WO2020004984A1 - Mutant pd-l1 ayant une affinité de liaison améliorée pour pd-1 - Google Patents

Mutant pd-l1 ayant une affinité de liaison améliorée pour pd-1 Download PDF

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WO2020004984A1
WO2020004984A1 PCT/KR2019/007829 KR2019007829W WO2020004984A1 WO 2020004984 A1 WO2020004984 A1 WO 2020004984A1 KR 2019007829 W KR2019007829 W KR 2019007829W WO 2020004984 A1 WO2020004984 A1 WO 2020004984A1
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variant
amino acid
seq
wild
type
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정상택
하지연
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국민대학교 산학협력단
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Priority to US17/255,629 priority patent/US20210324038A1/en
Priority claimed from KR1020190076918A external-priority patent/KR102216576B1/ko
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

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  • the present invention relates to a PD-L1 variant and a method for preparing the same, wherein PD-1 binding ability is increased to effectively inhibit binding between wild-type PD-L1 and PD-1.
  • Drugs for the treatment of cancer are largely divided into low-molecular weight drugs and high-molecular weight drugs, and due to their specificity, high-molecular weight drugs have specific attention as low-molecular weight drugs.
  • PD-1 and PD-L1 are expressed not only in cancer cells but also in human immune cells, antibody drugs can kill normal immune cells and cause autoimmune diseases.
  • TILs tumor-infiltrating lymphocytes
  • PD-L1 variants were screened through screening.
  • PD-L1 has few mutations because of the relatively low binding force and the introduction of many mutations. As a result, it is necessary to find a variant that binds to PD-1 with high binding force.
  • the present inventors made an effort to discover PD-L1 variants that can effectively inhibit the binding between wild-type PD-L1 and PD-1 due to their high binding capacity with PD-1, and at the same time minimize the possibility of immunogenicity. .
  • the present invention was completed.
  • Another object of the present invention to provide a nucleic acid molecule encoding the PD-L1 variant.
  • Still another object of the present invention is to provide a vector containing the nucleic acid molecule.
  • Another object of the present invention to provide a host cell comprising the vector.
  • Still another object of the present invention is to provide a composition comprising the variant, nucleic acid molecule or vector.
  • Another object of the present invention to provide a method for producing the variant.
  • Another object of the present invention is to provide a method for screening the variants.
  • the present invention provides a PD-L1 variant with an increased PD-1 binding force.
  • the present inventors made an effort to discover PD-L1 variants that can effectively inhibit the binding between wild-type PD-L1 and PD-1 due to their high binding capacity with PD-1, and at the same time minimize the possibility of immunogenicity. .
  • PD-L1 variant or “Programmed death-ligand 1 variant” includes variants in which one or two or more amino acids are substituted, deleted or added to the amino acid sequence of wild type PD-L1. Means variant.
  • the PD-L1 variant of the present invention includes a variant in which some amino acids in the amino acid sequence of the wild-type PD-L1 of SEQ ID NO: 123 are substituted, deleted or added.
  • the PD-L1 variant of the present invention preferably has at least 50% homology, more preferably at least 60% homology, even more preferably at least 70% phase with the amino acid sequence of wild type PD-L1 of SEQ ID NO: 123 Homology, even more preferably at least 80% homology, most preferably at least 90% homology.
  • the PD-L1 variant of the present invention comprises a part of the amino acid sequence of the wild type PD-L1, the 169th amino acid sequence of the wild type PD-L1 of SEQ ID NO: 123 Amino acid substituted with E169D.
  • the PD-L1 variant of the present invention is 41, 73, 117, 124, 130, 139, 195 of the amino acid sequence of the wild type PD-L1 of SEQ ID NO: 123 And one or more amino acids selected from the group consisting of the 1 st, 198 th, 201 th, 213 th and 218 th amino acids are substituted with a sequence different from that of the wild type amino acid.
  • the 195th amino acid sequence of the wild-type PD-L1 of SEQ ID NO: 123 is R195K, R195A, R195I, R195T, R195V, R195F, R195L, R195R Or substituted with R195M.
  • the PD-L1 variant of the present invention comprises the substitution of P198S, P198T or P198H with the 198th amino acid sequence of the wild type PD-L1 of SEQ ID NO: 123.
  • the 41st amino acid is M41V
  • the 117th amino acid is N117S
  • the 124th amino acid is L124S of the amino acid sequence of wild type PD-L1 of SEQ ID NO: 123.
  • 195th amino acid is substituted with R195A.
  • the PD-L1 variant of the present invention comprises that the 195th amino acid of the amino acid sequence of wild-type PD-L1 of SEQ ID NO: 123 is substituted with R195K.
  • the PD-L1 variant of the present invention comprises that the 73rd amino acid of the amino acid sequence of wild-type PD-L1 of SEQ ID NO: 123 is replaced with Q73R, and the 195th amino acid is replaced with R195I .
  • the PD-L1 variant of the present invention comprises the substitution of 130 amino acids to T130A and 195 amino acids to R195I of the amino acid sequence of wild type PD-L1 of SEQ ID NO: 123 .
  • the PD-L1 variant of the present invention comprises the substitution of 117th amino acid with N117S and 198th amino acid with P198H of the amino acid sequence of the wild-type PD-L1 of SEQ ID NO: 123 .
  • the PD-L1 variant of the present invention comprises that the 195th amino acid of the amino acid sequence of wild-type PD-L1 of SEQ ID NO: 123 is substituted with R195I, and the 213th amino acid is substituted with L213P .
  • the PD-L1 variant of the present invention has a 139th amino acid as A139S, 198th amino acid as P198T, and 201th amino acid in the amino acid sequence of wild type PD-L1 of SEQ ID NO: 123 It includes one substituted with N201S.
  • the PD-L1 variant of the present invention comprises the substitution of N218D amino acid 218 of the amino acid sequence of wild-type PD-L1 of SEQ ID NO: 123.
  • the PD-L1 variant of the present invention is SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: And SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 108 to SEQ ID NO: 122.
  • the present invention provides a nucleic acid molecule encoding the PD-L1 variant, a vector comprising the same or a host cell comprising the vector.
  • Nucleic acid molecules of the invention can be isolated or recombinant and include single and double stranded DNA and RNA as well as corresponding complementarity sequences.
  • An “isolated nucleic acid” is a nucleic acid isolated from a naturally occurring source, which is separated from the surrounding genetic sequence present in the genome of the individual from which the nucleic acid is isolated.
  • nucleic acids such as PCR products, cDNA molecules, or oligonucleotides synthesized enzymatically or chemically from a template
  • the nucleic acid resulting from this procedure can be understood as an isolated nucleic acid molecule.
  • Isolated nucleic acid molecules refer to nucleic acid molecules in the form of separate fragments or as components of larger nucleic acid constructs.
  • Nucleic acids are “operably linked” when placed in a functional relationship with other nucleic acid sequences.
  • the DNA of a presequence or secretion leader is operably linked to the DNA of a polypeptide when expressed as a preprotein, which is the form before the polypeptide is secreted, and the promoter or enhancer is a polypeptide sequence. Operably linked to a coding sequence when affecting the transcription of the ribosome binding site, or when the ribosome binding site is arranged to facilitate translation.
  • operably linked means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, they are present in the same reading frame adjacently. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional methods.
  • vector refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence.
  • Nucleic acid sequences can be exogenous or heterologous.
  • Vectors include, but are not limited to, plasmids, cosmids and viruses (eg bacteriophages).
  • plasmids include, but are not limited to, plasmids, cosmids and viruses (eg bacteriophages).
  • viruses eg bacteriophages.
  • One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994, etc.).
  • expression vector refers to a vector comprising a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, RNA molecules are then translated into proteins, polypeptides, or peptides. Expression vectors can include various regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors can also include nucleic acid sequences that provide additional functionality.
  • the term “host cell” refers to any transgenic organism that includes eukaryotes and prokaryotes and is capable of replicating the vector or expressing a gene encoded by the vector.
  • the host cell may be transfected or transformed by the vector, which means a process in which exogenous nucleic acid molecules are delivered or introduced into the host cell.
  • the host cell of the present invention is a bacterial cell, more preferably a Gram negative bacterial cell.
  • the cells are suitable for the practice of the present invention in that they have a periplasmic region between the inner membrane and the outer membrane.
  • Examples of preferred host cells of the present invention include E. coli , Pseudomonas aeruginosa , Vibrio cholera , Salmonella typhimurium , Shigella flexneri , Haemophilus influenza , Bordotella pertussi , Erwinia amylovora , Rhizobium sp. And the like, but are not limited thereto.
  • aglycosylated proteins are capable of mass production in bacteria and have excellent speed and cost.
  • the present invention provides a wild type PD-L1 (Programmed death-ligand 1) and PD-1 (Programmed cell) comprising the PD-L1 variant, nucleic acid molecule or vector as an active ingredient death protein-1) liver binding inhibitor.
  • PD-L1 Programmed death-ligand 1
  • PD-1 Programmed cell
  • the present invention provides a composition comprising the PD-L1 variant, nucleic acid molecule or vector as an active ingredient.
  • the composition is preferably a pharmaceutical composition, more preferably a pharmaceutical composition for preventing or treating cancer diseases or infectious diseases.
  • the present invention provides a wild type PD-L1 (Programmed death-ligand 1) comprising administering a pharmaceutically effective amount of the PD-L1 variant, nucleic acid molecule or vector to a subject.
  • PD-1 Programmed cell death protein-1
  • the present invention provides a method for increasing an immune response comprising administering to the subject a pharmaceutically effective amount of the PD-L1 variant, nucleic acid molecule or vector.
  • the present invention provides a method for treating a cancer disease or infectious disease, comprising administering a pharmaceutically effective amount of the PD-L1 variant, nucleic acid molecule or vector to a subject.
  • the pharmaceutical composition of the present invention comprises (a) the PD-L1 variant, nucleic acid molecule or vector; And (b) a pharmaceutically acceptable carrier.
  • the type of cancer to be prevented or treated by the present invention is not limited, leukemias and acute lymphocytic leukemia, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic lymphocytic leukemia Lymphomas, brain tumors, neuroblastoma, such as myelogenous leukemia, Hodgkin's Disease, non-Hodgkin's lymphomas, and multiple myeloma Childhood solid tumors such as retinoblastoma, Wilms Tumor, bone tumors and soft-tissue sarcomas, lung cancer, breast cancer cancer, prostate cancer, urinary cancers, uterine cancers, oral cancers, pancreatic cancer, melanoma and other skin cance rs, stomach cancer, ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroid cancer, esophageal cancer and testicular cancer It may be administered to treat a number of cancers, including common solid tumors of adults
  • the type of infectious disease to be prevented or treated by the present invention is not limited, and includes a viral infection, an influenza infection, a bacterial infection and a fungal infection.
  • Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen.
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, a kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mann
  • composition of the present invention may be administered orally or parenterally to a subject, preferably parenteral administration, for example, by intravenous infusion, topical infusion and intraperitoneal infusion.
  • the term “subject” or “subject” refers to an object to prevent or treat the disease through inhibition of binding between the PD-1 and PD-L1, and preferably includes humans and animals.
  • the term “pharmaceutically effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, doctor or other clinician. Amounts that induce alleviation of the symptoms of the disease or disorder in question. It will be apparent to those skilled in the art that the effective amount and administration frequency for the active ingredient of the present invention will vary depending on the desired effect.
  • Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis.
  • the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.
  • the pharmaceutical composition of the present invention may be used alone as a therapy, but may also be used in conjunction with other conventional biological, chemo, or radiation therapies, and such combination therapy may be used to treat cancer or infectious disease more effectively.
  • Chemotherapeutic agents that can be used with the composition when the present invention is used for the prevention and treatment of cancer are cisplatin, carboplatin, procarbazine, mechlorethamine, Cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin, daunorucin Daunorubicin, doxorubicin, bleomycin, plecomycin, mitomycin, etoposide, tamoxifen, tamoxifen, taxol, transflavol Transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, and the like.
  • Radiation therapy that can be used with the composition of the present
  • the present invention provides a method for preparing a PD-L1 variant, comprising the following steps:
  • the invention provides a method of screening for PD-L1 variants comprising the following steps:
  • the screening methods of the present invention can use fluorescence labeled cell separation (FACS) screening, or other automated flow cytometry techniques.
  • FACS fluorescence labeled cell separation
  • Instruments for performing flow cytometry are known to those skilled in the art. Examples of such devices are FACSAria, FACS Star Plus, FACScan and FACSort devices (Becton Dickinson, Foster City, CA), Epics C (Coulter Epics Division, Hialeah, FL), MOFLO (Cytomation, Colorado Springs, Colo.), MOFLO- XDP (Beckman Coulter, Indianapolis, IN).
  • Flow cytometry techniques generally include the separation of cells or other particles in a liquid sample.
  • a flow cytometer typically the purpose of a flow cytometer is to analyze the separated particles for their one or more properties (eg the presence of labeled ligands or other molecules). Particles are passed one by one by the sensor and are classified based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, and the like.
  • the present invention provides PD-L1 variants with increased PD-1 binding capacity.
  • the present invention also provides a method for producing and screening the PD-L1 variant.
  • the PD-L1 variant of the present invention significantly improves the binding ability with PD-1 by replacing some amino acid sequences of wild-type PD-L1 with other amino acid sequences, thereby generating immunogenicity through the implementation of minimization of mutation sites. The likelihood can be greatly reduced.
  • Figure 6 shows the result of the binding force analysis of the PD-L1 variants obtained with PD-1.
  • Figure 7 shows the results of the binding force analysis of PD-1 of PD-L1 variants obtained through major position mutations.
  • Example 1 Cloning to Display Human PD-L1 in Bacterial Cell Intima (Wild Type PD-L1)
  • human PD-L1 extracellular region portion (SEQ ID NO: 123) was cloned for PD-L1 engineering using bacterial displays.
  • human PD-L1 gene cDNA was purchased from Sino Biotech (Catalog number: HG10084-M), and then the DNA of amino acid sequence F19-R238, which is a PD-L1 extracellular region, was prepared by primer (JY # 1, JY # 2). The gene was amplified by PCR with Vent Polymerase (New England Biolab).
  • the ligated plasmid was transformed into Jude1 ((F-mcrA ⁇ (mrr-hsdRMS-mcrBC) ⁇ 80lacZ ⁇ M15 ⁇ lacX74 recA1 endA1 araD139 ⁇ (ara, leu) 7697 galU galK ⁇ -rpsL nupG) Escherichia coli and analyzed by individual colonies). The sequence was confirmed.
  • Each sample was incubated for 16 hours at 37 ° C., 250 rpm in 4 ml of TB 2% glucose medium containing 40 ⁇ g / ml of chloramphenicol, and then the cultured cells were 7 mL of TB containing 40 ⁇ g / ml of chloramphenicol.
  • Resuspension was performed by adding 1 ml of 10 mM Tris-HCl (pH 8.0) to each e-tube from which the cells were recovered, and the remaining medium was removed by repeating the cell collection again by centrifugation (14,000 rpm, 1 min). . After washing, the cells were resuspensioned with 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 extracellular membrane. The cells were collected again by centrifugation (14,000 rpm, 1 min), and then the supernatant was removed.
  • STE 0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)
  • 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8] was added to resuspension and centrifuged (14,000 rpm, 1 min) to remove the supernatant, followed by 1 mL of Solution A and 50 mg / ml lysozyme. 1 ml of a solution containing 20 ⁇ l of solution was resuspensioned and rotated at 37 ° C. for 15 minutes to remove the peptidoglycan layer.
  • PD-1 was cloned in order to use it as a fluorescent probe when screening PD-L1 with strong binding force to PD-1.
  • dimer was induced by expressing GST in C-terminal part of PD-1 for more efficient screening using avidity effect through PD-1 dimerization.
  • a linker consisting of Gly and Ser between PD-1 and GST for fluidity of each protein.
  • GST was also amplified by PCR with Vent Polymerase using primers (CKJ # 3, CKJ # 4).
  • the PD-1 and GST DNAs amplified in this way were subjected to assembly PCR using a vent polymerase to generate PD-1-GST-His tags, followed by restriction enzymes Bss HII and Xba I (New England Biolab). Ligation was performed with pMAZ, an enzyme-treated animal cell expression vector. The ligated plasmid was transformed into Jude1 Escherichia coli and confirmed the sequence through individual colony analysis.
  • the completed dimeric PD-1 expression vector was transfected into animal cells (HEK293F) and incubated for 6 days. Cell culture was centrifuged at 6,000 xg for 15 minutes, then the supernatant was taken and filtered through a 0.22 ⁇ m filter. The filtered solution was mixed with 1 mL of Ni-NTA resin (Qiagen) and bound at 4 ° C. for 16 hours. The combined solution was poured into a column, washed with 10 CV (column volume) of PBS solution containing 10 mM imidazole (SIGMA), and washed once more with 10 CV of 20 mM imidazole containing PBS solution.
  • the purified PD-1 dimer was fluorescently labeled using Alexa-488 labeling kit. As a result of analyzing the activity of the fluorescently labeled dimeric PD-1 by ELISA, it was confirmed that it has excellent binding capacity with PD-L1 (Fig. 2).
  • E. coli cell anchoring motifs were determined, and NlpA system (pMopac12-NlpA-PDL1_WT-FLAG) anchoring the N-terminal of protein and geneIII system (pAK200-PelB-PDL1_WT) anchoring the C-terminal part -gene III) will be compared. Since pMopac12-NlpA-PDL1_WT-FLAG plasmid has already been secured, only pAK200-PelB-PDL1_WT-gene III was further cloned.
  • DNAs of the amino acid sequences F19-R238, which are part of the extracellular region of PD-L1 were amplified by PCR with Vent Polymerase using primers (JY # 3, JY # 2). Processing the amplified gene into the restriction enzyme Sfi I and then, the process proceeds to the process with Sfi I pAK200-PelB-geneIII vector and the ligation was complete the pAK200-PelB-PDL1_WT-geneIII plasmid. This is to secrete the protein into the E. coli periplasmic region through a signal peptide called PelB, and then to anchor the C-terminal of PD-L1 by the gene III protein immobilized on the cell membrane. The ligated plasmid was transformed into Jude1 Escherichia coli and confirmed the sequence through individual colony analysis.
  • Example 6 Selection of display method and probe concentration by verifying the binding force between PD-L1 expressed in E. coli and PD-1-GST probe using flow cytometry
  • the completed pMopac12-NlpA-PDL1-FLAG and pAK200-PelB-PDL1-geneIII plasmids were transformed into Jude1 cells, respectively.
  • Each sample was incubated for 16 hours at 37 ° C., 250 rpm in 4 ml of TB 2% glucose medium containing 40 ⁇ g / ml chloramphenicol, and then the cultured cells were stored in 7 mL TB containing 40 ⁇ g / ml chloramphenicol.
  • the cells were resuspensioned with 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 extracellular membrane. The cells were collected again by centrifugation (14,000 rpm, 1 min), and then the supernatant was removed. 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8] was added to resuspension and centrifuged (14,000 rpm, 1 min) to remove supernatant, followed by 1 ml of Solution A and 50 mg / ml lysozyme solution.
  • STE 0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)
  • Example 7 Fabrication of large PD-L1 error prone library for using ultrafast screening technique
  • PD-1 and enables the random mutations into every part of PDL1 based on pAK200-PelB-PDL1-geneIII to search at a high speed for PDL1 variant with a high affinity containing Sfi I site on both Primers (JY # 4, JY # 5) were designed.
  • DNA was amplified using the designed primer, Taq Polymerase (TAKARA), dNTPs (Invitrogen), MgCl 2 , MnCl 2 (SIGMA) using Error Prone PCR. Processing the amplified gene into the restriction enzyme Sfi I and then, the process proceeds to the process with Sfi I pAK200-PelB-geneIII vector and ligation was Jude1 transformation in E. coli.
  • TAKARA Taq Polymerase
  • dNTPs Invitrogen
  • MgCl 2 MnCl 2
  • SIGMA Error Prone PCR
  • Example 8 PD-L1 variant screening using flow cytometer
  • 1 ml of the initial library was inoculated into 25 ml of TB 2% glucose medium to which 40 ⁇ g / ml of chloramphenicol was added, and then incubated at 37 ° C. at 250 rpm for 4 hours.
  • E. coli cultured in 100 ml of TB medium containing 40 ⁇ g / ml chloramphenicol was inoculated at a ratio of 1: 100.
  • OD 600 0.5 at 37 ° C. and 250 rpm
  • the mixture was cooled at 25 ° C. and 250 rpm for 15 minutes, followed by induction for 25 ° C., 250 rpm, and 5 hours by adding 1 mM IPTG.
  • the cells were recovered in the e-tube by centrifugation (14,000 rpm, 1 min) by OD 600 normalization. Resuspension was performed by adding 1 ml of 10 mM Tris-HCl (pH 8.0) to each e-tube from which the cells were collected, and the remaining medium was removed by repeating the cell collection through centrifugation (14,000 rpm, 1 minute) twice. . The washed cells were resuspensioned with 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 extracellular membrane.
  • STE 0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)
  • the cells were collected again by centrifugation (14,000 rpm, 1 min), and then the supernatant was removed.
  • 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8] was added to the solution and centrifuged (14,000 rpm, 1 min) to remove the supernatant, followed by 1 ml of Solution A and 50 mg / ml lysozyme.
  • 1 ml of a solution containing 20 ⁇ l of solution was added and resuspensioned to rotate at 37 ° C. for 15 minutes to remove the peptidoglycan layer.
  • coli were able to be obtained by amplifying a gene by PCR using a primer (JY # 5, JY # 6) again, and then processed by a restriction enzyme, Sfi I, treated with Sfi I pAK200-PelB-geneIII Ligation with the vector was transformed into Jude1 Escherichia coli. Spread it on a plate and incubated for 16 hours at 37 °C to recover all E. coli using TB 2% glucose medium and stored at -80 °C. The screening process as described above was carried out a total of six times while reducing the concentration of the probe.
  • Example 9 Escherichia coli culture for confirming enrichment of PD-L1 variants with increased binding force with PD-1
  • wild-type PD-L1 a control group
  • wild-type PD-L1 was incubated for 16 hours at 37 ° C. and 250 rpm in 4 ml of TB 2% glucose medium containing 40 ⁇ g / ml of chloramphenicol, and the cultured cells contained 40 ⁇ g / ml of chloramphenicol.
  • Resuspension was performed by adding 1 ml of 10 mM Tris-HCl (pH 8.0) to each e-tube from which the cells were recovered, and the remaining medium was removed by repeating the cell collection through centrifugation (14,000 rpm, 1 minute) twice. .
  • the washed cells were resuspensioned with 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 extracellular membrane.
  • the cells were collected again by centrifugation (14,000 rpm, 1 minute), and then the supernatant was removed.
  • Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8], resuspension, remove the supernatant by centrifugation (14,000 rpm, 1 min), and 1 ml of Solution A with 50 mg / ml lysozyme. 1 ml of a solution containing 20 ⁇ l of solution was resuspensioned and rotated at 37 ° C. for 15 minutes to remove the peptidoglycan layer.
  • PD-L1_L3B3 variants were used as primers (JY # 7, JY # 8, JY # 9, JY # 10, JY # 11, JY # 12, JY # 13, JY # 14, JY # 15, JY # 16, JY # 17, JY # 18, JY # 19, JY # 20) the after processing that have been created through the assembly PCR using 14 was amplified gene with restriction enzyme Sfi I, treated with Sfi I pAK200-PelB Ligation with -geneIII vector was performed to complete the pAK200-PelB-PDL1_L3B3-geneIII plasmid. The ligated plasmid was transformed into Jude1 Escherichia coli and confirmed the sequence through individual colony analysis.
  • Example 12 Obtaining PD-L1 Variants with Increased Adhesion with PD-1 by Flow Cytometry Analysis
  • the cells were recollected by centrifugation (14,000 rpm, 1 min), and then the supernatant was removed.
  • Add 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8], resuspension, remove the supernatant by centrifugation (14,000 rpm, 1 min), and 1 ml of Solution A and 50 mg / ml 1 ml of a solution of 20 ⁇ l of lysozyme solution was added, resuspensioned, and rotated at 37 ° C. for 15 minutes to remove the peptidoglycan layer.
  • the process proceeds to the process with Sfi I pAK200-PelB-geneIII vector and the ligation was complete the pAK200-PelB-PDL1_L3B3-geneIII plasmid.
  • the ligated plasmid was transformed into Jude1 Escherichia coli and confirmed the sequence through individual colony analysis.
  • the washed cells were resuspensioned with 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 extracellular membrane. The cells were collected again by centrifugation (14,000 rpm, 1 min), and then the supernatant was removed.
  • STE 0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)
  • Solution A [0.5 M sucrose, 20 mM MgCl 2 , 10 mM MOPS pH 6.8], resuspension, remove the supernatant by centrifugation (14,000 rpm, 1 min), and then remove 1 ml of Solution A with 50 mg / 1 ml of the solution containing 20 ⁇ l of the ml lysozyme solution was resuspensioned and rotated at 37 ° C. for 15 minutes to remove the peptidoglycan layer.
  • Fluorescence signal values were indirectly analyzed for binding of the variants to PD-1 (FIG. 7). Through this, the variants with amplification of fluorescence signal values more than four-fold amplification were identified. Among them, the affinity of DAS, DTS, DTT, DLT and DMS increased five times or more (FIG. 8).

Abstract

La présente invention concerne un mutant PD-L1 ayant une affinité de liaison améliorée pour PD-1. La présente invention concerne également un procédé de préparation du mutant PD-L1 et un procédé de criblage du mutant PD-L1. Le mutant PD-L1 selon la présente invention est conçu pour améliorer considérablement l'affinité de liaison pour PD-1 par l'intermédiaire de l'optimisation obtenue par substitution d'une partie de la séquence d'acides aminés de PD-L1 de type sauvage avec une séquence d'acides aminés différente et pour réduire largement la plausibilité de la génération d'immunogénicité par la minimisation des positions de mutation.
PCT/KR2019/007829 2018-06-29 2019-06-27 Mutant pd-l1 ayant une affinité de liaison améliorée pour pd-1 WO2020004984A1 (fr)

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WO2023143443A1 (fr) * 2022-01-25 2023-08-03 BRL Medicine Inc. Cellules modifiées et leurs utilisations

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WO2017201131A1 (fr) * 2016-05-18 2017-11-23 Albert Einstein College Of Medicine, Inc. Variants de polypeptides pd-l1, polypeptides multimères modulateurs des lymphocytes t et procédés d'utilisation correspondants
KR20180039182A (ko) * 2016-06-13 2018-04-17 아이-맵 항-pd-l1 항체 및 이것의 사용
KR20180050179A (ko) * 2016-11-04 2018-05-14 주식회사 제넥신 변형된 pd-l1 단백질 및 이의 용도

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WO2017201131A1 (fr) * 2016-05-18 2017-11-23 Albert Einstein College Of Medicine, Inc. Variants de polypeptides pd-l1, polypeptides multimères modulateurs des lymphocytes t et procédés d'utilisation correspondants
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