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, autoimmune diseases, virus diseases and monogenetic diseases, e.g. also in gene therapy. The present invention furthermore describes an in vitro transcription method, in vitro methods for expression of this antibody using the RNA according to the invention and an in vivo method.

Claims

1. An 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.

2. An RNA according to claim 1, wherein the RNA is single-stranded or double-stranded, linear or circular, in the form of rRNA, tRNA or mRNA.

3. An RNA according to claim 1 or 2, wherein the RNA is an mRNA.

4. An RNA according to claims 1 to 3, 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.

5. An RNA according to claims 1 to 4, wherein the coded antibody fragments are chosen from Fab, Fab′, F(ab′)2, Fc, Facb, pFc′, Fd, and Fv or scFv fragments of these antibodies.

6. An RNA according to claims 1 to 5, wherein the coded antibodies or antibody fragments specifically recognize and bind tumour-specific surface antigens chosen from (TSSA), 5T4, α5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin/m, Bcr-abl, MN/C IX-antigen, CA125, CAMEL, CAP-I, CASP-8, β-catenin/m, CD4, 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, GpIOO, HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, IGF-IR, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/Melan-A, MART-2/Ski, MC1R, myosin/m, MUC1, MUM-I, -2, -3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-I or SART-3, survivin, TEL/AML1, TGFβ, TPI/m, TRP-I, TRP-2, TRP-2/INT2, VEGF and WT1, NY-Eso-1 and NY-Eso-B.

7. An RNA according to one of claims 1 to 6, wherein the RNA is modified.

8. An RNA according to one of the preceding claims, 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.

9. An RNA according to claim 8, 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.

10. An RNA according to claim 8 or 9, 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 cell 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.

11. An RNA according to one of claims 8 to 10, wherein the RNA has a lipid modification.

12. An RNA according to one of claims 8 to 11, 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-N6-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-deazaguanosine, 5-methylcytosine and inosine.

13. An RNA according to one of claims 8 to 12, 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 5′-triphosphate, 5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate, 6-azauridine 5′-triphosphate, 6-chloropurine 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.

14. An RNA according to claim 13, wherein the base-modified nucleotides are chosen from the group consisting of 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate.

15. An RNA according to one of the preceding claims, 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.

16. An RNA according to one of the preceding claims, wherein the RNA additionally has a poly-A tail of from about 10 to 200 adenosine nucleotides.

17. An RNA according to one of the preceding claims, wherein the RNA additionally has a poly-C tail of from about 10 to 200 cytosine nucleotides.

18. An RNA according to one of the preceding claims, wherein the RNA additionally codes a tag for purification chosen from the group consisting of a hexahistidine tag (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 Myc tag, a Swal 1 epitope, a FLAG tag or an HA tag.

19. An RNA according to one of the preceding claims, wherein the RNA additionally codes a signal peptide and/or a localization sequence, in particular a secretion sequence.

20. An RNA according to claim 19, wherein the localization sequence is chosen from one of the sequences according to SEQ ID NO: 18 to 50.

21. An RNA according to one of the preceding claims, 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.

22. An RNA according to one of the preceding claims 1 to 20, wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 5.

23. An RNA according to one of the preceding claims 1 to 20, 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.

24. An RNA according to one of the preceding claims 1 to 20, wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 10.

25. An RNA according to one of the preceding claims 1 to 20, 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.

26. An RNA according to one of the preceding claims 1 to 20, wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 15.

27. An RNA according to one of the preceding claims 1 to 20, 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.

28. A pharmaceutical composition comprising an RNA according to one of claims 1 to 27.

29. Use of an RNA sequence as defined in claims 1 to 27 for the preparation of a pharmaceutical composition for treatment of cancer diseases, cardiovascular diseases, infectious diseases or autoimmune diseases.

30. Use according to claim 29, wherein the pharmaceutical composition is in the form of a passive vaccine for treatment of tumour or infectious diseases.

31. Use according to one of claim 29 or 30, wherein the cancer diseases or tumour diseases are chosen from the group consisting of melanomas, malignant melanomas, colon carcinomas, lymphomas, sarcomas, blastomas, kidney carcinomas, gastrointestinal tumours, gliomas, prostate tumours, bladder cancer, rectal tumours, stomach cancer, oesophageal cancer, pancreatic cancer, liver cancer, mammary carcinomas (=breast cancer), uterine cancer, cervical cancer, acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), hepatomas, diverse virus-induced tumours, such as e.g. papilloma virus-induced carcinomas (e.g. cervix carcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumours (e.g. Burkitt's lymphoma, EBV-induced B cell lymphoma), hepatitis B-induced tumours (hepatocell carcinomas), HTLV-I- and HTLV-2-induced lymphomas, acusticus neurinoma, lung carcinomas (=lung cancer=bronchial carcinoma), small cell lung carcinomas, throat cancer, anal carcinoma, glioblastoma, rectum carcinoma, astrocytoma, brain tumours, retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginal cancer, testicular cancer, thyroid carcinoma, Hodgkin's syndrome, meningeomas, Schneeberger's disease, pituitary tumour, mycosis fungoides, carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer, kidney cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's lymphomas, urethral cancer, CUP syndrome, head/neck tumours, oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma, oesophageal carcinoma (=oesophageal cancer), wart conditions, small intestine tumours, cra-niopharyngeomas, ovarian carcinoma, soft tissue tumours (sarcomas), ovarian cancer (=ovarian carcinoma), pancreatic carcinoma (=pancreatic cancer), endometrium carcinoma, liver metastases, penis cancer, tongue cancer, gallbladder cancer, leukaemia, plasmocytoma, lid tumour and prostate cancer (=prostate tumours).

32. Use according to one of claims 29 to 30, wherein the infectious diseases are chosen from the group consisting of influenza, malaria, SARS, yellow fever, AIDS, Lyme borreliosis, leishmaniasis, anthrax, meningitis, viral infectious diseases, such as AIDS, condyloma acuminata, 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 (poliomyelitis), pseuodcroup, infectious erythema, rabies, warts, West Nile fever, chicken-pox, cytomegalovirus (CMV), bacterial infectious diseases, such as abortion (infectious, septic), prostatitis (prostate inflammation), anthrax, appendicitis (inflammation of the caecum), 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 (chorioamnionitis), 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 (colpitis), soft chancre, and infectious diseases caused by parasites, protozoa or fungi, such as amoebic dysentery, bilharziosis, Chagas' disease, Echinococcus, fish tapeworm, ichthyotoxism (ciguatera), fox tapeworm, mycosis pedis, dog tapeworm, candiosis, ptyriasis, the itch (scabies), cutaneous leishmaniasis, lamblian dysentery (giadiasis), lice, malaria, onchocercosis (river blindness), fungal diseases, beef tapeworm, schistosomiasis, sleeping sickness, pork tapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness), visceral leishmaniasis, nappy dermatitis, or infections caused by the dwarf tapeworm.

33. Use according to one of claim 29 or 30, wherein the cardiovascular diseases are chosen from the group consisting of coronary heart disease, arteriosclerosis, apoplexy and hypertension, and neuronal diseases chosen from Alzheimer's disease, amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's disease.

34. Use according to one of claim 29 or 30, wherein the autoimmune diseases are chosen from the group consisting of autoimmune type I diseases or autoimmune type II diseases or auto-immune type III diseases or autoimmune type IV diseases, such as, for example, multiple sclerosis (MS), rheumatoid arthritis, diabetes, diabetes type I (diabetes mellitus), 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 or vasculitis.

35. An 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 claims 4 to 6; 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 all 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 claims 1 to 27; d) optionally purification of the antibody-coding, optionally modified RNA and removal of the non-incorporated nucleotides from the in vitro transcription medium.

36. An 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 claims 4 to 6; 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 all 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 claims 1 to 27; 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).

37. An 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 claims 4 to 6; 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 claims 1 to 27; 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).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0205] The following figures and examples are intended to explain in more detail and illustrate the above description, without being limited thereto.

[0206] FIG. 1 illustrates the structure of an IgG antibody. IgG antibodies are built up from in each case two identical light and two heavy protein chains which are bonded to one another via disulfide bridges. The light chain comprises the N-terminal variable domain V.sub.L and the C-terminal constant domain C.sub.L. The heavy chain of an IgG antibody can be divided into an N-terminal variable domain V.sub.H and three constant domains C.sub.H1, C.sub.H2 and C.sub.H3.

[0207] FIGS. 2A-D show the gene cluster for the light and the heavy chains of an antibody: [0208] (A): Gene cluster for the light chain κ. [0209] (B): Gene cluster for the light chain λ. [0210] (C): and (D): Gene cluster for the heavy chain. [0211] In this context, the variable region of a heavy chain is composed of three different gene segments. In addition to the V and J segments, additional D segments are also found here. The V.sub.H, D.sub.H and J.sub.H segments can likewise be combined with one another virtually as desired to form the variable region of the heavy chain.

[0212] FIG. 3 illustrates in the form of a diagram the differences in the light and heavy chains of murine (i.e. obtained in the mouse host organism), chimeric, humanized and human antibodies.

[0213] FIG. 4 shows an overview of the structure of various antibody fragments. The constituents of the antibody fragments are shown on a dark grey background.

[0214] FIGS. 5A-C show various variants of antibodies and antibody fragments in FIGS. 5A, 5B and 5C: [0215] (A) shows a diagram of an IgG antibody of two light and two heavy chains. [0216] (B) shows an Fab fragments from the variable and a constant domain in each case of a light and a heavy chain. The two chains are bonded to one another via a disulfide bridge. [0217] (C) shows an scFv fragment from the variable domain of the light and the heavy chain, which are bonded to one another via an artificial polypeptide linker.

[0218] FIG. 6 shows a presentation of an antibody-coding (modified) RNA according to the invention as an expression construct. In this: [0219] V.sub.H=variable domain of the heavy chain; [0220] C.sub.H=constant domain of the heavy chain; [0221] V.sub.L=variable domain of the light chain; [0222] C.sub.L=constant domain of the light chain; [0223] SIRES=internal ribosomal entry site (IRES, superIRES) [0224] muag=mutated form of the 3′ UTR of the alpha-globin gene; and [0225] A70C30=polyA-polyC tail.

[0226] FIG. 7 shows a diagram of the detection of an antibody coded by an RNA according to the invention by means of ELISA on the example of the antigen Her2.

[0227] FIG. 8 shows the wild-type DNA sequence of the heavy chain of the antibody rituximab (=Rituxan, MabThera) (wild-type: GC content: 56.5%, length: 1,344) (SEQ ID NO: 1).

[0228] FIG. 9 shows the GC-optimized DNA sequence of the heavy chain of the antibody rituximab (=Rituxan, MabThera) (GC content: 65.9%, length: 1,344) (SEQ ID NO: 2).

[0229] FIG. 10 shows the wild-type DNA sequence of the light chain of the antibody rituximab (=Rituxan, MabThera) (wild-type: GC content: 58.5%, length: 633) (SEQ ID NO: 3).

[0230] FIG. 11 shows the GC-optimized DNA sequence of the light chain of the antibody rituximab (=Rituxan, MabThera) (GC content: 67.2%, length: 633) (SEQ ID NO: 4).

[0231] FIG. 12 shows the total construct of the GC-optimized DNA sequence of the antibody rituximab (=Rituxan, MabThera) with the light and heavy chains (SEQ ID NO: 5). The total construct contains the following sequences and cleavage sites (see also alternative cleavage sites of FIG. 25, SEQ ID No. 51):

TABLE-US-00004  linker for an optimum Kozak sequence  HindIII  stop codon  SpeI  BglII  NsiI (SEQ ID NO: 60) CATCATCATCATCATCAT His tag Signal peptide, HLA-A*0201: GC-rich (SEQ ID NO: 61) ATGGCCGTGATGGCGCCGCG- GACCCTGGTCCTCCTGCTGAGCGGCGCCCTCGCCCTGACGCAGAC- CTGGGCCGGG. [0232] The coding region of the heavy chain sequence starts with the signal peptide as given above (italic). This region is G/C enriched as well. The subsequent sequence starting with CAG represents the actual antibody coding sequence (see FIG. 9) for the heavy chain, which ends with AAG and is followed by the above described His tag sequence. Finally, the open reading frame for the heavy chain ends with the stop codon TGA (). The coding region for the light chain sequence starts 3′ upstream with the signal peptide's ATG as given above followed by the light chain's coding region for the light chain starting with CAG running to the stop codon TGA ()(see FIG. 11). Both coding regions for the light and the heavy chain are separated by an IRES element (). The inventive RNA coded by the construct given in FIG. 12 may or may not contain a (His) tag sequence and may contain a signal peptide sequence different from the above peptide sequence or may even have no signal peptide sequence. Accordingly, the inventive RNA molecule contains preferably the coding region (with or without a signal peptide sequence at its beginning) of the heavy and/or the light chain (e.g. as shown in FIG. 12), preferably in combination with at least one ribosomal entry site.

[0233] FIG. 13 shows the wild-type DNA sequence of the heavy chain of the antibody cetuximab (=Erbitux) (wild-type: GC content: 56.8%, length: 1,359) (SEQ ID NO: 6).

[0234] FIG. 14 shows the GC-optimized DNA sequence of the heavy chain of the antibody cetuximab (=Erbitux) (GC content: 65.9%, length: 1,359) (SEQ ID NO: 7).

[0235] FIG. 15 shows the wild-type DNA sequence of the light chain of the antibody cetuximab (=Erbitux) (wild-type: GC content: 58.2%, length: 642) (SEQ ID NO: 8).

[0236] FIG. 16 shows the GC-optimized DNA sequence of the light chain of the antibody cetuximab (=Erbitux) (GC content: 65.7%, length: 642) (SEQ ID NO: 9).

[0237] FIG. 17 shows the total construct of the GC-optimized DNA sequence of the antibody cetuximab (=Erbitux) with the light and heavy chains (SEQ ID NO: 10). The total construct contains the following sequences and cleavage sites (see also alternative cleavage sites of FIG. 26, SEQ ID No 52):

TABLE-US-00005  linker for an optimum Kozak sequence  HindIII  stop codon  SpeI  BglII  Nsil (SEQ ID NO: 60) CATCATCATCATCATCAT His tag Signal peptide, HLA-A*0201: GC-rich (SEQ ID NO: 61) ATGGCCGTGATGGCGCCGCG- GACCCTGGTCCTCCTGCTGAGCGGCGCCCTCGCCCTGACGCAGAC- CTGGGCCGGG. [0238] The coding region of the heavy chain sequence starts with the signal peptide as given above (italic). This region is G/C enriched as well. The subsequent sequence starting with CAG represents the actual antibody coding sequence (see FIG. 14) for the heavy chain, which ends with AAG and is followed by the above described His tag sequence. Finally, the open reading frame for the heavy chain ends with the stop codon TGA (). The coding region for the light chain sequence starts 3′ upstream with the signal peptide's ATG as given above followed by the light chain's coding region for the light chain starting with GAC running to the stop codon TGA ()(see FIG. 16). Both coding regions for the light and the heavy chain are separated by an IRES element (). The inventive RNA coded by the construct given in FIG. 17 may or may not contain a (His) tag sequence and may contain a signal peptide sequence different from the above peptide sequence or may even have no signal peptide sequence. Accordingly, the inventive RNA molecule contains preferably the coding region (with or without a signal peptide sequence at its beginning) of the heavy and/or the light chain (e.g. as shown in FIG. 17), preferably in combination with at least one ribosomal entry site.

[0239] FIG. 18 shows the wild-type DNA sequence of the heavy chain of the antibody trastuzumab (=Herceptin) (wild-type: GC content: 57.8%, length: 1,356) (SEQ ID NO: 11).

[0240] FIG. 19 shows the GC-optimized DNA sequence of the heavy chain of the antibody trastuzumab (=Herceptin) (GC content: 67.0%, length: 1,356) (SEQ ID NO: 12).

[0241] FIG. 20 shows the wild-type DNA sequence of the light chain of the antibody trastuzumab (=Herceptin) (wild-type: GC content: 56.9%, length: 645) (SEQ ID NO: 13).

[0242] FIG. 21 shows the GC-optimized DNA sequence of the light chain of the antibody trastuzumab (=Herceptin) (GC content: 66.4%, length: 645) (SEQ ID NO: 14).

[0243] FIG. 22 shows the total construct of the GC-optimized DNA sequence of the antibody trastuzumab (=Herceptin) with the light and heavy chains (SEQ ID NO: 15). The total construct contains the following sequences and cleavage sites (see also alternative cleavage sites of FIG. 27, SEQ ID No. 53):

TABLE-US-00006  linker for an optimum Kozak sequence  HindIII  stop codon  SpeI  BglII  NsiI (SEQ ID NO: 60) CATCATCATCATCATCAT His tag Signal peptide, HLA-A*0201: GC-rich (SEQ ID NO: 61) ATGGCCGTGATGGCGCCGCG- GACCCTGGTCCTCCTGCTGAGCGGCGCCCTCGCCCTGACGCAGAC- CTGGGCCGGG. [0244] The coding region of the heavy chain sequence starts with the signal peptide as given above (italic). This region is G/C enriched as well. The subsequent sequence starting with GAG represents the actual antibody coding sequence (see FIG. 19) for the heavy chain, which ends with AAG and is followed by the above described His tag sequence. Finally, the open reading frame for the heavy chain ends with the stop codon TGA (). The coding region for the light chain sequence starts 3′ upstream with the signal peptide's ATG as given above followed by the light chain's coding region for the light chain starting with GAC running to the stop codon TGA ()(see FIG. 21). Both coding regions for the light and the heavy chain are separated by an IRES element (). The inventive RNA coded by the construct given in FIG. 22 may or may not contain a (His) tag sequence and may contain a signal peptide sequence different from the above peptide sequence or may even have no signal peptide sequence. Accordingly, the inventive RNA molecule contains preferably the coding region (with or without a signal peptide sequence at its beginning) of the heavy and/or the light chain (e.g. as shown in FIG. 22), preferably in combination with at least one ribosomal entry site.

[0245] FIG. 23 shows RNA-mediated antibody expression in cell culture. CHO or BHK cells were transfected with 20 μg of antibody-encoding mRNA according to the invention which was produced (RNA, G/C enriched, see above) or mock-transfected. 24 hours after transfection protein synthesis was analysed by Western blotting of cell lysates. Cells harboured about 0.5 μg of protein as assessed by Western Blot analysis. Each lane represents 10% of total lysate. Humanised antibodies served as control and for a rough estimate of protein levels. The detection antibody recognises both heavy and light chains; moreover, it shows some unspecific staining with cell lysates (three distinct bands migrating much slower than those of the antibodies). A comparison with control antibodies clearly demonstrates that heavy and light chains were produced in equal amounts.

[0246] FIGS. 24A-E show that RNA-mediated antibody expression gives rise to a functional protein (antibody). Functional antibody formation was addressed by FACS staining of antigen-expressing target cells. In order to examine the production of functional antibodies, cell culture supernatants of RNA-transfected (20 μg of Ab-RNA as defined above in Example 1) cells were collected after 48 to 96 hours. About 8% of total supernatant was used to stain target cells expressing the respective antigen. Humanised antibodies served as control and for a rough estimate of protein levels. Primary antibody used for cell staining: a) humanised antibody; b) none; c,d) supernatant from RNA-transfected cells expressing the respective antibody; e) supernatant from mock-transfected CHO cells. Calculations on the basis of the analysis shown in FIG. 24 reveal that cells secreted more than 12-15 μg of functional antibody within 48-96 hours. Accordingly, the present invention proves that RNA encoding antibodies may enter into cell, may be expressed within the cell and considerable amounts of RNA encoded antibodies are then secreted by the cell into the surrounding medium/extracellular space. Cell transfection in vivo or in vitro by the inventive RNA may therefore be used to provide antibodies acting e.g. therapeutically in the extracellular space.

[0247] FIG. 25 shows an alternative sequence of the construct of FIG. 12 (antibody rituximab), wherein the restriction sites have been modified as compared to SEQ ID No. 5 of FIG. 12 (SEQ ID No.: 51). For closer information with regard to the description of various sequence elements it is referred to FIG. 12.

[0248] FIG. 26 shows an alternative sequence of the construct of FIG. 17 (antibody cetuximab), wherein the restriction sites have been modified as compared to SEQ ID No. 10 of FIG. 17 (SEQ ID No.: 52). For closer information with regard to the description of various sequence elements it is referred to FIG. 17.

[0249] FIG. 27 shows an alternative sequence of the construct of FIG. 22 (antibody trastuzumab), wherein the restriction sites have been modified as compared to SEQ ID No. 15 of FIG. 22 (SEQ ID No.: 53). For closer information with regard to the description of various sequence elements it is referred to FIG. 22.

[0250] The following examples explain the present invention in more detail, without limiting it.

EXAMPLES

1. Example

1.1 Cell Lines and Cell Culture Conditions Used:

[0251] The cell lines HeLa (human cervix carcinoma cell line; Her2-positive), HEK293 (human embryonal kidney; Her2-negative) and BHK21 (Syrian hamster kidney; Her2-negative) were obtained from the DMSZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH) in Braunschweig and cultured in RPMI medium enriched with 2 mM L-glutamine (Bio Whittaker) and 10 μg/ml streptomycin and 10 U/ml of penicillin at 37° C. under 5% CO.sub.2.

1.2 Preparation of Expression Vectors for Modified RNA Sequences According to the Invention:

[0252] For the production of modified RNA sequences according to the invention, the GC-enriched and translation-optimized DNA sequences which code for a heavy chain and a light chain of an antibody (e.g. cetuximab (ERBITUX®), trastuzumab (HERCEPTIN®) and rituximab (RITUXAN®), cf. SEQ ID NO: 1-15, where SEQ ID NO: 1, 3, 6, 8, 11 and 13 represent the particular coding sequences which are not GC-optimized of the heavy and the light chains of these antibodies and SEQ ID NO: 2, 4, 5, 7, 9, 10, 12, 14 and 15 represent the coding GC-enriched sequences (see above)) were cloned into the pCV19 vector (CureVac GmbH) by standard molecular biology methods. To ensure equimolar expression of the two chains, an IRES (internal ribosomal entry site) was introduced. The mutated 3′ UTR (untranslated region) of the alpha-globin gene and a polyA-polyC tail at the 3′ end serve for additional stabilizing of the mRNA. The signal peptide of the HLA-A*0201 gene is coded for secretion of the antibody expressed. A His tag was additionally introduced for detection of the antibody. FIG. 6 shows the expression constructs used.

1.3 Preparation of the G/C-Enriched and Translation-Optimized Antibody-Coding mRNA

[0253] An in vitro transcription was carried out by means of T7 polymerase (T7-Opti mRNA Kit, CureVac, Tubingen, Germany), followed by purification with Pure Messenger™ (CureVac, Tubingen, Germany). For this, a DNase digestion was first carried out, followed by an LiCl precipitation and thereafter an HPLC using a porous reverse phase as the stationary phase (PURE Messenger).

1.4 Detection of RNA-Antibody by Means of Flow Cytometry:

[0254] 1 million cells were transfected with the mRNA according to one of SEQ ID NO: 5, 10 or 15 (see above), which codes for an antibody as described above, by means of electroporation and were then cultured in the medium for 16 h. The antibody expressed was detected by means of an FITC-coupled His tag antibody. Alternatively, the secreted antibody from the supernatant of transfected cells was added to non-transfected, antigen-expressing cells and, after incubation, detected by the same method.

[0255] 1.5 In Vitro Detection of an Antibody Coded by an RNA According to the Invention by Means of ELISA:

[0256] A microtitre plate was loaded with a murine antibody (1) against a first antigen (HER-2). Cell lysate of antigen-expressing cells was then added to the plate. The antigen was bound here by the murine antigen-specific antibody (1). The supernatant of cells which were transfected with a modified mRNA according to the invention which codes for an HER-2-specific antibody was then added to the microtitre plate. The HER-2-specific antibody (2) contained in the supernatant likewise binds to the antibody-bound antigen, the two antibodies recognizing different domains of the antigen. For detection of the bound antibody (2), anti-human IgG coupled to horseradish peroxidase (3-HRP) was added, the substrate TMB being converted and the result determined photometrically.

1.6 In Vivo Detection of an Antibody Coded by an RNA According to the Invention:

[0257] An antibody-coding (m)RNA according to the invention as described above was injected intradermally or intramuscularly into BALB/c mice. 24 h thereafter, the corresponding tissues were removed and protein extracts were prepared. The expression of the antibody was detected by means of ELISA as described here.

1.7 Detection of an Antibody Coded by an RNA According to the Invention by Means of Western Blotting:

[0258] The expressed antibodies from the supernatant of cells which were transfected with a modified mRNA which codes for an antibody as described above were separated by means of a polyacrylamide gel electrophoresis and then transferred to a membrane. After incubation with anti-His tag antibody and a second antibody coupled to horseradish peroxidase, the antibody expressed was detected by means of chemoluminescence.

1.8 Tumour Model:

[0259] SKOV-3 cells were injected subcutaneously into BALB/c mice. Within the following 28 days, eight portions of 10 μg of a modified mRNA which codes for an antibody as described above were injected into the tail vein of the mice. The tumour growth was monitored over a period of 5 weeks.

2. Example

2.1. Cell Lines

[0260] RNA-based expression of humanised antibodies was done in either CHO-K1 or BHK-21 cells. The tumour cell lines BT-474, A-431 and Raji strongly expressing HER2, EGFR and CD20, respectively, were used to record antibody levels. All cell lines except CHO were maintained in RPMI supplemented with FCS and glutamine according to the supplier's information. CHO cells were grown in Ham's F12 supplemented with 10% FCS. All cell lines were obtained from the German collection of cell cultures (DSMZ).

2.2. Antibody Expression

[0261] Various amounts of antibody-RNA (G/C enriched as defined by FIGS. 12, 17, 22, 25, 26, 27) encoding the humanised antibodies Herceptin, Erbitux, and Rituxan, respectively, (see the description given above for Example 1) were transfected into either CHO or BHK cells by electroporation. Conditions were as follows: 300 V, 450 μF for CHO and 300 V, 150 μF for BHK. After transfection, cells were seeded onto 24-well cell culture plates at a density of 2-400.000 cells per well. For collection of secreted protein, medium was replaced by 250 μl of fresh medium after cell attachment to the plastic surface. Secreted protein was collected for 24-96 hours and stored at 4° C. In addition, cells were harvested into 50 μl of phosphate buffered saline containing 0.5% BSA and broken up by three freeze-thaw cycles. Cell lysates were cleared by centrifugation and stored at −80° C.

2.3. Western Blot Analysis

[0262] In order to detect translation of transfected RNA, proteins from either cell culture supernatants or cell lysates were separated by a 12% SDS-PAGE and blotted onto a nitrocellulose membrane. Humanised antibodies Herceptin (Roche), Erbitux (Merck KGAA), and Mabthera=Rituxan (Roche) were used as controls. After blotting was completed, membranes were consecutively incubated with biotinylated goat anti-human IgG (Dianova), streptavidin coupled to horseradish peroxidase (BD), and a chemiluminescent substrate (SuperSignal West Pico, Pierce). Staining was detected with a Fuji LAS-1000 chemiluminescence camera.

2.4. FACS Analysis

[0263] 200.000 target cells expressing the respective antigen were incubated with either control antibodies (Herceptin, Erbitux, Mabthera) or cell culture supernatants. For detection of bound antibodies, cells were stained with biotinylated goat anti-human IgG (Dianova) and PE-labelled streptavidin (Invitrogen). Cells were analysed on a FACSCalibur (BD).