PD-1 variants with improved binding affinity to PD-L1

Abstract

The present disclosure relates to PD-1 variants with improved binding affinity to PD-L1. In addition, the present disclosure relates to methods for preparing PD-1 variants and methods for screening PD-1 variants. The PD-1 variants disclosed herein effectively inhibit the binding between wild-type PD-1 and PD-L1, and therefore it is expected that the PD-1 variants have significantly higher immune cell penetration ability and anti-cancer effects or therapeutic effects on infectious diseases compared to conventional therapeutic agents for immune checkpoint inhibition. At the same time, the PD-1 variants can minimize the possibility of immunogenicity. In addition, the implementation of the glycosylated PD-1 variants can facilitate the convenient development of biopharmaceuticals.

Description

Technical Field

[0001] The present disclosure relates to PD-1 variants having improved binding affinity to PD-L1 and thus effectively inhibiting the binding between wild-type PD-1 and PD-L1, and methods for preparing the same. Background Art

[0002] Drugs used to treat cancer are mainly divided into low molecular weight drugs and high molecular weight drugs. High molecular weight drugs with specificity are more popular than low molecular weight drugs that lack specificity and therefore have relatively more side effects.

[0003] Recently, it has been reported that inhibition of immune checkpoint inhibitor proteins, especially the combination of PD-1 / PD-L1, is very effective in treating cancer and has fewer side effects compared to other immune checkpoint inhibitor proteins (J. Naidoo et al. (2015) Annals of Oncology, Lucia Gelao et al. (2014) Toxins, Gorge K. Philips et al. (2015) International Immunology).

[0004] Large pharmaceutical companies such as Bristol-Myers Squibb are working hard to develop therapeutics, for example by inhibiting the PD-1 / PD-L1 immune checkpoint inhibitors, and anti-cancer drugs such as Yervoy (ipilimumab) and Opdivo (nivolumab) are being developed using antibody formats.

[0005] In order to remove the binding of tumor infiltrating lymphocytes (TIL) to PD-1 / PD-L1, a therapeutic agent with excellent cell infiltration ability is required. However, antibodies have the disadvantage of penetrating cells because they are macromolecular proteins with a very large size of about 150 kDa.

[0006] To treat cancer through effective immune checkpoint inhibition, PD-1, which has excellent cell infiltration ability due to its smaller size, seems to be more suitable than PD-L1. However, the existing PD-1 engineering is based on screening by yeast display and has the risk of immunogenicity due to glycan heterogeneity and various mutations.

[0007] At the same time, aglycosylated protein drugs can be easily prepared in bacteria on a large scale and have great advantages in biomedical preparation, without the glycan heterogeneity problems associated with cell lines, culture processes or purification procedures.

[0008] Therefore, it is necessary to screen for aglycosylated PD-1 variants with fewer mutations and high binding affinity to PD-L1.

[0009] The foregoing description of the background art is given only to enhance the understanding of the background art of the present disclosure and should not be taken as an admission that it is the prior art well known to those of ordinary skill in the art.

[0010] public

[0011] Technical issues

[0012] The inventors of the present disclosure have been devoted to screening for PD-1 variants that have high binding affinity to PD-L1 and are therefore able to effectively inhibit the binding between wild-type PD-1 and PD-L1 and simultaneously minimize the possibility of immunogenicity. As a result, they found that by replacing a portion of the amino sequence of wild-type PD-1 with another amino sequence, the binding affinity to PD-L1 can be greatly improved, and the possibility of immunogenicity can be reduced by minimizing mutation sites and / or aglycosylated PD-1, and completed the present disclosure.

[0013] The present disclosure relates to PD-1 variants that provide improved binding affinity to PD-L1.

[0014] The present disclosure also provides nucleic acid molecules encoding PD-1 variants.

[0015] The present disclosure also provides vectors comprising nucleic acid molecules.

[0016] The present disclosure also provides a host cell comprising the vector.

[0017] The present disclosure also provides compositions comprising PD-1 variants, nucleic acid molecules or vectors.

[0018] The present disclosure also provides methods for preparing PD-1 variants.

[0019] The present disclosure also relates to providing an inhibitor of the binding between wild-type PD-1 and PD-L1, which comprises a PD-1 variant, a nucleic acid molecule or a vector as an active ingredient.

[0020] The present disclosure also provides a method for inhibiting the binding between wild-type PD-1 and PD-L1, comprising the step of administering an effective amount of a PD-1 variant, a nucleic acid molecule or a vector to a subject.

[0021] The present disclosure also provides a method for treating cancer or infectious diseases, comprising the step of administering a therapeutically effective amount of a PD-1 variant, a nucleic acid molecule or a vector to a subject.

[0022] The present disclosure also provides methods for screening PD-1 variants.

[0023] Other objects and advantages of the present disclosure will become more apparent from the following detailed description, claims and drawings.

[0024] Technical Solution

[0025] In one aspect of the present disclosure, the present disclosure provides PD-1 variants with improved binding affinity to PD-L1.

[0026] The inventors of the present disclosure have been working on screening for PD-1 variants that have high binding affinity to PD-L1 and are therefore able to effectively inhibit the binding between wild-type PD-1 and PD-L1 while minimizing the possibility of immunogenicity. As a result, they found that by replacing a portion of the amino sequence of wild-type PD-1 with another amino sequence, the binding affinity to PD-L1 can be greatly improved, and the possibility of immunogenicity can be reduced by minimizing mutation sites and / or aglycosylated PD-1, and completed the present disclosure.

[0027] As used herein, the term "PD-1 variant" or "programmed cell death protein 1 variant" refers to a variant of wild-type PD-1, wherein one or two or more than two amino acids in the amino acid sequence include a substitution, deletion or addition mutation.

[0028] In a specific exemplary embodiment of the present disclosure, the amino sequence of wild-type PD-1 includes the amino sequence of SEQ ID NO:61.

[0029] In a specific exemplary embodiment of the present disclosure, a PD-1 (programmed cell death protein 1) variant with improved binding affinity to PD-L1 (programmed death ligand 1) comprises a portion of the amino sequence of wild-type PD-1, and includes the 69th amino acid of the amino sequence of wild-type PD-1 of SEQ ID NO: 61 replaced with a sequence different from the wild-type amino acid.

[0030] In a specific exemplary embodiment of the present disclosure, the 69th amino acid is substituted with C69S, C69T, C69Y, C69G or C69A.

[0031] In a specific exemplary embodiment of the present disclosure, the PD-1 variant further comprises the 36th amino acid of the amino acid sequence of the wild-type PD-1 of SEQ ID NO: 61 substituted with S36P.

[0032] In a specific exemplary embodiment of the present disclosure, the PD-1 variant further comprises the 114th amino acid of the amino acid sequence of the wild-type PD-1 of SEQ ID NO: 61 substituted with L114P.

[0033] In a specific exemplary embodiment of the present disclosure, the PD-1 variant further comprises one or more than one amino acid selected from the 12th, 34th, 92nd, 107th, 131st, 132nd and 142nd amino acids of the amino acid sequence of wild-type PD-1 of SEQ ID NO: 61, substituted with a sequence different from the wild-type amino acid.

[0034] In a specific exemplary embodiment of the present disclosure, the PD-1 variant comprises one or more than one amino acid substitution selected from T12S, N34T, N92K or N92S, K107N, H131R, P132L and F142L.

[0035] In a specific exemplary embodiment of the present disclosure, the PD-1 variant further comprises the 13th amino acid of the amino acid sequence of the wild-type PD-1 of SEQ ID NO: 61 substituted with F13I or F13L, and the 46th amino acid substituted with M46I.

[0036] In a specific exemplary embodiment of the present disclosure, the PD-1 variant further comprises one or more than one amino acid selected from the 1st, 17th, 36th, 50th, 79th, 100th, 114th, 127th and 139th amino acids of the amino acid sequence of wild-type PD-1 of SEQ ID NO: 61, substituted with a sequence different from the wild-type amino acid.

[0037] In a specific exemplary embodiment of the present disclosure, the PD-1 variant comprises one or more than one amino acid substitution selected from L1S, L17M, S36P, N50S, G79R, G100V, L114P, V127A and A139L.

[0038] In a specific exemplary embodiment of the present disclosure, the PD-1 variant further comprises the 25th amino acid of the amino sequence of the wild-type PD-1 of SEQ ID NO: 61 substituted with N25D, and the 92nd amino acid substituted with N92S or N92K.

[0039] In certain exemplary embodiments of the present disclosure, the PD-1 variant further comprises the 13th amino acid of the amino acid sequence of the wild-type PD-1 of SEQ ID NO: 61 substituted with F13I or F13L.

[0040] In a specific exemplary embodiment of the present disclosure, the PD-1 variant further comprises one or more than one amino acid selected from the 9th, 88th, 101st, 125th, 137th amino acid of the amino acid sequence of wild-type PD-1 of SEQ ID NO: 61, which is replaced with a sequence different from the wild-type amino acid.

[0041] In a specific exemplary embodiment of the present disclosure, the PD-1 variant comprises one or more than one amino acid substitution selected from N9D, R88K, A101V, A125S and R137K.

[0042] In certain exemplary embodiments of the present disclosure, the PD-1 variant is an aglycosylated PD-1 variant.

[0043] Currently, most commercially available therapeutic proteins are produced by animal cell culture. When proteins are produced, they are modified by various sugar (carbohydrate) variants. In this regard, polysaccharide heterogeneity causes changes in the efficacy and stability of therapeutic proteins, and purification, analysis and quality control (QC) during protein production require a lot of costs.

[0044] When compared to glycosylated proteins that require expensive animal cell culture systems, aglycosylated proteins can be produced on a large scale in bacteria and exhibit remarkable advantages in terms of production speed and cost.

[0045] In another aspect of the present disclosure, the present disclosure provides a nucleic acid molecule encoding a PD-1 variant, a vector comprising the same, or a host cell comprising the vector.

[0046] In certain exemplary embodiments of the present disclosure, the host cell is a bacterial cell.

[0047] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of separation or reorganization, and include not only single-stranded and double-stranded DNA and RNA, but also complementary sequences corresponding thereto. When "isolated nucleic acid" is a nucleic acid isolated from a natural source, the nucleic acid is a nucleic acid isolated from a nearby gene sequence present in the isolated genome of an individual. For nucleic acids synthesized or chemically synthesized from a template enzyme, such as PCR products, cDNA molecules or oligonucleotides, nucleic acids prepared by these processes can be understood as isolated nucleic acid molecules. Isolated nucleic acid molecules can be components of fragments or larger nucleic acid constructs. When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably connected". For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably connected to the DNA of the polypeptide. If a promoter or enhancer affects the transcription of a polypeptide sequence, it is operably connected to a coding sequence. And, if a ribosome binding site is positioned so as to facilitate translation, it is operably connected to a coding sequence. Generally, "operably connected" means that the DNA sequence connected is continuous, and for a secretory leader, it is continuous and present in the same reading frame. However, enhancers need not be continuous. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

[0048] As used herein, the term "vector" refers to a vector capable of inserting a nucleic acid sequence to be introduced into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. The vector may be a plasmid, a cosmid or a virus (e.g., a bacteriophage), but is not limited thereto. Those skilled in the art can construct vectors according to standard recombinant techniques (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc., NY, 1994, etc.).

[0049] As used herein, the term "expression vector" refers to a vector comprising a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is translated into a protein, polypeptide or peptide. The expression vector may contain various regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, the vector or expression vector may also contain a nucleic acid sequence that provides another function.

[0050] As used herein, the term "host cell" refers to any transformable organism, including cells of eukaryotic and prokaryotic organisms, that is capable of replicating a vector or expressing a gene encoded by a vector. A host cell can be transfected or transformed by a vector, which refers to the process by which an exogenous nucleic acid molecule is delivered or introduced into a host cell.

[0051] In a specific exemplary embodiment of the present disclosure, the host cell of the present disclosure is a bacterial cell, more specifically a Gram-negative bacterial cell. The cell is sufficient for implementing the present disclosure because it has a periplasmic region between the inner membrane and the outer membrane. Specific examples of host cells according to the present disclosure include cells of Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhimurium, Shigella flexneri, Haemophilus influenzae, Bordetella pertussis, Erwinia amylovora, Rhizobium, etc., but are not limited thereto.

[0052] In another aspect of the present disclosure, the present disclosure provides an inhibitor of binding between wild-type PD-1 (programmed cell death protein 1) and PD-L1 (programmed death ligand 1), comprising a PD-1 variant, a nucleic acid molecule or a vector as an active ingredient.

[0053] In another aspect of the present disclosure, the present disclosure provides a composition comprising a PD-1 variant, a nucleic acid molecule or a vector as an active ingredient.

[0054] The composition is particularly a pharmaceutical composition, more particularly a pharmaceutical composition for preventing or treating cancer or infectious diseases.

[0055] In another aspect of the present disclosure, the present disclosure provides a method for inhibiting the binding between wild-type PD-1 (programmed cell death protein 1) and PD-L1 (programmed death ligand 1), comprising the step of administering an effective amount of a PD-1 variant, nucleic acid molecule or vector to a subject.

[0056] In another aspect of the present disclosure, the present disclosure provides a method for enhancing an immune response, comprising the step of administering an effective amount of a PD-1 variant, a nucleic acid molecule or a vector to a subject.

[0057] In another aspect of the present disclosure, the present disclosure provides a method for treating cancer or infectious diseases, comprising the step of administering a therapeutically effective amount of a PD-1 variant, a nucleic acid molecule or a vector to a subject.

[0058] The pharmaceutical composition of the present disclosure may include: (a) a PD-1 variant, a nucleic acid molecule or a vector; and (b) a pharmaceutically acceptable carrier.

[0059] The type of cancer to be prevented or treated in the present disclosure is not limited. The pharmaceutical composition can be administered to treat various cancers, including leukemias, such as acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia and chronic myeloid leukemia, lymphomas, such as Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, etc., solid tumors in children, such as brain tumors, neuroblastomas, retinoblastomas, Wilms' tumors, bone tumors, soft tissue sarcomas, etc., and common solid tumors in adults, such as lung cancer, breast cancer, prostate cancer, urinary cancer, uterine cancer, oral cancer, pancreatic cancer, melanoma and other skin cancers, gastric cancer, ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroid cancer, esophageal cancer, testicular cancer, etc.

[0060] The type of infectious disease to be prevented or treated in the present disclosure is not limited, and includes viral infection, influenza infection, bacterial infection, and fungal infection.

[0061] The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present disclosure is a kind of carrier that is usually used for preparation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate, mineral oil etc., but is not limited to this. In addition to the above-mentioned ingredients, the pharmaceutical composition of the present disclosure may also include lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives etc. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0062] The pharmaceutical composition of the present disclosure can be administered orally or parenterally, particularly parenterally. For example, it can be administered by intravenous injection, local injection, intraperitoneal injection, etc.

[0063] As used herein, the term "subject" refers to an object in which the above-mentioned diseases are prevented or treated by inhibiting the binding between PD-1 and PD-L1, and specifically includes humans and animals.

[0064] The appropriate dosage of the pharmaceutical composition of the present disclosure varies according to factors such as formulation method, administration method, age, body weight, gender and pathological condition of the patient, diet, administration time, administration route, excretion rate and response sensitivity. Ordinary technicians can easily determine and prescribe an effective dosage for the desired treatment or prevention. In a specific exemplary embodiment of the present disclosure, the daily dosage of the pharmaceutical composition of the present disclosure is 0.0001 mg / kg to 100 mg / kg.

[0065] According to the method that can be easily implemented by ordinary technicians in the field of the present disclosure, the pharmaceutical composition of the present disclosure can be prepared as a single dose or multi-dose preparation using pharmaceutically acceptable carriers and / or excipients. The preparation can be in the form of a solution, suspension, emulsion, extract, powder, granule, tablet or capsule in an oily or aqueous medium, and may also contain a dispersant or stabilizer.

[0066] The pharmaceutical composition of the present disclosure can be used alone or in combination with common biological therapy, chemotherapy or radiotherapy. Combination therapy may be more effective in treating cancer or infectious diseases. Chemotherapeutic agents that can be used together with the composition of the present disclosure include cisplatin, carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, actinomycin, daunorubicin, adriamycin, bleomycin, plicamycin, mitomycin, etoposide, tamoxifen, paclitaxel, anti-platinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, etc. Radiotherapy that can be used together with the composition of the present disclosure includes X-ray radiation, gamma ray radiation, etc.

[0067] In another aspect of the present disclosure, the present disclosure provides a method for preparing a PD-1 variant, comprising the following steps:

[0068] a) a step of culturing a host cell comprising a vector comprising a nucleic acid molecule encoding a PD-1 variant; and

[0069] b) A step of recovering the PD-1 variant expressed by the host cells.

[0070] In another aspect, the present invention discloses a method for screening PD-1 variants, comprising the following steps:

[0071] a) the step of establishing a library of PD-1 variants into which random point mutations or nucleic acid molecules encoding random point mutations have been introduced; and

[0072] b) A step of screening the library for PD-1 variants that inhibit the binding between wild-type PD-1 (programmed cell death protein 1) and PD-L1 (programmed death ligand 1).

[0073] According to the screening method disclosed herein, fluorescence activated cell sorting (FACS) screening or other automatic flow cytometry techniques can be used. Instruments for flow cytometry are known to those skilled in the art. Examples of instruments include FACSAria, FACS Star Plus, FACScan and FACSort (Becton Dickinson, Foster City, California), EpicsC (Coulter Epics Division, Hialeah, Florida), MOFLO (Cytomation, Colorado Springs, Colorado) and MOFLO-XDP (Beckman Coulter, Indianapolis, Indiana). Generally, flow cytometry techniques involve separating cells or other particles from liquid samples. Generally, the purpose of flow cytometry is to analyze one or more than one feature (e.g., the presence of a labeled ligand or other molecule) of the separated particles. Particles are classified by sensors when the particles flow one by one according to the size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc. of the particles.

[0074] Beneficial Effects

[0075] The features and advantages of the present disclosure can be summarized as follows:

[0076] (i) The present disclosure provides PD-1 variants with improved binding affinity to PD-L1.

[0077] (ii) The present disclosure also provides a method for preparing PD-1 and a method for screening PD-1 variants.

[0078] (iii) The PD-1 variant disclosed herein effectively inhibits the binding between wild-type PD-1 and PD-L1, and is therefore expected to have significantly higher immune cell penetration affinity and anti-cancer effects or therapeutic effects on infectious diseases compared to existing immune checkpoint inhibitors. At the same time, the possibility of immunogenicity can be minimized. In addition, the development of biopharmaceuticals can be facilitated by aglycosylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1Shown are SDS-PAGE images of wild-type PD-1 and four PD-1 glycovariants that have been produced in animal cells and then purified.

[0080] Figure 2 Shown are the results of studying the binding affinity of wild-type PD-1 and four PD-1 variants to PD-L1.

[0081] Figure 3 Shown are the results of preparing fluorescently labeled tetrameric PD-L1 and studying its activity.

[0082] Figure 4 Shown are the results of analyzing the expression of aglycosylated PD-1 in E. coli using anti-FLAG-FITC.

[0083] Figure 5 Shown are the results of measuring the activation of aglycosylated PD-1.

[0084] Figure 6 The amino acid sequences of the established primary libraries are shown.

[0085] Figure 7 Results of library enrichment testing by flow cytometry are shown.

[0086] Figure 8 Shown are the results of analyzing the binding affinity of E. coli cells displaying aglycosylated PD-1 variants to PD-L1.

[0087] Fig. 9 Shown are the results of analyzing protein expression levels in E. coli cells displaying aglycosylated PD-1 variants using anti-FLAG-FITC.

[0088] Fig.10 Shown are the results of analyzing the binding affinity of E. coli cells displaying aglycosylated PD-1 variants to PD-L1.

[0089] Fig.11 Shown are the results of analyzing protein expression levels in E. coli cells displaying aglycosylated PD-1 variants using anti-FLAG-FITC.

[0090] Fig.12 The results of screening for variants with novel mutations based on CKJ 41 are shown.

[0091] Fig.13 Analysis of the amino acid sequence of the secondary library established using CKJ 41T as template is shown.

[0092] Fig.14 Results of library enrichment testing by flow cytometry are shown.

[0093] Fig.15 The results of analyzing the binding affinity of E. coli cells displaying aglycosylated PD-1 variants screened by secondary screening to PD-L1 are shown.

[0094] Fig.16 Results comparing the binding affinity of PD-1 variants and consensus sequence variants to PD-L1 are shown.

[0095] Fig.17 SDS-PAGE images of purified HAC-V and two PD-1 variant proteins are shown.

[0096] Fig.18 Results of studying the binding affinity of wild-type PD-1, HAC-V, and two PD-1 variants to PD-L1 are shown.

[0097] Best Mode for Carrying Out the Invention

[0098] Hereinafter, the present disclosure will be described in detail by way of examples. The examples are provided only for the purpose of specifically describing the present disclosure, and it is obvious to those skilled in the art that the scope of the present disclosure is not limited by the examples. Example

[0099] Example 1: Verification of N-linked glycosylation sites that affect binding to human PD-L1

[0100] In order to verify that the glycosylation of human PD-1 actually plays an important role in the binding affinity with PD-L1, the effect of each glycosylation on the binding affinity was tested by deglycosylation of the four glycosylation sites present in PD-1. Because PD-1 has four N-glycosylation sites, four genes (N25A, N34A, N50A, N92A) were prepared by replacing each asparagine at the four sites with alanine. The DNA fragment encoding the PD-1 outer membrane site (amino sequence L25-Q167) was amplified by PCR using the template gene (catalog number: HG10377-M) and primers (CKJ#1, CKJ#2) of PD-1 and Vent polymerase. The amplified DNA fragment was treated with BssHII and XbaI enzymes and ligated into the pMaz vector, which is a vector for expression in animal cells, which was treated with the same enzymes. Transformation into Jude1((F-mcrAΔ(mrr-hsdRMS-mcrBC) ZΔM15ΔlacX74recA1 endA1 araD139Δ(ara,leu)7697galU galKλ-rpsL nupG)E. coli, the ligated plasmid sequence was analyzed by single colony analysis to identify the desired plasmid. Primers (CKJ#3, CKJ#4, CKJ#5, CKJ#6, CKJ#7, CKJ#8, CKJ#9, CKJ#10) were designed to easily obtain variants containing asparagine replaced with alanine by QuikChange PCR site-directed mutagenesis using plasmids. The DNA fragment containing the gene was amplified using the designed primers and Pfu Turbo polymerase (Agilent). The amplified gene was sequenced and confirmed after transformation into Jude1. After animal cells (HEK293F) were transfected with prepared vectors for expressing wild-type PD-1 (pMaz-wild-type PD-1-His tag) and PD-1 glycovariants (pMaz-N25A PD-1-His tag, pMaz-N34A PD-1-His tag, pMaz-N50A PD-1-His tag, pMaz-N92A PD-1-His tag) and cultured for 6 days, the cell culture was centrifuged at 6000 rpm for 20 minutes, and then the supernatant was filtered through a 0.22 μm filter. The filtered supernatant was incubated with 1 mL Ni-NTA resin (Qiagen) at 4°C for 16 hours. The protein-bound resin was washed with 10CV (column volume) of PBS solution containing 10mM imidazole (Sigma), and then washed once with 10CV of PBS solution containing 20mM imidazole. Finally, the eluate was recovered with a PBS solution containing 250mM imidazole ( Figure 1 The binding affinity of wild-type PD-1 and four PD-1 glycoforms to PD-L1 was studied by ELISA. Each protein was stained with 0.05 M Na 2 CO 3(pH 9.6, Junsei) was diluted to 200 ng / well and then bound to a high-binding 96-well plate (Costar) at 4°C for 16 hours. After removing the protein, each 96-well plate was incubated in a 5% (w / v) PBST (PBS containing 0.5% Tween 20) solution containing 5% skim milk solution at room temperature for 1 hour. After discarding the upper solution and washing 4 times with 200 μL of PBS solution containing 0.5% Tween 20, the PD-L1 tetramer was diluted with PBS solution and then added to each 96-well plate and incubated at room temperature for 1 hour. After washing 4 times with 200 μL of 0.5% PBST solution, anti-streptavidin-HRP (Genetex) diluted to 1:2000 with PBS was added to each 96-well plate and incubated at room temperature for 1 hour. After discarding the upper layer and washing four times with 200 μL of 0.5% PBST solution, the reaction was started by adding 50 μL of TMB (Genetex), and 4NH 2 SO 4 Stop the reaction ( Figure 2 The results showed that while the N92A variant did not show a significant difference in binding affinity to wild-type PD-1, the remaining three glycovariants showed a significant decrease in binding affinity, indicating that glycosylation of N25, N34, and N50 has a significant effect on the binding affinity of PD-L1.

[0101] Table 1

[0102]

[0103] Primers used in the experiments

[0104] Example 2: Cloning of tetrameric human PD-L1 (PD-L1-streptavidin) for PD-1 engineering

[0105] The cDNA of human PD-L1 gene was purchased from Sino Biotech (Catalog No.: HG10084-M), and the gene of the cell outer membrane of PD-L1 (amino sequence F19-R238) was amplified by PCR using primers (HW#1, HW#2) and Vent polymerase. Since the dissociation constant between wild-type PD-1 and wild-type PD-L1 is not good enough (equilibrium dissociation constant, K D= about 8.7 μM), thus inducing an affinity effect by effectively screening the tetramerization of the glycosylated PD-1 variants. Tetramerization was induced by expressing streptavidin at the C-terminus of PD-L1, and a GS linker was inserted between streptavidin and PD-L1 to ensure the flexibility of each protein. After amplifying the gene using primers (HW#3, HW#4) and Vent polymerase (New England Biolab), assembly PCR was performed using the previously amplified PD-L1 gene and Vent polymerase. The prepared gene was treated with BssHII and XbaI restriction enzymes (New England Biolab). The restriction enzyme-treated PD-L1-streptavidin-His tag gene was ligated into the pMaz vector, which is a vector for mammalian expression that has been treated with the same restriction enzymes. The ligated plasmid was transformed into Jude1 Escherichia coli. A single clone was obtained, and sequencing was then performed to confirm that the PD-L1-streptavidin-His tag had been successfully inserted into the pMaz vector.

[0106] Example 3: Expression, purification and fluorescent labeling of tetrameric PD-L1-streptavidin in animal cells

[0107] The prepared vector for tetrameric PD-L1 expression was transfected into animal cells (HEK293F). After the cells were cultured for 6 days, the cell culture was centrifuged at 6000rpm for 20 minutes, and the supernatant was taken and filtered through a 0.22μm filter. The filtered supernatant was bound to 1mL Ni-NTA resin (Qiagen) at 4°C for 16 hours. The bound resin was washed with 10CV of PBS solution containing 10mM imidazole (Sigma), and then washed again with 10CV of PBS solution containing 20mM imidazole. Finally, the protein was eluted and recovered with a PBS solution containing 250mM imidazole. The purified PD-L1 tetramer was fluorescently labeled using an Alexa-488 labeling kit. The results of the ELISA test were that the fluorescently labeled tetrameric PD-L1 showed excellent binding affinity to PD-1 ( Figure 3 ).

[0108] Example 4: Construction of wild-type PD-1 and HAC-V PD-1 expression plasmids displaying human PD-1 in bacterial cell membranes

[0109] To display the outer membrane site of PD-1, the DNA of PD-1 (amino sequence L25-Q167) was amplified by PCR using primers (JY#1, JY#2) and Vent polymerase, and then treated with SfiI restriction enzyme. In order to fix the PD-1 protein on the intracellular membrane during transport to the periplasmic region of Escherichia coli through the NlpA signal peptide, the pMopac12-NlpA-WTPD-1-FLAG vector was prepared by connecting the SfiI-treated PD-1 DNA fragment with the SfiI fragment of the pMopac12-NlpA-FLAG vector. For the HAC-V variant as a control group, the synthesized HAC-V DNA fragment was also cloned into the Sfil site of the pMopac12-NlpA-FLAG vector to construct pMopac12-NlpA-HAC-V PD-1-FLAG. Then, after transformation into E. coli Jude1 and obtaining single clones, it was confirmed by sequencing that wild-type PD-1 and HAC-VPD-1 had been successfully inserted into the pMopac-12 vector.

[0110] Example 5: Verification of the expression of PD-1 and PD-1 variants (wild-type PD-1, HAC-VPD-1) in Escherichia coli and their binding affinity to PD-L1 by flow cytometry

[0111] Each plasmid prepared by cloning (pMopac12-NlpA-PD-1-FLAG, pMopac12-NlpA-HAC-VPD-1-FLAG, pMopac12-NlpA-Fc-FLAG) was transformed into Jude1. Each prepared sample was cultured in TB medium containing 2% glucose and 40 μg / mL chloramphenicol at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated into 6 mL of TB medium containing 40 μg / mL chloramphenicol at a ratio of 1:50. After culturing to OD 600 = 0.5, then cooled at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours. 600Normalization The same amount of protein-overexpressing E. coli was added to tube e, and the cells were recovered by centrifugation at 14,000 rpm for 1 minute. To remove the residual culture medium, the cells were washed twice by resuspending the precipitate in 1 mL of 10 mM Tris-HCl (pH 8.0) and centrifuging 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 membrane of the cells. E. coli was collected by centrifugation at 13,500 rpm for 1 minute, and the supernatant was removed. The E. coli cell pellet was resuspended in 1 mL of solution A [0.5 M sucrose, 20 mM MgCl 2 , 10mM MOPS, pH 6.8], and then centrifuged at 13500rpm for 1 minute. After resuspending the cell pellet by adding 1mL of a solution prepared by mixing 1mL of solution A with 20μL of a 50mg / mL lysozyme solution, the peptidoglycan layer was removed by rotating at 37°C for 15 minutes. The supernatant was removed after centrifugation. After the precipitate was resuspended in 1mL of PBS, 300μL was taken, and then 700μL of PBS, 12.5nM tetramer PD-L1-Alexa488 probe and 33nM anti-FLAG-FTIC (Sigma) were added. Then, the spheroplasts were labeled with fluorescent probes by rotating at room temperature. After the labeling treatment was performed for 1 hour, centrifuged at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged at 13500rpm for 1 minute again. The cells were resuspended in 1mL of PBS and analyzed using a Guava instrument (Merck Millipore). The results confirmed that aglycosylated PD-1 was well expressed in E. coli ( Figure 4 ), but it showed no binding affinity to PD-L1. It was also confirmed that aglycosylated HAC-V PD-1 showed weak binding affinity to PD-L1 ( Figure 5 ).

[0112] Example 6: Construction of an error-prone PD-1 library for high-throughput screening

[0113] For high-throughput screening of aglycosylated PD-1 showing high binding affinity to PD-L1, primers (JY#3, JY#4) with SfiI sites on both sides were designed to allow insertion errors at all sites of PD-1 during PCR-based amplification using pMopac12-NlpA-PD-1-FLAG as a template. 2 and MnCl2 (Sigma) to construct the library, and the gene was amplified by error-prone PCR. The PCR product was treated with SfiI enzyme, ligated to the SfiI fragment of pMopac12-NlpA-FLAG vector, and then transformed into Jude1 cells. The transformed E. coli cells were spread on square plates and cultured at 37°C for 16 hours. Then, the primary library was obtained by recovering the E. coli cells with TB containing 2% glucose ( Figure 6 ).

[0114] Example 7: Screening of PD-1 variants by flow cytometry sorting

[0115] After adding 40 μg / mL chloramphenicol to 25 mL of TB medium containing 2% glucose, the constructed E. coli library was inoculated into a 250 mL flask and incubated at 37°C and 250 rpm for 4 hours. Then, the E. coli cells were inoculated into 100 mL of TB medium containing 40 μg / mL chloramphenicol at a ratio of 1:100. After culturing to OD 600 =0.5, then after cooling at 25°C and 250rpm for 20 minutes, protein overexpression was induced at 25°C and 250rpm for 5 hours by adding 1mM IPTG, and the cells were recovered by centrifugation at 14000rpm for 2 minutes. In order to remove the residual culture medium, the cells were resuspended in 1mL of 10mMTris-HCl (pH 8.0), centrifuged at 13500rpm for 1 minute, and then washed. Repeat this process 2 times. The cells were resuspended in 1mL of STE [0.5M sucrose, 10mM Tris-HCl, 10mM EDTA (pH8.0)] solution and rotated at 37°C for 30 minutes to remove the outer membrane of the cells. Escherichia coli was collected by centrifugation at 13500rpm for 1 minute, and then the supernatant was removed. The cells were resuspended in 1mL of solution A [0.5M sucrose, 20mM MgCl 2, 10mM MOPS, pH 6.8] and then centrifuged at 13500rpm for 1 minute. After resuspending the cells by adding 1mL of a solution prepared by mixing 1mL of solution A with 20μL of a 50mg / mL lysozyme solution, the peptidoglycan layer was removed by rotating at 37°C for 15 minutes. The supernatant was removed after centrifugation. After resuspending the cells in 1mL of PBS, 300μL was taken and 700μL of PBS and 12.5nM of tetramer PD-L1-Alexa488 probe were added. Then, the spheroplasts were labeled with fluorescent probes by rotating at room temperature. After 1 hour of labeling, centrifugation was performed at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. Escherichia coli cells with high binding affinity to PD-L1 were selected from cells resuspended in 1mL of PBS and recovered using an S3 sorter (Bio-Rad). The gene was obtained from the recovered E. coli by PCR using primers (JY#1, JY#2). The gene was treated with SfiI restriction enzyme and connected to the SfiI fragment of pMopac12-NlpA-FLAG vector. The resulting plasmid was transformed into Jude1 and the cells were spread on square plates. After incubation at 37°C for 16 hours, the cells were recovered and stored in a deep freezer. The screening process was repeated 5 times.

[0116] Example 8: Cultivation of E. coli to study the enrichment of PD-1 variants with increased affinity for PD-L1

[0117] After adding 40 μg / mL chloramphenicol to 25 mL of TB containing 2% glucose, the primary library, round 1 library, round 2 library, round 3 library, round 4 library, round 5 library, and round 6 library were added to a 250 mL flask. After incubation at 37°C and 250 rpm for 4 hours, E. coli cultured in 100 mL of TB containing 40 μg / mL chloramphenicol was inoculated at a ratio of 1:100. After culturing to OD 600 = 0.5, then after cooling at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours. In addition, cells expressing wild-type PD-1 and HAC-V-PD-1 as a control group were inoculated into 4 mL of TB medium containing 2% glucose and 40 μg / mL chloramphenicol and grown at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated into 6 mL of TB medium containing 40 μg / mL chloramphenicol at a ratio of 1:50. After culturing to OD 600 = 0.5, then cooled at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours.600 Normalization The same amount of E. coli was added to tube e, and the cells were recovered by centrifugation at 14000 rpm for 1 min.

[0118] Example 9: Enrichment of PD-1 variants with increased affinity for PD-1 by flow cytometry

[0119] To remove the residual culture medium, resuspend the cells added to tube e in 1 mL of 10 mM Tris-HCl (pH 8.0), centrifuge at 13500 rpm for 1 minute, and then wash. Repeat this process twice. Resuspend the cells in 1 mL of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution and rotate at 37°C for 30 minutes to remove the outer membrane of the cells. Collect E. coli by centrifugation at 13500 rpm for 1 minute, and then remove the supernatant. Resuspend the cell pellet in 1 mL of solution A [0.5 M sucrose, 20 mM MgCl 2 , 10mM MOPS, pH 6.8], and then centrifuged at 13500rpm for 1 minute. After resuspending the cells by adding 1mL of a solution prepared by mixing 1mL of solution A with 20μL of a 50mg / mL lysozyme solution, the peptidoglycan layer was removed by rotating at 37°C for 15 minutes. The supernatant was removed after centrifugation. After resuspending in 1mL of PBS, 300μL was taken and 700μL of PBS and 12.5nM tetramer PD-L1-Alexa488 probe were added together. Then, the spheroplasts were labeled with fluorescent probes by rotating at room temperature. After the labeling treatment was performed for 1 hour, centrifuged at 13500rpm for 1 minute. After discarding the supernatant, the cell pellet was washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. After the cells were resuspended in 1mL of PBS, the binding affinity to PD-L1 was analyzed by measuring the mean fluorescence intensity (MFI) using a flow cytometer (Guava, Millipore). It was confirmed that as the screening progressed, variants with higher binding affinity to PD-L1 were amplified from the library ( Figure 7 ).

[0120] Example 10: Determination of PD-1 variants with increased binding affinity to PD-L1 by flow cytometry

[0121] The single colony of the last round was inoculated into TB medium containing 2% glucose and 40 μg / mL chloramphenicol, and then cultured at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated into 6 mL of TB medium containing 40 μg / mL chloramphenicol by diluting to a ratio of 1:50. After culturing to OD 600= 0.5, then cooled at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours. 600 Normalization: Add the same number of E. coli to tube e, and recover the cells by centrifugation at 14,000 rpm for 1 minute. In order to remove the residual culture medium, resuspend the cells added to tube e in 1 mL of 10 mM Tris-HCl (pH 8.0), centrifuge at 13,500 rpm for 1 minute, and then wash. Repeat this process twice. Resuspend the cells in 1 mL of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution and rotate at 37°C for 30 minutes to remove the outer membrane of the cells. After collecting the cells by centrifugation at 13,500 rpm for 1 minute, remove the supernatant. Resuspend the cell pellet in 1 mL of solution A [0.5 M sucrose, 20 mM MgCl 2 , 10mM MOPS, pH 6.8] and then centrifuged at 13500rpm for 1 minute. After resuspending the cells by adding 1mL of a solution prepared by mixing 1mL of solution A with 20μL of a 50mg / mL lysozyme solution, the peptidoglycan layer was removed by rotating at 37°C for 15 minutes. The supernatant was removed after centrifugation. After resuspending in 1mL of PBS, 300μL was taken and 700μL of PBS and 12.5nM of tetramer PD-L1-Alexa488 probe were added together. The spheroplasts were then labeled with a fluorescent probe by rotating at room temperature. After the labeling treatment was performed for 1 hour, centrifuged at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. The cells were resuspended in 1mL of PBS and the binding affinity to PD-L1 was analyzed by measuring the fluorescence signal using a Guava instrument ( Figure 8 ). In addition, since the expression level of PD-1 protein displayed by E. coli can affect the fluorescence intensity, it is necessary to determine the expression level of PD-1. To this end, 300 μL of E. coli remaining after resuspending in PBS was added to 700 μL of PBS and 1 μL of anti-FLAG-FITC and rotated at room temperature to label the spheroplasts with the fluorescent probe. After 1 hour of labeling treatment, centrifuge at 13500 rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1 mL of PBS and then centrifuged again at 13500 rpm for 1 minute. The cells were resuspended in 1 mL of PBS, and the binding affinity to anti-FLAG-FITC was analyzed by measuring the fluorescence signal by flow cytometry ( Fig. 9 ).

[0122] Table 2

[0123] SEQ ID NO PD-1 variants PD-1 variants and positions of substituted amino acids SEQ ID NO 48 No.41(CKJ 41) S36P / C69S / L114P SEQ ID NO 50 No.45(CKJ 45) S36P / C69S / K107N / L114P / F142L SEQ ID NO51 No.46 (CKJ 46) S36P / C69S / L114P / P132L SEQ ID NO 56 No.56 (CKJ 56) S36P / C69S / N92K / L114P / H 131R SEQ ID NO 58 No.67 (CKJ 67) N34T / S36P / C69S / L114P SEQ ID NO 59 No.70(CKJ 70) S36P / C69S SEQ ID NO:60 No.78 (CKJ 78) T12S / S36P / C69S / L114P SEQ ID NO 49 No.44 (CKJ 44) F13I / M46I / N50S / C69Y / V127A SEQ ID NO 54 No.52 (CKJ 52) F13I / M46I / C69Y SEQ ID NO 52 No.49 (CKJ 49) F13L / N25D / C69S / N92S / R137K SEQ ID NO 53 No.50(CKJ 50) F13L / N25D / C69S / R88K / N92S / A125S SEQ ID NO 55 No.55(CKJ 55) N25D / C69S / N92S / A101V SEQ ID NO 57 No.66 (CKJ 66) N9D / N25D / C69S / N92S / A101V

[0124] PD-1 variants with improved binding affinity to PD-L1

[0125] Example 11. Cloning for comparison with other control groups of PD-1 variants (PD-1 S.6.8.3 and S.5.1T)

[0126] As other control groups, S.6.8.3 and S.5.1T variants were cloned by primer assembly PCR. First, after assembly PCR using primers (S.6.8.3: JY#5, 6, 7, 8, 9, 10, 11, 12 / S.5.1T: JY#13, 14, 15, 16, 17, 18, 19, 20) and Vent polymerase, the assembled genes were amplified by amplification PCR. The assembled genes were treated with Sfil and then connected with the Sfil fragment of the pMopac12-NlpA-FLAG vector to construct pMopac12-NlpA-PD-1S.6.8.3-FLAG and pMopac12-NlpA-PD-1S.5.1T-FLAG vectors. Then, after obtaining individual clones by transformation into E. coli Jude1, they were confirmed to have been successfully inserted by sequencing.

[0127] Table 3

[0128]

[0129]

[0130] Primers used in other experiments

[0131] Example 12. Comparison of the Binding Affinity of the Screened PD-1 Variants and the Control Group to PD-L1 by Flow Cytometry

[0132] In order to compare the binding affinity of PD-1 variants with high binding affinity to PD-L1 with that of other control groups, each of wild-type PD-1, PD-1HAC-V, S.6.8.3 and S.5.1T was used as a control group, and CKJ 49 and CKJ 50 were inoculated into TB medium containing 2% glucose and 40 μg / mL chloramphenicol, and then cultured at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated into 6 mL of TB medium containing 40 μg / mL chloramphenicol by diluting to a ratio of 1:100. After culturing to OD 600 = 0.5, then after cooling at 25°C and 250 rpm for 20 min, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 h. 600Normalization The same number of E. coli was added to the e-tube, and the cells were recovered by centrifugation at 14000rpm for 1 minute. After preparing spheroplasts in the same manner as described in Example 5, 12.5nM of tetramer PD-L1-Alexa488 probe was added to the prepared spheroplasts, and they were labeled with fluorescent probes by rotating at room temperature. After 1 hour of labeling, centrifugation was performed at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. The cells were resuspended in 1mL of PBS and analyzed for binding affinity to PD-L1 by measuring fluorescence using a Guava instrument ( Fig.10 ). In addition, since the expression level of PD-1 protein displayed by E. coli can affect the fluorescence intensity, the expression level of PD-1 was determined. To this end, take the 300 μL of E. coli remaining after resuspending in PBS, add 700 μL of PBS and 1 μL of anti-FLAG-FITC together, and label the spheroplasts with the fluorescent probe by rotating at room temperature. After 1 hour of labeling treatment, centrifuge at 13500 rpm for 1 minute. After discarding the supernatant, wash the cells once with 1 mL of PBS and then centrifuge again at 13500 rpm for 1 minute. Resuspend the cells in 1 mL of PBS, and analyze the binding affinity to anti-FLAG-FITC by measuring the fluorescence signal by flow cytometry ( Fig.11 ).

[0133] Example 13. Cloning to screen for variants with new mutations

[0134] In order to screen variants with new mutations of CKJ 41 from the selected variants, which has the latest mutation and high binding affinity, C69S in CKJ 41 was replaced with several amino acids (C, T, Y, A, G). To this end, the gene was amplified by Quikchange PCR using designed primers and Pfu Turbo polymerase (Agilent). The amplified gene was converted to Jude1 and then analyzed by sequencing.

[0135] Example 14. Screening of variants with new mutations by flow cytometry

[0136] CKJ41, HAC-V variant (control group), and CKJ41C, CKJ41T, CKJ41Y, CKJ41A, and CKJ41G in which the 69th amino acid of CKJ41 was changed were inoculated into TB medium containing 2% glucose and 40 μg / mL chloramphenicol, and cultured at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated into 6 mL of TB medium containing 40 μg / mL chloramphenicol by diluting to 1:100. After culturing to OD600 = 0.5, then cooled at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours. 600 Normalization The same amount of E. coli was added to the e-tube, and the cells were recovered by centrifugation at 14000rpm for 1 minute. After preparing spheroplasts in the same manner as described in Example 5, 12.5nM of tetramer PD-L1-Alexa488 probe was added to the prepared spheroplasts, and they were labeled with fluorescent probes by rotating at room temperature. After 1 hour of labeling, centrifugation was performed at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. The cells were resuspended in 1mL of PBS and analyzed for binding affinity to PD-L1 by measuring fluorescence using a Guava instrument ( Fig.12 ).

[0137] Example 15. Construction of a secondary error-prone PD-1 library for identification of PD-1 variants with improved binding affinity

[0138] For high-throughput screening of PD-1 that exhibits higher binding affinity to PD-L1, insert fragments were prepared using pMopac12-NlpA-PD-1CKJ 41T-FLAG, which exhibits fewer mutations but high fluorescence intensity, as a template to allow insertion of errors into all regions. The primers (JY#3, JY#4) with SfiI sites on both sides, Taq polymerase (Takara), dNTPs (Invitrogen), MgCl 2 and MnCl 2 (Sigma), and the library was constructed by amplifying genes by error-prone PCR. The amplified genome was treated with SfiI enzyme and ligated with the SfiI fragment of the pMopac12-NlpA-FLAG vector, and then transformed into Jude1 cells. The transformed E. coli cells were spread on square plates and cultured at 37°C for 16 hours. Then, the primary library was prepared by recovering the E. coli cells with TB containing 2% glucose ( Fig.13 ).

[0139] Example 16. Secondary screening of PD-1 variants by flow cytometry sorting

[0140] After adding 40 μg / mL chloramphenicol to 25 mL of TB medium containing 2% glucose, the constructed library was inoculated into a 250 mL flask and incubated at 37°C and 250 rpm for 4 hours. Then, E. coli was inoculated into 100 mL of TB medium containing 40 μg / mL chloramphenicol at a ratio of 1:100. After culturing to OD600 =0.5, then cooled at 25 ° C and 250rpm for 20 minutes, induced protein overexpression at 25 ° C and 250rpm for 5 hours by adding 1mM IPTG, and recovered cells by centrifugation at 14000rpm for 1 minute. After preparing spheroplasts in the same manner as described in Example 5, 12.5nM of tetramer PD-L1-Alexa488 probe was added and rotated at room temperature to mark the prepared spheroplasts. After labeling for 1 hour, centrifuge at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. The cells were resuspended in 1mL of PBS, and Escherichia coli cells with higher binding affinity for PD-L1 were recovered using an S3 sorter (Bio-Rad). Genes were prepared from the recovered Escherichia coli by PCR using primers (JY#3, JY#4). The gene was treated with SfiI restriction enzyme and then ligated to the SfiI fragment of pMopac12-NlpA-FLAG vector. The resulting plasmid was transformed into Jude1 and E. coli was spread on square plates. After incubation at 37°C for 16 hours, the cells were recovered and stored in a deep freezer. The screening process was repeated 3 more times while gradually reducing the concentration of the probe.

[0141] Example 17. Cultivation of E. coli to study the enrichment of secondary PD-1 variants with increased affinity for PD-L1

[0142] After adding 40 μg / mL chloramphenicol to 25 mL of TB containing 2% glucose, the primary library, round 1 library, round 2 library, round 3 library, and round 4 library were added to a 250 mL flask. After incubation at 37°C and 250 rpm for 4 hours, E. coli cultured in 100 mL of TB containing 40 μg / mL chloramphenicol was inoculated at a ratio of 1:100. After culturing to OD 600 = 0.5, then after cooling at 25°C and 250rpm for 20 minutes, protein overexpression was induced by adding 1mM IPTG at 25°C and 250rpm for 5 hours. In addition, as a control group, 40μg / mL chloramphenicol was added to 4mL TB medium containing 2% glucose, and after inoculating wild-type PD-1 and HAC-V-PD-1 cells, the cells were cultured at 37°C and 250rpm for 16 hours. The cultured cells were inoculated into 6mL TB medium containing 40μg / mL chloramphenicol at a ratio of 1:100. After culturing to OD 600 = 0.5, then cooled at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours. 600Normalization The same amount of E. coli was added to tube e, and the cells were recovered by centrifugation at 14000 rpm for 1 min.

[0143] Example 18. Enrichment of PD-1 variants with increased affinity for PD-1 determined by flow cytometry

[0144] After preparing spheroplasts in the same manner as described in Example 5, 4nM of tetrameric PD-L1-Alexa488 probe was added to the prepared spheroplasts and rotated at room temperature for labeling. After 1 hour of labeling, centrifuge at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. The cells were resuspended in 1mL of PBS and analyzed for binding affinity to PD-L1 by measuring the mean fluorescence intensity (MFI) using a flow cytometer (Guava, Millipore). It was confirmed that as the screening progressed, variants with higher binding affinity to PD-L1 were amplified from the library ( Fig.14 ).

[0145] Example 19. Other PD-1 variants with increased binding affinity to PD-L1 determined by flow cytometry

[0146] Each single colony from the last round (APD1-CKJ 41T: aglycosylated form of CKJ 41T, APD1-JY 101: aglycosylated form of JY 101), wild-type PD-1, and HAC-V were inoculated into TB medium containing 2% glucose and 40 μg / mL chloramphenicol, and then cultured at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated into 6 mL of TB medium containing 40 μg / mL chloramphenicol by diluting to a ratio of 1:100. After culturing to OD 600 = 0.5, then cooled at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours. 600 Normalization The same amount of E. coli was added to the e-tube, and the cells were recovered by centrifugation at 14000rpm for 1 minute. After preparing spheroplasts in the same manner as described in Example 5, 4nM of tetramer PD-L1-Alexa488 probe was added to the prepared spheroplasts, and they were labeled with fluorescent probes by rotating at room temperature. After 1 hour of labeling, centrifugation was performed at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. The cells were resuspended in 1mL of PBS and the binding affinity to PD-L1 was analyzed by measuring the fluorescence signal using a Guava instrument ( Fig.15 ).

[0147] Example 20. Cloning variants to compare mutation sites of screened PD-1 variants

[0148] By cloning PD-1_LDSS variants with overlapping mutations of CKJ 49 and CKJ 50, we investigated whether introducing new mutations of CKJ 49 and CKJ50 into LDSS affects binding affinity. To prepare PD-1_LDSS, Quikchange PCR was performed using the CKJ 49 vector as a template. To this end, the gene was amplified using primers (JY#21, JY#22) and Pfu Turbo polymerase (Agilent). The amplified gene was converted to Jude1 and then sequenced.

[0149] Example 21. Comparison of the binding affinity of PD-1 variants to PD-L1 by flow cytometry

[0150] After inoculating each of wild-type PD-1, HAC-V variant (control group), CKJ 49, CKJ 50, and LDSS into TB medium containing 2% glucose and 40 μg / mL chloramphenicol, the cells were cultured at 37°C and 250 rpm for 16 hours. The cultured cells were inoculated into 6 mL of TB medium containing 40 μg / mL chloramphenicol by diluting to 1:100. After culturing to OD 600 = 0.5, then cooled at 25°C and 250 rpm for 20 minutes, protein overexpression was induced by adding 1 mM IPTG at 25°C and 250 rpm for 5 hours. 600 Normalization The same number of E. coli was added to the e-tube, and the cells were recovered by centrifugation at 14000rpm for 1 minute. After preparing spheroplasts in the same manner as described in Example 5, 12.5nM of tetramer PD-L1-Alexa488 probe was added to the prepared spheroplasts and rotated at room temperature for labeling. After 1 hour of labeling, centrifuge at 13500rpm for 1 minute. After discarding the supernatant, the cells were washed once with 1mL of PBS and then centrifuged again at 13500rpm for 1 minute. The cells were resuspended in 1mL of PBS and the binding affinity to PD-L1 was analyzed by measuring the fluorescence signal using a Guava instrument ( Fig.16). In addition, since the expression level of PD-1 protein displayed by E. coli can affect the fluorescence intensity, the expression level of PD-1 was determined. To this end, take the 300 μL of E. coli remaining after resuspending in PBS, add 700 μL of PBS and 1 μL of anti-FLAG-FITC together, and label the spheroplasts with the fluorescent probe by rotating at room temperature. After 1 hour of labeling treatment, centrifuge at 13500 rpm for 1 minute. After discarding the supernatant, wash the cells once with 1 mL of PBS and then centrifuge again at 13500 rpm for 1 minute. Resuspend the cells in 1 mL of PBS, and analyze the binding affinity to anti-FLAG-FITC by measuring the fluorescence signal by flow cytometry ( Fig.16 ).

[0151] Table 4

[0152]

[0153] Additional screened PD-1 variants with improved binding affinity to PD-L1

[0154] Example 22. Expression, purification and binding affinity verification of PD-1 variants in animal cells

[0155] In order to verify the binding affinity of PD-1 variants expressed in animal cells, PD-1 variants were cloned into mammalian expression vectors. The genes of HAC-V (control group) and the screened variants CKJ 49 and CKJ50 were amplified by PCR using primers (CKJ#1, CKJ#2) and Vent polymerase. The amplified genes were treated with BssHII and XbaI enzymes and connected to the BssHII and XbaI fragments of the pMaz vector. The sequence of the connected plasmid was studied by single colony analysis transformed into Jude1 Escherichia coli. The prepared vectors expressing PD-1 sugar variants (pMaz-PD1 HAC-V-His tag, pMaz-PD1 CKJ 49-His tag, pMaz-PD1 CKJ 50-His tag) were transfected into animal cells (HEK293F). After culturing the cells for 6 days, the cell culture was centrifuged at 6000rpm for 20 minutes, and the supernatant was taken and filtered through a 0.22μm filter. The filtered supernatant was applied to 1 mL Ni-NTA resin (Qiagen) at 4°C for 16 hours. The protein-bound resin was washed with 10 CV (column volume) of PBS solution containing 10 mM imidazole (Sigma), and then washed again with 10 CV of PBS solution containing 20 mM imidazole. Finally, the eluate was recovered with PBS solution containing 250 mM imidazole ( Fig.17The binding affinity of wild-type PD-1, a control group, and two variants (GPD1-CKJ 49: glycosylated form of CKJ 49, GPD1-CKJ 50: glycosylated form of CKJ 50) was investigated by ELISA. Each protein was stained with 0.05 M Na 2 CO 3 (pH 9.6, Junsei) was diluted to 200 ng / well and then bound to a high-binding 96-well plate (Costar) at 4°C for 16 hours. After removing the protein, a blocking solution (PBST (PBS containing 0.5% Tween 20) solution containing 5% skim milk) was added to each 96-well plate at room temperature for 1 hour. After discarding the upper solution and washing 4 times with 200 μL of PBS solution containing 0.5% Tween 20, the PD-L1 tetramer was diluted with a PBS solution and then added to each 96-well plate at room temperature for 1 hour. After washing 4 times with 200 μL of 0.5% PBST solution, anti-streptavidin-HRP (Genetex) diluted to 1:2000 with PBS was added to each 96-well plate at room temperature for 1 hour. After discarding the upper solution and washing 4 times with 200 μL of 0.5% PBST solution, the reaction was started by adding 50 μL TMB (Thermo Scientific) and washed 20 minutes later with 4NH 2 SO 4 The reaction was terminated. The results confirmed that although glycosylation resulted in changes in binding affinity to PD-L1, CKJ 49 showed the best binding affinity ( Fig.18 ).

[0156] Although specific embodiments of the present disclosure have been described in detail above, it is obvious to those skilled in the art that they are only provided as preferred exemplary embodiments and do not limit the scope of the present disclosure. Therefore, the essential scope of the present disclosure should be defined by the appended claims and their equivalents.

[0157] [National research and development project supporting this disclosure]

[0158] [Project ID] 1711053534.

[0159] [Competent department] Ministry of Science and ICT.

[0160] [R&D management agency] National Research Foundation of Korea.

[0161] [Research project name] Rising Research Support Project.

[0162] [Research Name] Development of protein therapeutics and bispecific antibodies using antigen-binding aglycosylated Fc variants.

[0163] [Contribution coefficient] 1 / 2.

[0164] [Research institution] National University Industry-Academic Partnership.

[0165] [Research period] 2017.06.01 to 2018.03.31.

[0166] [National research and development project supporting this disclosure]

[0167] [Project ID] 1711058394.

[0168] [Competent department] Ministry of Science and ICT.

[0169] [R&D management agency] National Research Foundation of Korea.

[0170] [Research project name] New drug development pipeline management.

[0171] [Study Name] Identification of serum-persistent next-generation Fc-based anticancer antibodies targeting endothelin GPCR.

[0172] [Contribution coefficient] 1 / 2.

[0173] [Research institution] National University Industry-Academic Partnership.

[0174] [Research period] 2017.06.30 to 2018.03.29.

Claims

1. A PD-1 variant having improved binding affinity to PD-L1, wherein the amino acid sequence of the PD-1 variant is the amino acid sequence of the wild-type PD-1 of SEQ ID NO: 61, wherein the amino acid sequence of the wild-type PD-1 of SEQ ID NO: 61 is (i) the 69th amino acid is substituted by C69S and the 36th amino acid is substituted by S36P; (ii) the 69th amino acid is substituted with C69S, C69T, C69Y, C69G or C69A, the 36th amino acid is substituted with S36P and the 114th amino acid is substituted with L114P; (iii) the 69th amino acid is substituted with C69T or C69Y, the 13th amino acid is substituted with F13I and the 46th amino acid is substituted with M46I; or (iv) the 69th amino acid is substituted with C69S, the 25th amino acid is substituted with N25D, and the 92nd amino acid is substituted with N92S.

2. The PD-1 variant according to claim 1, wherein when the 69th amino acid is substituted by C69S, the 36th amino acid is substituted by S36P, and the 114th amino acid is substituted by L114P, one or more amino acids selected from the 12th, 34th, 92nd, 107th, 131st, 132nd and 142nd amino acids of the amino acid sequence of the PD-1 variant are also substituted with a sequence different from the wild-type amino acid, wherein one or more amino acid substitutions are selected from T12S, N34T, N92K or N92S, K107N, H131R, P132L and F142L.

3. The PD-1 variant according to claim 1, wherein when the 69th amino acid is substituted by C69S, the 25th amino acid is substituted by N25D, and the 92nd amino acid is substituted by N92S, the 13th amino acid of the amino acid sequence of the PD-1 variant is also substituted by F13L.

4. The PD-1 variant according to claim 1, wherein when the 69th amino acid is substituted by C69T, the 13th amino acid is substituted by F13I, and the 46th amino acid is substituted by M46I, one or more amino acids selected from the 1st, 17th, 50th, 79th, 100th, 114th, 127th and 139th amino acids of the amino acid sequence of the PD-1 variant are also substituted with a sequence different from the wild-type amino acid, wherein the one or more amino acid substitutions are selected from L1S, L17M, G79R, G100V, L114P and A139L.

5. The PD-1 variant according to claim 1, wherein when the 69th amino acid is substituted by C69S, the 36th amino acid is substituted by S36P, and the 114th amino acid is substituted by L114P, the 92nd amino acid and the 131st amino acid of the PD-1 variant are also substituted by N92K and H131R.

6. The PD-1 variant according to claim 1, wherein when the 69th amino acid is substituted by C69S, the 25th amino acid is substituted by N25D, and the 92nd amino acid is substituted by N92S, one or more amino acids selected from the 9th and 101st amino acids of the amino acid sequence of the PD-1 variant are also substituted with a sequence different from the wild-type amino acid, wherein the one or more amino acid substitutions of the PD-1 variant are selected from N9D and A101V.

7. The PD-1 variant according to claim 3, wherein one or more amino acids selected from the 88th, 125th and 137th amino acids of the amino acid sequence of the PD-1 variant are also substituted with a sequence different from the wild-type amino acid, wherein the one or more amino acid substitutions of the PD-1 variant are selected from R88K, A125S and R137K.

8. The PD-1 variant according to claim 1, wherein when the 69th amino acid is substituted by C69Y, the 13th amino acid is substituted by F13I, and the 46th amino acid is substituted by M46I, one or more amino acids selected from the 50th and 127th amino acids of the amino acid sequence of the PD-1 variant are also substituted with a sequence different from the wild-type amino acid, wherein the one or more amino acid substitutions of the PD-1 variant are selected from N50S and V127A. 9 . The PD-1 variant according to any one of claims 1 to 8 , wherein the PD-1 variant is an aglycosylated PD-1 variant.

10. A nucleic acid molecule encoding the PD-1 variant according to any one of claims 1 to 8. A vector comprising the nucleic acid molecule according to claim 10 . A host cell comprising the vector according to claim 11 .

13. The host cell of claim 12, wherein the host cell is a bacterial cell. 14 . A composition comprising the PD-1 variant according to any one of claims 1 to 8 , the nucleic acid molecule according to claim 10 , or the vector according to claim 11 as an active ingredient.

15. A method for preparing a PD-1 variant, comprising: a) a step of culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the PD-1 variant according to any one of claims 1 to 8; and b) A step of recovering the PD-1 variant expressed by the host cells.

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