# US 11,110,156 B2 · Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded tumour antigen

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- Number: US 11,110,156 B2
- Document ID: US11110156B2
- Status: Granted
- Kind: B2
- Application number: 16/002,695
- Filed: 2018-06-07
- Published: 2018-09-27
- Granted: 2021-09-07
- Earliest claimed: 2012-02-15
- Assignees: CureVac AG
- Applicants: CureVac AG
- Inventors: Andreas Thess; Thomas Schlake; Jochen Probst
- Web page: https://patentgrep.com/patent/US11110156B2

## CPC

- A61K 39/0011: {Cancer antigens}
- A61K 39/001104: {Epidermal growth factor receptors [EGFR]}
- A61K 39/001106: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4}
- A61K 39/001109: {Vascular endothelial growth factor receptors [VEGFR]}
- A61K 39/001112: {CD19 or B4}
- A61K 39/001113: {CD22, BL-CAM, siglec-2 or sialic acid- binding Ig-related lectin 2}
- A61K 39/001119: {Receptors for interleukins [IL]}
- A61K 39/001122: {Ephrin Receptors [Eph]}
- A61K 39/001124: {CD20}
- A61K 39/001129: {Molecules with a "CD" designation not provided for elsewhere}
- A61K 39/001132: {Fibroblast growth factors [FGF]}
- A61K 39/001134: {Transforming growth factor [TGF]}
- A61K 39/001139: {Colony stimulating factors [CSF]}
- A61K 39/001149: {Cell cycle regulated proteins, e.g. cyclin, CDC, CDK or INK-CCR}
- A61K 39/00115: {Apoptosis related proteins, e.g. survivin or livin}
- A61K 39/001151: {p53}
- A61K 39/001153: {Wilms tumor 1 [WT1]}
- A61K 39/001156: {Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]}
- A61K 39/001157: {Telomerase or TERT [telomerase reverse transcriptase]}
- A61K 39/001158: {Proteinases}
- A61K 39/001162: {Kinases, e.g. Raf or Src}
- A61K 39/001164: {GTPases, e.g. Ras or Rho}
- A61K 39/001166: {Adhesion molecules, e.g. NRCAM, EpCAM or cadherins}
- A61K 39/001168: {Mesothelin [MSLN]}
- A61K 39/00117: {Mucins, e.g. MUC-1}
- A61K 39/001176: {Heat shock proteins}
- A61K 39/001182: {Carcinoembryonic antigen [CEA]}
- A61K 39/001184: {Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE}
- A61K 39/001186: {MAGE}
- A61K 39/001188: {NY-ESO}
- A61K 39/001189: {PRAME}
- A61K 39/001191: {Melan-A/MART}
- A61K 39/001192: {Glycoprotein 100 [Gp100]}
- A61K 39/001193: {Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR}
- A61K 39/001194: {Prostate specific antigen [PSA]}
- A61K 39/001195: {Prostate specific membrane antigen [PSMA]}
- A61K 39/001197: {Breakpoint cluster region-abelson tyrosine kinase [BCR-ABL]}
- A61P 35/00: Antineoplastic agents
- C07K 14/47: from mammals
- C07K 14/4703: {Inhibitors; Suppressors}
- C07K 14/4748: {Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/67: General methods for enhancing the expression
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/53: {DNA (RNA) vaccination}
- A61K 2039/64: {characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units}
- C12N 2830/50: regulating RNA stability, not being an intron, e.g. poly A signal
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a tumour antigen or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic acid for increasing the expression of said encoded peptide or protein. It also discloses its use for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the treatment of cancer or tumour diseases. The present invention further describes a method for increasing the expression of a peptide or protein comprising a tumour antigen or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal.

## Claims

### Claim 1

A nucleic acid molecule comprising:
(I) a DNA molecule coding for, from 5 to 3′:
a) a polypeptide coding region, encoding a tumour antigen or an epitope of a tumour antigen;
b) a poly(A) sequence or a polyadenylation signal, and
c) at least one histone stem-loop that encodes a RNA that specifically binds to stem-loop binding protein (SLBP) without a histone downstream element (HDE); or
(II) a RNA molecule comprising, from 5′ to 3′:
a) a polypeptide coding region, encoding a tumour antigen or an epitope of a tumour antigen;
b) a poly(A) sequence, and
c) at least one histone stem-loop that specifically binds to SLBP without a HDE.

### Claim 2 (depends on 1)

The nucleic acid molecule according to claim 1, wherein the tumour antigen or the epitope of a tumour antigen is selected from 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, B AGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDCl27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, and or WT1.

### Claim 3 (depends on 1)

The nucleic acid molecule of claim 1, wherein the molecule does not comprise a sequence encoding a reporter protein, a marker, or a selection protein.

### Claim 4 (depends on 1)

The nucleic acid molecule of claim 1, wherein the nucleic acid is an RNA.

### Claim 5 (depends on 1)

The nucleic acid molecule of claim 1, wherein the poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides.

### Claim 6 (depends on 1)

The nucleic acid molecule of claim 1, wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA.

### Claim 7 (depends on 1)

The nucleic acid molecule of claim 1, wherein at least one guanosine, uridine, adenosine, thymidine, or cytidine position of the nucleic acid molecule is substituted with an analogue of these nucleotides selected from 2-amino-6-chloropurineriboside-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-chloropurineriboside-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, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, and xanthosine-5′-triphosphate.

### Claim 8 (depends on 1)

The nucleic acid sequence molecule of claim 1, wherein the G/C content of the polypeptide coding region is increased compared with the G/C content of the coding region of a wild-type nucleic acid encoding the tumour antigen.

### Claim 9 (depends on 4)

The nucleic acid molecule of claim 4, wherein the RNA comprises a 5′ cap structure and a poly(A) sequence of about 25 to about 400 adenosine nucleotides.

### Claim 10 (depends on 1)

The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a sequence of at least 10 consecutive cytidines.

### Claim 11 (depends on 1)

The nucleic acid molecule of claim 1, wherein the nucleic acid molecule further comprises a stabilizing sequence from the alpha globin 3′ UTR, positioned 3′ relative to the polypeptide coding region of the nucleic acid molecule.

### Claim 12 (depends on 1)

A pharmaceutical composition comprising a nucleic acid molecule of claim 1 and a pharmaceutically acceptable carrier.

### Claim 13 (depends on 12)

The pharmaceutical composition of claim 12, further comprising an adjuvant.

### Claim 14 (depends on 12)

The pharmaceutical composition of claim 12, wherein the composition further comprises a cationic or polycationic compound in complex with the nucleic acid molecule.

### Claim 15 (depends on 12)

The pharmaceutical composition of claim 12, wherein the composition further comprises a polycationic polypeptide in complex with the nucleic acid molecule.

### Claim 16 (depends on 4)

The nucleic acid molecule of claim 4, wherein RNA comprises a polypeptide coding region, encoding an epitope of a tumour antigen.

### Claim 17 (depends on 16)

The nucleic acid molecule of claim 16, wherein the epitope of a tumour antigen is a T-cell epitope.

### Claim 18 (depends on 16)

The nucleic acid molecule of claim 16, wherein the epitope is at least one epitope from KRAS.