Rna-coded antibody

Abstract

The present application describes an antibody-coding, non-modified or modified RNA and the use thereof for expression of this antibody, for the preparation of a pharmaceutical composition, in particular a passive vaccine, for treatment of tumours and cancer diseases, cardiovascular diseases, infectious diseases, auto-immune diseases, virus diseases and monogenetic diseases, e.g. also in gene therapy. The present invention furthermore describes and in vitro transcription method, in vitro methods for expression of this antibody using the RNA according to the invention and an in vivo method.

1 - 37 . (canceled) 
     
     
         38 . A method of treating a cancer disease, cardiovascular disease, infectious disease or autoimmune disease, the method comprising administering a pharmaceutical composition, the pharmaceutical composition comprising a RNA for intracellular expression of an antibody, wherein the RNA contains at least one coding region, wherein at least one coding region codes for at least one antibody. 
     
     
         39 . The method of  claim 38 , wherein the pharmaceutical composition is a passive vaccine for treatment of tumour or infectious diseases. 
     
     
         40 . The method of  claim 38 , wherein the cancer disease or tumour disease is selected from the group consisting of a melanoma, malignant melanoma, colon carcinoma, lymphoma, sarcoma, blastoma, kidney carcinoma, gastrointestinal tumour, glioma, prostate tumour, bladder cancer, rectal tumour, stomach cancer, oesophageal cancer, pancreatic cancer, liver cancer, mammary carcinoma, uterine cancer, cervical cancer, acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), hepatomas, diverse virus-induced tumour, adenocarcinoma, herpes virus-induced tumour, hepatitis B-induced tumour, HTLV-1- and HTLV-2-induced lymphoma, acusticus neurinoma, lung carcinoma, small cell lung carcinoma, throat cancer, anal carcinoma, glioblastoma, rectum carcinoma, astrocytoma, brain tumour, retinoblastoma, basalioma, brain metastasis, medulloblastoma, vaginal cancer, testicular cancer, thyroid carcinoma, Hodgkin's syndrome, meningeomas, Schneeberger's disease, pituitary tumour, mycosis fungoide, carcinoid, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer. kidney cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's lymphoma, urethral cancer, CUP syndrome, head/neck tumour, oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma, oesophageal carcinoma, wart condition, small intestine tumour, craniopharyngeomas, ovarian carcinoma, soft tissue tumour, ovarian cancer, pancreatic carcinoma, endometrium carcinoma, liver metastasis, penis cancer, tongue cancer, gallbladder cancer, leukaemia, plasmocytoma, lid tumour and prostate cancer. 
     
     
         41 . The method of  claim 38 , wherein the infectious disease is selected from the group consisting of influenza, malaria, SARS, yellow fever, AIDS, Lyme borreliosis, leishmaniasis, anthrax, meningitis, viral infectious diseases, such as AIDS, condyloma acuminate, molluscum contagiosum, dengue fever, three-day fever, Ebola virus, colds, early summer meningoencephalitis (ESME), influenza, shingles, hepatitis, herpes simplex type I, herpes simplex type II, herpes zoster, influenza, Japanese encephalitis, Lassa fever, Marburg virus, measles, foot and mouth disease, mononucleosis, mumps, Norwalk virus infection, Pfeiffer's glandular fever, smallpox, polio, pseuodcroup, infectious erythema, rabies, warts, West Nile fever, chicken-pox, cytomegalovirus (CMV), bacterial infectious diseases, anthrax, appendicitis, borreliosis, botulism, Campylobacter, Chlamydia trachomatis (inflammation of the urethra, conjunctiva), cholera, diphtheria, donavonosis, epiglottitis, louse-borne typhus, typhoid fever, gas gangrene, gonorrhoea, hare plague, Helicobacter pylori, whooping cough, climatic bubo, osteomyelitis, legionnaires' disease, leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis, anthrax, inflammation of the middle ear, Mycoplasma hominis, neonatal sepsis, noma, paratyphoid fever, plague, Reiter's syndrome, Rocky Mountain spotted fever,  Salmonella  paratyphoid fever,  Salmonella  typhoid fever, scarlet fever, syphilis, tetanus, gonorrhoea, tsutsugamushi fever, tuberculosis, typhus, vaginitis, soft chancre, and infectious diseases caused by parasites, protozoa or fungi, such as amoebic dysentery, bilharziosis, Chagas' disease,  Echinococcus , fish tapeworm, ichthyotoxism, fox tapeworm, mycosis pedis, dog tapeworm, candiosis, ptyriasis, scabies, cutaneous leishmaniasis, lamblian dysentery, lice, malaria, onchocercosis, fungal diseases, beef tapeworm, schistosomiasis, sleeping sickness, pork tapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis, visceral leishmaniasis, nappy dermatitis, or infections caused by the dwarf tapeworm. 
     
     
         42 . The method of  claim 38 , wherein the cardiovascular disease is selected from the group consisting of coronary heart disease, arteriosclerosis, apoplexy and hypertension, and neuronal disease. 
     
     
         43 . The method of  claim 42 , wherein the neuronal disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's disease. 
     
     
         44 . The method of  claim 38 , wherein the autoimmune disease is selected from the group consisting of autoimmune type I disease, autoimmune type H disease, autoimmune type III disease and autoimmune type IV disease. 
     
     
         45 . The method of  claim 38 , wherein the autoimmune disease is selected from the group consisting of multiple sclerosis (MS), rheumatoid arthritis, diabetes, diabetes type I, systemic lupus erythematosus (SLE), chronic polyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis, colitis ulcerosa, allergy type I diseases, allergy type II diseases, allergy type III diseases, allergy type IV diseases, fibromyalgia, hair loss, Bechterew's disease, Crohn's disease, myasthenia gravis, neurodermatitis, polymyalgia rheumatica, progressive systemic sclerosis (PSS), psoriasis, Reiter's syndrome, rheumatic arthritis, psoriasis and vasculitis. 
     
     
         46 . The method of  claim 38 , wherein the RNA is single-stranded or double-stranded. 
     
     
         47 . The method of  claim 38 , wherein the RNA Is linear or circular, 
     
     
         48 . The method of  claim 38 , wherein the RNA is rRNA, tRNA or mRNA. 
     
     
         49 . The method of  claim 48 , wherein the RNA is an mRNA. 
     
     
         50 . The method of  claim 38 , wherein the antibody coded is chosen from monoclonal and polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies and fragments of these antibodies. 
     
     
         51 . The method of  claim 38 , wherein the coded antibody fragments are chosen from Fab, Fab′, F(ab′) 2 , Fe, Facb, pFc′, Fd, and FIT or scFv fragments of these antibodies. 
     
     
         52 . The method of  claim 38 , wherein the coded antibodies or antibody fragments specifically recognize and bind tumour-specific surface antigens chosen from (TSSA), 5T4, a5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin/m, Bcr-abl, MN/C IX-antigen, CA125, CAMEL, CAP-1, CASP-8, β-catenin/m, CB4, CD19, CD20, CD22, CD25, CDC27/m, CD 30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HLAA*0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, IGF-1R, IL2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1 /Melan-A, MART-2/Ski, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pm1/RARα, FRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, survivin, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF and WT1, NY-Eso-1 and NY-Eso-B. 
     
     
         53 . The method of  claim 38 , wherein the RNA is modified. 
     
     
         54 . The method of  claim 53 , wherein the modification is chosen from modifications of the nucleotide sequence compared with a precursor RNA sequence by introduction of non-native nucleotides and/or by covalent coupling of the RNA with another group. 
     
     
         55 . The method of  claim 54 , wherein the RNA has a G/C content in the coding region of the base-modified RNA which is greater than the G/C content of the coding region of the native RNA sequence, the coded amino acid sequence being unchanged with respect to the wild-type or, respectively, the precursor RNA. 
     
     
         56 . The method of  claim 54 , wherein the coding region of the modified RNA is modified compared with the coding region of the native RNA such that at least one codon of the native RNA which codes for a tRNA which is relatively rare in the ceil is exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which carries the same amino acid as the relatively rare tRNA. 
     
     
         57 . The method of  claim 54 , wherein the RNA has a lipid modification. 
     
     
         58 . The method of  claim 54 , wherein the RNA contains on at least one nucleotide of the RNA a modification of a nucleotide, wherein the nucleotides are chosen from 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -methyl-inosine, dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), pseudouracil, 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazagnanosines 5-methylcytosine and inosine. 
     
     
         59 . The method of  claim 54 , wherein the RNA contains on at least one nucleotide of the RNA a modification of a nucleotide, wherein the nucleotides are base-modified nucleotides chosen from the group consisting of 2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine 5-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine 51-triphosphate, 5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate, 6-azauridine 5′-triphosphate, 6-cholorpurine riboside 5′-triphosphate, 7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate, 8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate, benzimidazole riboside 5′-triphosphate, N1-methyladenosine 5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine 5′-triphosphate, O6-methylguanosine 5′-triphosphate, pseudouridine 5′-triphosphate, puromycin 5′-triphosphate or xanthosine 5′-triphosphate. 
     
     
         60 . The method of  claim 59 , wherein the base-modified nucleotides are chosen from the group consisting of 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate. 
     
     
         61 . The method of  claim 38 , wherein the RNA additionally has a 5′ cap structure chosen from the group consisting of m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G. 
     
     
         62 . The method of  claim 38 , wherein the RNA additionally has a poly-A tail of from about 10 to 200 adenosine nucleotides (SEQ ID NO: 62). 
     
     
         63 . The method of  claim 38 , wherein the RNA additionally has a poly-C tail of from about 10 to 200 cytosine nucleotides (SEQ ID NO: 54). 
     
     
         64 . The method of  claim 38 , wherein the RNA additionally codes a tag for purification chosen from the group consisting of a hexahistidine tag (SEQ ID NO: 59) (HIS tag, polyhistidine tag), a streptavidin tag (Strep tag), an SBP tag (streptavidin-binding tag) or a GST (glutathione S-transferase) tag, or codes for a tag for purification via an antibody epitope chosen from the group consisting of antibody-binding tags, a Mye tag, a Swa11 epitope, a FLAG tag or an HA tag. 
     
     
         65 . The method of  claim 38 , wherein the RNA additionally codes a signal peptide and/or a localization sequence, in particular a secretion sequence. 
     
     
         66 . The method of  claim 65 , wherein the localization sequence is chosen from one of the sequences according to SEQ ID NO: 18 to 50. 
     
     
         67 . The method of  claim 38 , wherein the RNA contains an antibody-coding sequence which codes for the heavy chains according to SEQ ID NO: 2 and the light chains according to SEQ ID NO: 4. 
     
     
         68 . The method of  claim 38 , wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 5. 
     
     
         69 . The method of  claim 38 , wherein the RNA contains an antibody-coding sequence which codes for the heavy chains according to SEQ ID NO: 7 and the light chains according to SEQ ID NO: 9. 
     
     
         70 . The method of  claim 38 , wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 10. 
     
     
         71 . The method of  claim 38 , wherein the modified RNA contains an antibody-coding sequence which codes for the heavy chains according to SEQ ID NO: 12 and the light chains according to SEQ ID NO: 14. 
     
     
         72 . The method of  claim 38 , wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 15. 
     
     
         73 . The method of  claim 38 , wherein the modified RNA contains an antibody-coding sequence which has a sequence identity of at least 70% to the sequence SEQ ID NO: 5,10 or 15 over the total length of the nucleic acid sequence of SEQ ID NO: 5, 10 or 15. 
     
     
         74 . In vitro transcription method for the preparation of an antibody-coding, optionally modified RNA, comprising the following steps:
 a. provision of a nucleic acid which codes for an antibody, as defined in claim  13 ;   b. addition of the nucleic acid to an in vitro transcription medium comprising an RNA polymerase, a suitable buffer, a nucleic acid mix comprising one or more modified nucleotides in exchange for one or more of the naturally occurring nucleotides A, G, C or U, and optionally one or more naturally occurring nucleotides A, G, C or U, if not ail the naturally occurring nucleotides A, G, C or U are to be exchanged, or optionally only naturally occurring nucleotides and optionally an RNase inhibitor;   c. incubation of the nucleic acid in the in vitro transcription medium and in vitro transcription of the nucleic acid to give an antibody-coding, optionally modified RNA according to  claim 38 ;   d. optionally purification of the antibody-coding, optionally modified RNA and removal of the non-incorporated nucleotides from the in vitro transcription medium.   
     
     
         75 . In vitro transcription and translation method for expression of an antibody, comprising the following steps:
 a. provision of a nucleic acid which codes for an antibody, as defined in claim  13 ;   b. addition of the nucleic acid to an in vitro transcription medium comprising an RNA polymerase, a suitable buffer, a nucleic acid mix comprising one or more modified nucleotides in exchange for one or more of the naturally occurring nucleotides A, O, C or U, and optionally one or more naturally occurring nucleotides A, G, C or U, if not ail the naturally occurring nucleotides A, G, C or U are to be exchanged, or optionally only naturally occurring nucleotides and optionally an RNase inhibitor;   c. incubation of the nucleic acid in the in vitro transcription medium and in vitro transcription of the nucleic acid to give an antibody-coding, optionally modified RNA according to  claim 38 ;   d. optionally purification of the antibody-coding, optionally modified RNA and removal of the non-incorporated nucleotides from the in vitro transcription medium,   e. addition of the optionally modified RNA obtained in step c) (and optionally in step d) to an in vitro translation medium;   f. incubation of the optionally modified RNA in the in vitro translation medium and in vitro translation of the antibody coded by the optionally modified RNA;   g. optionally purification of the antibody translated in step f).   
     
     
         76 . In vitro transcription and translation method for expression of an antibody in a host cell, comprising the following steps:
 a. provision of a nucleic acid which codes for an antibody, as defined in claim  13 ;   b. addition of the nucleic acid to an in vitro transcription medium comprising an RNA polymerase, a suitable buffer, one or more modified nucleotides in exchange for one or more of the naturally occurring nucleotides A, G, C or U and optionally one or more naturally occurring nucleotides A, G, C or U, if not all the naturally occurring nucleotides A, G, C or U are to be exchanged, or only naturally occurring nucleotides and optionally an RNase inhibitor;   c. incubation of the nucleic acid in the in vitro transcription medium and in vitro transcription of the nucleic acid to give an antibody-coding, optionally modified RNA according to  claim 38 ;   d. optionally purification of the antibody-coding, optionally modified RNA according to the invention and removal of the non-incorporated nucleotides from the in vitro transcription medium,   e. transfection of the optionally modified RNA obtained in step c) (and optionally d)) into a host cell;   f. incubation of the optionally modified nucleic acid in the host cell and translation of the antibody coded by the optionally modified RNA in the host cell;   g. optionally isolation and/or purification of the antibody translated in step f).