Conditional control of universal CAR T cells by stimulus-responsive adapters

Abstract

This invention provides site-specific antibody conjugate compounds for use in the construction of conditional universal chimeric antigen receptor (CAR) systems. [Solution] A molecule is provided comprising the following three components: 1) a site-specific antibody conjugate group X, 2) a SNAPtag substrate, and 3) a stimulus-responsive saging group R, wherein the site-specific antibody conjugate group X is preferably a bis-sulfone or dibromopyrazine, the SNAPtag substrate is preferably benzylguanine (BG), and the molecule carrying the stimulus-responsive saging group R preferably contains a photoreactive saging group including nitrobenzyl, coumarin, BODIPY, or cyanine.

Claims

1. A conditional universal chimeric antigen receptor (CAR) system, i) a conditional adapter - Receptors that target molecular tag ligands, hinge domains, and signaling domains. CAR including the body; and ii) antigen recognition element, stimulating reaction group, and tag ligand The conditional universal CAR system, which includes a conditional adapter molecule.

2. The tag ligand on the conditional adapter molecule is benzylguanine (BG), Dylcytosine (BC), chloroalkane, fluorescein (FITC), SpyTag leucine zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc domain, pe A conditional application according to claim 1, comprising a ptidoneoepitope (PNE) or biotin. Universal car type.

3. The aforementioned conditional adapter molecule is an NHS-ester conjugation, disulfide Restaping, glycan conjugate chemistry, via one or more short peptide tags Recombinant antibodies with tag ligand incorporation, saltase-mediated ligation, THIO MAB, chemical ligation, split-intein, and / or non-natural amino acids A conditional universal CAR system according to claim 1, including the following.

4. Claim 3, wherein the antigen recognition element comprises an antibody or an antigen recognition fragment thereof. Universal car system with the conditions described.

5. The antigen recognition element is rituximab, FMC63, Herceptin, cetuximab Nimotuzumab, Panitumumab, Omalizumab, Tositumomab, Trastuzumab, Gemutz Zumab, alemtuzumab, bevacizumab, or the same The conditional universal CA according to claim 4, comprising either one antigen-binding fragment R series.

6. The antigen recognition element is a protein-binding domain, a lectin, a DNA aptamer, RNA aptamers, small molecule ligands for cell surface receptors, or peptides for innate protein receptors The conditional universal CAR system according to claim 3, comprising a butyl / protein ligand.

7. The stimulus in which the aforementioned stimulating reactive group is reactive is light, enzyme activity, low molecular weight, pH, H ２ O ２ , low acid Conditional universal according to any one of claims 1 to 6, including elements and / or ROS. CAR type.

8. The stimulating reaction group includes a cleavage-type linker, as described in any one of claims 1 to 7. Universal Car series.

9. Claims that the stimulating reaction group includes a photocleavage type linker or a phosphine cleavage type linker. The conditional universal car system described in item 8.

10. The stimulating reactive group blocks the binding of the CAR to the tag ligand. A conditional universal CAR system according to any one of claims 1 to 9, comprising a casing group.

11. The aforementioned stimulating reaction group includes nitrobenzyl, coumarin, BODIPY, or cyanine. A conditional universal CAR system according to claim 10, comprising a photoreactive casing group.

12. The stimulating reactive group includes a photoreactive group, and the wavelength of the photostimulation is 365, 405, 544, A conditional universal CAR according to any one of claims 1 to 10, including 780 nm system.

13. The stimulating reaction group includes an enzyme reaction group, and the enzyme in which the reaction group is reactive is regmain. Matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde Dodehydrogenase 7 family, member A1, (ALDH7A1), lipase C, liver Visceral type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate Acid kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexyl Kinase A (KHK-A), hexokinase (HK), nucleoside diphosphate kinase ( NDPK or NDK), and 6-phosphofructo-2-kinase / fructose-2, 6-Biphosphatase 4 (PFKFB4), Mitochondrial α-Ketoglutarate Dehydro Genase (α-KGDH), lysine acetyltransferase 2A (KAT2A), A Cetyl CoA synthetase short chain family member 2 (ACSS2), ATP-citric acid Lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutamyl lyase Contains fumarase, alpha-kGDH, CD39, CD73, or fumarase. A conditional universal car system according to any one of claims 1 to 7.

14. The aforementioned stimulating reactive group is reactive to low molecules containing phosphine or tetrazine. A conditional universal CAR system according to any one of claims 1 to 7.

15. The conditional version according to any one of claims 1 to 14, further comprising one or more co-stimulatory domains Universal Car series.

16. The one or more of the aforementioned co-stimulatory domains are CD27, CD28, ICOS, 4-1BB, and The conditional universal C according to claim 15, comprising the signal transduction domain of OX40. AR (Augmented Reality) type.

17. Tag ligands that target CAR are used in CAR T cells, CAR NK cells, and CAR N K1-1, contained on K1 T cells, CAR B cells, or CAR macrophages A conditional universal car system as described in any of item 5.

18. Conditional adapter molecule containing a stimulating reactive group and a tag, not containing one or more cleavage sites A conditional universal synthesis Notch ( containing a ch core and one or more transcription factors) SYN(Notch) receptor.

19. The aforementioned conditional adapter molecule is benzylguanine (BG), benzylcytosine (BC ), chloroalkane, fluorescein (FITC), SpyTag, leucine zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc domain, peptide neoepitope The conditional user according to claim 18, comprising (PNE), or a tag ligand containing biotin. Universal synNotch.

20. The adapter molecule performs NHS-ester conjugation and disulfide restatement. Pulling, glycan conjugate chemistry, tag removal via one or more short peptide tags Recombinant antibodies with ligation, saltase-mediated ligation, chemical ligation, Claim 1, comprising split-intein, thio-mab, and / or a non-natural amino acid. Conditional universal synNotch as described in 8 or 19.

21. The stimulus for which the aforementioned stimulating reactive group is reactive is light, enzyme, low molecular weight, pH, hypoxia, H ２ O ２ , and / or ROS, a conditional universal according to any one of claims 18 to 20 synNotch.

22. The stimulating reactive group comprises a stimulating cleavage type linker, according to any one of claims 18 to 21. Conditional universal synNotch.

23. Claims that the stimulating reaction group includes a photocleavage type linker or a phosphine cleavage type linker. Conditional universal synNotch as described in 22.

24. The stimulating group blocks the receptor from binding to the tag ligand. A conditional universal syn according to any one of claims 18 to 21, comprising a casing group. Notch.

25. The aforementioned stimulating reaction group includes nitrobenzyl, coumarin, BODIPY, or cyanine. A conditional universal according to any one of claims 18 to 21, comprising a photoreactive casing group. Lu synNotch.

26. The stimulating reactive group includes a photoreactive group, and the wavelength of the photostimulation is 365, 405, 544, A conditional universal synNotch according to claim 25, including 780 nm.

27. The aforementioned stimulating reaction group includes an enzyme reaction group, and the enzyme that the reaction group reacts with is regmain, ma Trix metalloproteinase, pyridoxal kinase (PDXK), aldehyde Hydrogenase 7 family, member A1, (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate Nase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexoxin -ase A (KHK-A), hexokinase (HK), nucleoside diphosphate kinase (ND PK or NDK), and 6-phosphofructo-2-kinase / fructose-2,6- Biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase -ase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl CoA synthetase short chain family member 2 (ACSS2), ATP-citric acid -ase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutaric acid Contains dehydrogenase (α-KGDH), CD39, CD73, or fumarase. A conditional universal synNotch as described in any of requirements 18-21.

28. The aforementioned stimulating reactive group is reactive to low molecules containing phosphine or tetrazine. A conditional universal synNotch according to any one of claims 18 to 21.

29. The aforementioned transcription factors include Gal4-VP64, Gal4-VP16, TetR-VP64, and Or a conditional universe according to any one of claims 18 to 28, including LacI-VP64 Monkey syn Notch.

30. Further comprising an antigen recognition element, wherein the antigen recognition element is the conditional universe The monkey adapter molecule may be covalently bonded to it, according to claims 18 to 29. Conditional universal synNotch as described below.

31. Claim 18, wherein the antigen recognition element comprises an antibody or an antigen recognition fragment thereof. Conditional universal synNotch as described in any of the above 30.

32. The antigen recognition element is rituximab, FMC63, Herceptin, cetuximab Nimotuzumab, Panitumumab, Omalizumab, Tositumomab, Trastuzumab, Gemutz Zumab, alemtuzumab, bevacizumab, or the same A conditional according to any one of claims 18 to 31, comprising any one antigen-binding fragment Universal Sync Notch.

33. The antigen recognition element is a protein-binding domain, a lectin, a DNA aptamer, RNA aptamers, small molecule ligands for cell surface receptors, or peptides for innate protein receptors A conditional unit according to any one of claims 18 to 32, comprising a peptide / protein ligand. Versal synNotch.

34. A conditional universal CAR according to any one of claims 1 to 17 and / or claim 1 Manipulated cells containing the conditional universal synNotch described in any of 8 to 33.

35. A transcriptional signal operatively linked to a promoter that drives the expression of one or more cellular response genes. The vector further comprises the response element and the transcription factor on the synNotch receptor. The operation according to claim 34, wherein one or more of the offspring are specific to the transcription response element. production cells.

36. The one or more of the above response genes are IL-4, IL-10, FASL, IFN-γ, TNF -α, Granzyme A, Granzyme B, Granulysin, and / or Perforin The manipulated cells according to claim 34 or 35, including the manipulated cells according to claim 34 or 35.

37. One or more transcription factors of the conditional universal synNotch receptor, The operating method according to any one of claims 34 to 36, which activates the expression of the innate cell response gene. Cell.

38. The one or more of the aforementioned native cell response genes are IL-4, IL-10, FASL, IFN-γ , TNF-α, Granzyme A, Granzyme B, Granulisin, and / or Par The manipulated cell according to claim 37, comprising folin.

39. The cells are immune cells, neurons, epithelial cells, and endothelial cells, or stem cells. The manipulated cell according to any one of claims 34 to 36.

40. A method for treating a disease or disorder in the subject, wherein the method is as described in claims 1 to 17 A conditional universal chimeric antigen receptor (CAR) system as described in any of the claims 18- A conditional SynNotch as described in any of claims 33, and / or as described in claims 34 to 39. This includes administering the manipulated cells to the subject, wherein the disease or disorder is This includes autoimmune diseases, autoinflammatory diseases, viral infections, bacterial infections, or fungal infections. The aforementioned method.

41. The aforementioned diseases include lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer NCE), squamous cell carcinoma of the head and neck, lung cancer, small cell lung cancer and non-small cell lung cancer, neuroblastoma / Glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, and lungs, uterine cancer cervical cancer oma), breast cancer, epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer (head a nd neck cancer), large bowel cancer r), hematopoietic cancer; testicular cancer; colorectal cancer, rectal cancer, prostate cancer, A disease or disorder according to claim 40, which is a cancer selected from the group consisting of pancreatic cancer. Methods of treatment.

42. The aforementioned diseases include achalasia, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, Painful steatosis, adult Still's disease, agammaglobulinemia, alopecia areata, Alzheimer's disease, Amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid antibody syndrome Aplastic anemia, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune cerebrospinal fluid inflammation, autoimmune bowel disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease ( AIED, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis Autoimmune polyendocrine syndrome, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal nerve disorders Disorder (AMAN), Balo's disease, Behçet's disease, benign mucosal pemphigoid, Bickerst AFF-type brainstem encephalitis, bullous pemphigoid, Castleman disease (CD), celiac disease, Shar Gas sickness, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing polyneuropathy Osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), Eosinophilic granulomatosis (EG PA), scarring pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsat Key myocarditis, CREST syndrome, Crohn's disease, herpetiform dermatitis, dermatomyositis, Devic's disease (visual) Neuromyelitis, type 1 diabetes mellitus, lupus discoid, Dressler syndrome, endometriosis Enthesitis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed type erythema Rioglobulinemia, Evans syndrome, Felty syndrome, fibromyalgia, pulmonary fibrosis, mega Cellular arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes zoster of pregnancy, or gestational purpura Smallpox (PG), hidradenitis suppurativa (HS) (reverse acne), hypogammaglobulinemia, IgA nephropathy , IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM ), interstitial cystitis (IC), inflammatory bowel disease (IBD), juvenile arthritis, juvenile diabetes (1 Type 1 diabetes, juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytosis Vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus nephritis, Lupus vasculitis, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed Connective tissue disease (MCTD), Mollen's ulcer, Mucha-Haberman disease, multiple motor neuropathy MMN (Many Myasthenia Gravis) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, Neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring pemphigoid, optic neuritis, oatitis Ord's schyroiditis, relapsing rheumatoid arthritis (PR), PANDA S, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), hemifacial disfigurement Atrophy, squamous cellulitis (peripheral uveitis), personage-Turner syndrome, pemphigus, peripheral nerve Perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa Polyglandular syndrome type I, type II, type III, polymyalgia rheumatica, polymyositis, myocardial infarction Post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone Psoriasis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs RLS syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatoid vasculitis, Lucoidosis, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjögren's syndrome Lennell's syndrome, sperm and testicular autoimmunity, Stiff Person Syndrome (SPS), subacute bacterial Endocarditis (SBE), Susac syndrome, Sydenham chorea, sympathetic ophthalmitis (SO), systemic Lupus erythematosus, systemic sclerosis, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenia Minor purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, Ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), urticaria, urticarial vasculitis, grapes Femoritis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and Wegener's granulomatosis (or multiple) Claim 40, an autoimmune disease selected from the group consisting of granulomatous vasculitis (GPA). Methods for treating the diseases or disorders described above.

43. The aforementioned diseases include asthma, graft-versus-host disease, allergy, graft rejection, and familial cold autoitis. FCAS syndrome, Muckle-Wells syndrome (MWS), neonatal multiorgan syndrome Autoinflammatory diseases (NOMIDs) (also known as chronic infantile neurocutaneous arthral syndrome (CINCA)) (It can be), Familial Mediterranean Fever (FMF), Tumor Necrosis Factor (TNF)-Associated Periodic Syndrome (T) RAPS, TNFRSF11A-associated hereditary fever disorder (TRAPS11), periodic fever Hyperimmune globulinemia D with syndrome (HIDS), mevalonaciduria (MA), mevalonate Kinase deficiency (MKD), interleukin-1β (IL-1β) receptor antagonist DIRA deficiency (osteomyelitis, pustular periostitis) Majeed syndrome, also known as aseptic multifocal skeletal dysplasia (SIS), chronic nonbacterial bone syndrome Myelitis (CNO), early-onset inflammatory bowel disease, diverticulitis, interleukin-36 receptor antagonism Factor deficiency (DITRA), familial psoriasis (PSORS2), pustular psoriasis (15), suppurative Aseptic arthritis, pyoderma gangrenosum, and acne syndrome (PAPA), immunodeficiency, fever, and developmental changes. Hereditary sideroblastic anemia with delayed symptoms (SIFD), granulomatous arthritis in children (PGA), familial vein dysplasia Chet-like autoinflammatory syndrome, NLRP12-associated periodic fever syndrome, proteasome-related Autoinflammatory syndrome (PRAAS), spondylochondrodysplasia with immunomodulatory disorders (SPENC) DI, infant-onset STING-associated vasculitis (SAVI), Ecardi-Goutier syndrome , acute febrile neutrophilic dermatosis, X-linked familial hemophagocytic lymphohistiocytosis, and Lynquina It is an autoinflammatory disease selected from the group consisting of -ase-related autoinflammatory diseases (LAIDs). A method for treating the disease or disorder described in claim 40.

44. The aforementioned diseases include herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus. Viruses, Epstein-Barr virus, cytomegalovirus, human herpesvirus-6 Smallpox virus, varicella stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus Hepatitis viruses, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus Influenza A virus, Influenza B virus, Measles virus, Polyovirus Myvirus, human papillomavirus, respiratory syncytial virus, adenovirus, coxavirus Viruses such as buckeye, dengue virus, mumps virus, poliovirus, and rabies virus. Rous sarcoma virus, reovirus, yellow fever virus, Ebola virus, Marburg Viruses, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus Inflammatory viruses, Murray Valley fever virus, West Nile virus, Rift Valley fever virus Rotavirus A, Rotavirus B, Rotavirus C, Sindbisvirus, Monkey Immunovirus Inferiority virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, monkey virus A group consisting of epidemic deficiency virus, human immunodeficiency virus type 1, and human immunodeficiency virus type 2. A person who treats the disease or disorder described in claim 40, which is a viral infection selected from among Law.

45. The aforementioned disease is Mycobaterium tuberculosis, Mycobat erium bovis, Mycobaterium bovis strain BCG, BCG substrain , Mycobaterium avium, Mycobaterium intratrace llular, Mycobaterium africanum, Mycobateri um kansasii, Mycobaterium marinum, Mycobat erium ulcerans, Mycobaterium avium subspecies cies paratuberculosis, Nocardia asteroid s, Legionella pneumophila, Salmonella typ i, Salmonella enterica, Shigella boydii, Sh igella dysenteriae, Shigella sonnei, Shige lla flexneri, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, Actin obacillus pleuropneumoniae, Listeria mono cytogenes, Listeria ivanovii, Brucella abo rtus, Cowdria ruminantium, Borrelia burgdo rferi, Bordetella avium, Bordetella pertus sis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella p terii, Bordetella parapertussis, Bordetella ansorpi, Burkholderia mallei, Burkholder ia pseudomallei, Burkholderia cepacian, Ch lamydia pneumoniae, Chlamydia trachomatis 、Chlamydia psittaci, Coxiella burnetii, Ri ckettsial species, Ehrlichia species, Staphylococcus aur eus, Staphylococcus epidermidis, Streptoco ccus pneumoniae, Streptococcus pyogenes, S treptococcus agalactiae, Escherichia coli 、Vibrio cholerae, Campylobacter species, Neisserri a meningitidis, Neisseria gonorrhea, Pseud omonas aeruginosa, other Pseudomonas species, Haemoph ilus influenzae, Haemophilus ducreyi, Clost ridium tetani, and Yersinia enterocolitica from A person who treats the disease or disorder according to claim 40, which is a bacterial infection selected from the group. Law.

46. The aforementioned diseases include Candida albicans and Cryptococcus neon. formans, Histoplama capsulatum, Aspergillus S fumigatus, Coccidiodes immitis, Paracocc idiodes brasiliensis, Blastomyces dermiti dis, Pneumocystis carnii, Penicillium marn A true value selected from the group consisting of effi and Alternaria alternata. A method for treating a disease or disorder according to claim 40, which is a bacterial infection.

47. The method is the first conditional universal CAR system according to any one of claims 1 to 17. and the second conditional universal CAR system described in any of claims 1 to 17 is the target. The first conditional universal CAR system is administered to a stimulant cleavage type linker The second CAR system includes a stimulating reactive group, and the CAR binds to the tag. The obstruction according to any one of claims 40 to 46, comprising a stimulus-reactive saging group that blocks Or, a method of treating a disease.

48. The first conditional universal CAR system and the second conditional universal CAR The system is responsive to the same stimulus, the disorder or Methods for treating diseases.

49. The first conditional universal CAR system and the second conditional universal CAR The system is responsive to different stimuli, the disorder described in any of claims 40 to 46 This refers to methods for treating diseases.

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