centrifuged at 13,500 rpm for 1 minute. 1 ml of a mixture of 1 ml of Solution A and 20 μl of 50 mg/ml lysozyme solution was added to resuspend the centrifuged E. coli and rotated at 37°C for 15 minutes to remove the peptidoglycan layer. After centrifugation, the supernatant was removed, resuspended in 1 ml of PBS, 300 μl was taken, 700 μl of PBS and 25 nM dimeric PD-L1-Alexa488 probe were added together, and the spheroplast was labeled with a fluorescent probe by rotating at room temperature. did. After the labeling process was performed for 1 hour, centrifugation was performed at 13,500 rpm for 1 minute, and the supernatant was discarded. The resulting E. coli was washed once with 1 ml of PBS and centrifuged again at 13,500 rpm for 1 minute. After resuspending the centrifuged E. coli with 1 ml of PBS, E. coli with high binding affinity to PD-L1 were recovered using S3 sorter (Bio-Rad) equipment. The recovered E. coli genes were secured through PCR amplification using primers, and the obtained genomes were treated with sfi I restriction enzyme and ligated into the restriction enzyme-treated pMopac12-NlpA-FLAG vector. After transforming each plasmid into Jude1, E. coli were spread on a square plate and incubated at 37°C for 16 hours, then recovered and stored frozen. The above screening process was repeated four additional times (rounds).

Example 6. Confirmation of amplification of non-glycosylated PD-1 variants with increased PD-L1 affinity

After adding 40 μg/ml of chloramphenicol to 25 ml of TB containing 2% glucose, the initial library of Example 5 (library prepared in Example 4), 1st round library, 2nd round library, 3 The round library, 4-round library, and 5-round library were each placed in a 250 mL flask. After culturing it at 37°C and 250 rpm for 4 hours, the cultured E. coli was inoculated at 1:100 into 100 ml of TB containing 40 μg/ml chloramphenicol. E. coli were cultured until OD ₆₀₀ = 0.5, then cooled at 25°C and 250 rpm for 20 minutes, and 1 mM IPTG was added to overexpress the protein at 25°C and 250 rpm for 5 hours. Additionally, to use as a control, wild-type PD-1 and HAC-V-PD-1 cells were each inoculated and cultured at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated 1:50 into 6 ml of TB medium containing 40 μg/ml chloramphenicol, cultured until OD ₆₀₀ = 0.5, and then overexpressed the protein. Cells were recovered by putting equal amounts of each E. coli into an e-tube and centrifuging at 14,000 rpm for 1 minute. To remove 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. The cells were resuspended in 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution and rotated at 37°C for 30 minutes to remove the outer cell membrane. E. coli were collected by centrifugation at 13,500 rpm for 1 minute, and the supernatant was removed. The centrifuged E. coli was resuspended with 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl ₂ , 10mM MOPS pH6.8] and then centrifuged at 13500 rpm for 1 minute. 1 ml of a mixture of 1 ml of Solution A and 20 μl of 50 mg/ml lysozyme solution was added to resuspend the centrifuged E. coli and rotated at 37°C for 15 minutes to remove the peptidoglycan layer. After centrifugation, the supernatant was removed, resuspended in 1 ml of PBS, then 300 μl was taken, 700 μl of PBS and 3 nM dimeric PD-L1-Alexa488 probe were added together, and the spheroplast was labeled with a fluorescent probe by rotating at room temperature. did. After labeling for 1 hour, it was centrifuged at 13,500 rpm for 1 minute. The supernatant was discarded, the centrifuged E. coli was washed once with 1 ml of PBS, and centrifuged again at 13,500 rpm for 1 minute. The centrifuged E. coli was resuspended in 1 ml of PBS, and the binding force with PD-L1 was analyzed by measuring the mean fluorescence intensity (MFI) using a flow cytometer (Calibur, BD Biosciences).

As a result, it was confirmed that as the screening progressed, variants with high binding affinity to PD-L1 were amplified from the library (Figure 6).

Example 7. Obtaining non-glycosylated PD-1 variants with improved PD-L1 binding ability

After culturing the single colonies of the last round as in the above examples, overexpressing the protein and recovering E. coli, the peptidoglycan layer was removed, the spheroplasts were fluorescently labeled, and PD-L1 was isolated using FACSCalibur equipment. The binding force was analyzed by measuring the fluorescence signal value. As a result, Q10 (F13I, N25Q, F32L, N34Q, M46I, N50Q, T52M, C69T, V87D, N92Q, T96S, G100V, A108V) (SEQ ID NO: 3), Q12 (W8L, N9D, F13I, N25Q, N34Q, E37K , M46I, N50Q, C69T, N92Q, G100V, A108V, G140C) (SEQ ID NO: 4), Q18 (F13I, N25Q, S31G, F32L, S33P, N34Q, M46I, S47G, N50Q, T52M, C69T, N92Q, G100V, A108 V , P128R, P138S) (SEQ ID NO: 5) and Q33 (F13I, N25Q, N34R, M46I, N50Q, C69T, R88G, R90Q, N92Q, G100V, A108V) (SEQ ID NO: 6) have high binding affinity to PD-L1. It was found that it was selected (Figure 7).

Example 8. Expression and purification of variants with improved PD-L1 binding ability

The top four non-glycosylated PD-1 variants (Q10, Q12, Q18, and Q33), which were shown to have high binding affinity to PD-L1 in Example 7, were expressed and purified in animal cells, and then cloned to verify their binding affinity. proceeded. For this purpose, the genes of wild-type Q_PD1, in which all N-glycosylation sites of wild-type PD-1 were changed to Q, AHAC, a control group, and the mutants Q_IITV, Q10, Q12, Q18, and Q33 discovered in the present invention were subjected to PCR using primers and Vent polymerase. It was amplified. The amplified genome was treated with Bss HII and The ligated plasmid was transformed into Jude1 E. coli, and the sequence was confirmed through individual colony analysis. Vectors for expressing PD-1 variants (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 each transfected into Expi293F animal cells using PEI. Afterwards, it was cultured in a CO ₂ shaking incubator at 37°C, 125 rpm, and 8% CO ₂ for 7 days, then centrifuged, and only the supernatant was collected. Afterwards, equilibration was achieved using 25x PBS. It was filtered using a 0.2 μm filter (Merck Millipore) using a bottle top filter, and 0.5 ml of Ni-NTA resin was added to the filtered culture solution, stirred at 4°C for 16 hours, and then flowed through a column to recover the resin. The recovered resin was washed with 10 CV (column volume) of a PBS solution containing 10 mM imidazole (Sigma) and then washed once more with 10 CV (column volume) of a PBS solution containing 20 mM imidazole. Afterwards, the solution was eluted with a PBS solution containing 250 mM imidazole, and the buffer was changed using centrifugal filter units 3K (Merck Millipore). Afterwards, the expressed and purified non-glycosylated PD-1 variant proteins were confirmed using SDS-PAGE gel.

As a result, wild-type Q_PD-1 was not expressed, and the control AHAC mutant had a very low yield, while the mutants discovered in the present invention had increased stability despite the absence of glycosylation, and were obtained with high purity and yield. (Figure 8).

Example 9. Measurement of binding affinity for non-glycosylated PD-1 variants

CKJ 49 (F13L/N25D/C69S/N92S/R137K), JY 101 (N1S/F13I/L17M/S36P/M46I/C69T/G79R/G100V/L114P/A139L), and The binding affinity of the non-glycosylated PD-1 variants (Q10, Q12, Q18, and Q33) discovered in the present invention to PD-L1 was measured. Specifically, 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, and then activated with 1 M ethanolamine for 5 minutes. It was quenched for a minute. After that, a baseline was set for 30 seconds with 1 There was dissociation. As a result of analyzing the sensorgram at each concentration of each variant (FIG. 9) and analyzing the final K _D value, it was found that Q12 had the highest binding affinity to PD-L1 (FIG. 10).

Example 10. Comparison of binding ability to PD-L1 between the discovered non-glycosylated PD-1 variant (JY_Q12) and the existing glycosylated PD-1 variant (HAC)

In the case of PD-1, glycosylation is very important for PD-L1 binding ability, and unglycosylated unglycosylated PD-1 almost loses PD-L1 binding ability, so the unglycosylated PD-1 variant discovered in the present invention The binding ability of and glycosylated PD-1 variants to PD-L1 was compared and analyzed. For this purpose, the binding affinity to PD-L1 of HAC, a glycosylated PD-1 variant known in previous studies through biolayer interferometry assay (BLItz, Pall Fortebio), and JY_Q12 (Q12), an unglycosylated variant that showed the highest binding affinity in Example 9. was measured. Specifically, as in the above example, the AR2G biosensor (Pall Fortebio) was first activated with 20 mM EDC and 10 mM s-NHS for 5 minutes, and then immobilized with 20 μg/ml PD-L1-streptavidin for 5 minutes. Afterwards, it was quenched with 1 M ethanolamine for 5 minutes. After that, a baseline was set for 30 seconds with 1 It was dissociated. In this way, the sensorgrams for each concentration of each variant were analyzed (FIG. 11), and the final K _D value was analyzed (FIG. 12).

As a result, the non-glycosylated PD-1 variant Q12 (JY_Q12) discovered in the present invention showed PD-L1 binding ability almost similar to that of the conventional glycosylated PD-1 variant (HAC) (Figures 11 and 12). Through this, it can be confirmed that the binding ability of the non-glycosylated variant of the present invention is significantly improved to a similar extent as the glycosylated variant without the glycosylation that is important for binding ability to PD-L1, and because it is an unglycosylated form, it can be produced in bacteria and in animal cells. There is no problem of glycan heterogeneity during production, and it can be seen that the problem of non-specific receptor binding due to glycation can be solved for various purposes such as treatment and diagnosis.

<110> Korea University Research and Business Foundation <120> PD-1 VARIANTS WITH INCREASED BINDING TO PD-L1 <130> DP-2020-0218 <160> 6 <170> KoPatentIn 3.0 <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 Gln Phe Gln 130 135 140 <210> 2 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q_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 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> 3 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q10 <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 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 Gln Asp Ser Gly Ser 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> 4 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q12 <400> 4 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 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> 5 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q18 <400> 5 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 Gly Leu 20 25 30 Pro Gln Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Ile Gly 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 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 Arg 115 120 125 Thr Ala His Pro Ser Pro Ser Pro Arg Ser Ala Gly Gln Phe Gln 130 135 140 <210> 6 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> JY_Q33 <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 Glu Gly Asp Gln 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

Claims (24)

8th, 9th, 13th, 25th, 31st, 32nd, 33rd, 34th, 37th, 46th, 47th of the amino acid sequence of wild type PD-1 shown in SEQ ID NO: 1 , any one or more amino acids selected from the group consisting of the 50th, 52nd, 69th, 87th, 88th, 90th, 92nd, 96th, 100th, 108th, 128th, 138th and 140th amino acids. This is a PD-1 (Programmed cell death protein-1) variant with increased binding affinity to PD-L1 (Programmed death-ligand 1), substituted with a sequence different from the wild-type amino acid.
(1)F13I, N25Q, F32L, N34Q, M46I, N50Q, T52M, C69T, V87D, N92Q, T96S, G100V and A108V;
(2)W8L, N9D, F13I, N25Q, N34Q, E37K, M46I, N50Q, C69T, N92Q, G100V, A108V and G140C;
(3)F13I, N25Q, S31G, F32L, S33P, N34Q, M46I, S47G, N50Q, T52M, C69T, N92Q, G100V, A108V, P128R and P138S; or
(4) Mutants in which amino acids of F13I, N25Q, N34R, M46I, N50Q, C69T, R88G, R90Q, N92Q, G100V and A108V are substituted. delete delete delete delete delete The PD-1 variant with increased binding affinity to PD-L1 according to claim 1, which is an aglycosylated variant. delete delete delete delete delete A nucleic acid molecule encoding the PD-1 variant of claim 1. A vector containing the nucleic acid molecule of claim 13. A host cell containing the vector of claim 14. A binding inhibitor of PD-L1 and PD-1 comprising the PD-1 variant of claim 1, the nucleic acid molecule of claim 13, or the vector of claim 14. A composition for detecting PD-L1, comprising the PD-1 variant of claim 1. The composition for detecting PD-L1 according to claim 17, wherein the PD-1 variant is labeled with one selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromophore, a luminescent substance, and a fluorescent substance. A pharmaceutical composition for the treatment or prevention of cancer comprising the PD-1 variant of claim 1, the nucleic acid molecule of claim 13, or the vector of claim 14 as an active ingredient. The method of claim 19, 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, and colon cancer. , small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer. , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma. and a pharmaceutical composition for the treatment or prevention of cancer, which is at least one selected from the group consisting of pituitary adenoma. A composition for diagnosing cancer comprising the PD-1 variant of claim 1. a) contacting the PD-1 variant of claim 1 with a biological sample isolated from the subject;
b) confirming the binding level of the PD-1 variant and PD-L1; and
c) A method of providing information for cancer diagnosis, comprising comparing the binding level of the PD-1 variant and PD-L1 in a normal control sample. A method for specific detection of PD-L1, comprising contacting a sample with the PD-1 variant of claim 1 and detecting the binding of the PD-1 variant to PD-L1. a) cultivating 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 aglycosylated PD-1 with increased binding affinity to PD-L1, comprising the step of recovering the PD-1 variant expressed by the host cell.