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US-12618060-B2 - Nucleic acid molecules

US12618060B2US 12618060 B2US12618060 B2US 12618060B2US-12618060-B2

Abstract

The present invention provides novel artificial nucleic acid molecules encoding at least one antigenic peptide or protein and at least one additional sequence preferably targeting the antigenic peptides or proteins to cellular compartments of interest. Further, the invention provides (pharmaceutical) compositions or vaccines and kits comprising said nucleic acid molecules. The nucleic acid molecules, (pharmaceutical) compositions or vaccines and kits are useful for treating a variety of diseases such as cancer, infectious diseases, autoimmune diseases, allergies or graft-versus host disease.

Inventors

  • Mariola Fotin-Mleczek
  • Katja Fiedler
  • Aleksandra KOWALCZYK
  • Regina HEIDENREICH

Assignees

  • CUREVAC AG

Dates

Publication Date
20260505
Application Date
20210315

Claims (19)

  1. 1 . An artificial RNA molecule comprising at least one coding region encoding at least one antigenic protein comprising (i) at least one signal peptide, (ii) at least two epitopes from two different tumor antigens; (iii) a linker peptide linking said at least two epitopes, and (iv) at least one transmembrane domain from CTLA4 (Cytotoxic T-lymphocyte protein 4).
  2. 2 . The artificial RNA molecule according to claim 1 , wherein said one of said at least two epitopes is from a tumor antigen selected from the group consisting of BRAF, PIK3CA, KRAS, IDH1, TP53, NRAS, AKTI, SF3B1, CDKN2A, RPSAP58, EGFR, NY-ESO1, MUC-1, 5T4, Her2, MAGE-A3, LY6K, CEACAM6, CEA, MCAK, KK-LC1, Gastrin, VEGFR2, MMP-7, MPHOSPH1, MAGE-A4, MAGE-A1, MAGE-C1, PRAME, Survivin, MAGE-A9, MAGE-C2, FGFR2, WT1, PSA, PSMA, Prostate-specific antigen precursor, Kita-kyushu lung cancer antigen 1, Trophoblast glycoprotein, Cyclin-dependent kinase inhibitor 2A, Cyclin-dependent kinase inhibitor 2A, isoforms 1/2/3, multiple tumor suppressor 1/cyclin-dependent kinase 4 inhibitor p16, GTPase, and NRas.
  3. 3 . The artificial RNA molecule according to claim 1 , wherein said at least one transmembrane domain from CTLA4 is identical to the transmembrane domain from SEQ ID NO: 169.
  4. 4 . The artificial RNA molecule according to claim 1 , wherein the linker peptide is 1-20 amino acids in length.
  5. 5 . The artificial RNA according to claim 4 , which comprises, in 5′ to 3′ direction, the following elements: a) a 5′-CAP structure, b) a 5′-UTR element, c) said at least one coding region sequence, d) a 3′-UTR element, and e) a poly(A) tail.
  6. 6 . The artificial RNA according to claim 5 , wherein the linker peptide comprises the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 76409).
  7. 7 . The artificial RNA according to claim 6 , wherein the linker peptide consists of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 76409).
  8. 8 . The artificial RNA according to claim 5 , wherein the at least one signal peptide is from CTLA4.
  9. 9 . The artificial RNA according to claim 8 , wherein said coding region further comprises at least one cytoplasmic domain of CTLA4.
  10. 10 . The artificial RNA according to claim 9 , wherein one of said at least two epitopes is from a tumor antigen selected from the group consisting of BRAF, PIK3CA, KRAS, IDH1, TP53, NRAS, AKTI, SF3B1, CDKN2A, RPSAP58, EGFR, NY-ESO1, MUC-1, 5T4, Her2, MAGE-A3, LY6K, CEACAM6, CEA, MCAK, KK-LC1, Gastrin, VEGFR2, MMP-7, MPHOSPH1, MAGE-A4, MAGE-A1, MAGE-C1, PRAME, Survivin, MAGE-A9, MAGE-C2, FGFR2, WT1, PSA, PSMA, Prostate-specific antigen precursor, Kita-kyushu lung cancer antigen 1, Trophoblast glycoprotein, Cyclin-dependent kinase inhibitor 2A, Cyclin-dependent kinase inhibitor 2A, isoforms 1/2/3, multiple tumor suppressor 1/cyclin-dependent kinase 4 inhibitor p16, GTPase, and NRas.
  11. 11 . The artificial RNA according to claim 9 , wherein one of said at least two epitopes is from a MAGE tumor antigen.
  12. 12 . The artificial RNA according to claim 9 , wherein the at least one coding region further comprises an epitope from a Hepatitis B virus antigen.
  13. 13 . The artificial RNA molecule according to claim 1 , wherein said at least one coding region further encodes (v) at least one T helper epitope.
  14. 14 . The artificial RNA molecule according to claim 1 , wherein said artificial RNA molecule is a mRNA.
  15. 15 . A composition comprising at least one artificial RNA molecule according to claim 1 and a pharmaceutically acceptable carrier and/or excipient.
  16. 16 . The artificial RNA according to claim 1 , wherein said coding region further comprises at least one cytoplasmic domain of CTLA4.
  17. 17 . The artificial RNA according to claim 16 , wherein the at least one signal peptide comprises or consists of an amino acid sequence corresponding to any one of SEQ ID NOs: 1-156 or 76948-76951.
  18. 18 . The artificial RNA according to claim 1 , wherein one of said at least two epitopes is from KRAS.
  19. 19 . The artificial RNA according to claim 18 , wherein one of said at least two epitopes is a KRAS epitope having a mutation selected from the group consisting of A146T; G12A; G12C; G12D; G12F; G12R; G12S; G12V; G13C; G13D; K117N; Q61H; Q61K; Q61L; and Q61R.

Description

The present application is a divisional of U.S. application Ser. No. 16/026,729, filed Jul. 3, 2018, which is a continuation of International Application No. PCT/EP2017/066676, filed Jul. 4, 2017, the entire contents of each of which are hereby incorporated by reference. Unlike conventional protein-based vaccines, nucleic acid vaccines are based on nucleic acid molecules—either DNA or RNA—encoding vaccine antigens. DNA vaccines typically consist of antigen-encoding gene(s) inserted into a bacterial plasmid under the control of a eukaryotic promoter, whereas RNA vaccines may usually employ messenger RNAs (mRNA) or other antigen-encoding RNA molecules. Like protein vaccines, nucleic acid vaccines can be delivered through a variety of different routes, including intramuscular, subcutaneous, mucosal, or transdermal delivery. However, unlike protein antigens, the nucleic acid vaccine to be effective must gain entry to the cytoplasm of cells at the injection site in order to induce antigen expression in vivo, thereby enabling antigen presentation on major histocompatibility molecules (MHC) and T-cell recognition (Li and Petrovsky Expert Rev Vaccines. 2016; 15(3): 313-329, McNamara et al. J Immunol Res. 2015; 2015: 794528). DNA vaccines are administered to the host and internalized by host cells, where the antigen-encoding DNA plasmid is transcribed in the nucleus and translated in the cytoplasm by host cellular machinery. Unlike DNA vaccines, antigen-encoding mRNAs only needs to gain entry into the cytoplasm, where translation occurs, in order to transfect a cell. Either way, the resulting proteins are processed into peptides, which are ultimately presented on the surface of host cells in the context of major histocompatibility complex (MHC) molecules. The peptide-MHC complex is recognized by antigen-specific T cells, resulting in a cellular host immune response (McNamara et al. J Immunol Res. 2015; 2015: 794528). There are two branches of MHC receptor presentation. MHC class I molecules-abundantly expressed in all nucleated cells and also in platelets-bind endogenously produced peptides including viral peptides and tumor antigens in the endoplasmatic reticulum (ER). Specifically, MHC class I molecules present peptides that result from proteolytic cleavage of endogenous proteins. Cleaved peptide fragments bind to an antigen peptide transporter (TAP) of the endoplasmic reticulum (ER), where they undergo further trimming of N-terminal residues and then bind to MHC class I complexes (Murphy K (2011) Janeways Immunobiology. New York: Garland Science). In contrast, MHC class II molecules are predominantly expressed by professional antigen-presenting cells (APCs) such as macrophages, B cells, and especially dendritic cells (DCs). MHC class II molecules acquire their peptides from endocytosed antigens in endocytic vesicles. Specifically, the class II molecules primarily present peptides of exogenous or plasma membrane proteins that are taken up by APCs during the course of endocytosis. The antigen is processed through a series of endosomal compartments with denaturing environment and a set of proteolytic and denaturing enzymes (Bryant et al. Adv. Immunol. 2002; 80:71-114). As the major proportion of MHC class II epitopes is generated by cleavage and processing of peptides by endosomal and lysosomal proteases, MHC class II epitopes are mainly derived from endocytosed proteins and antigens, which reside in or travel through the endocytic pathway. Proteins without direct access to the endocytic pathway (e.g., antigens naturally located in the cytoplasm, in nonendocytic organelles, or in the nucleus) are in general poorly presented in a MHC class II context. Antigen/MHC complexes are recognized by T-lymphocytes bearing the antigen-specific TCRs (T-cell receptors). Antigenic peptides presented in a MHC class I context are recognized by CD8+ cytotoxic T-lymphocytes (CTLs), whereas complexes of antigenic peptides and MHC class I molecules are recognized by CD4+-T helper-cells. While CD8+ CTLs mediate important cell-mediated effector functions including cytotoxic activity directed against cancerous or virus-infected cells, CD4+ T helper cells play a key role in orchestrating CTL effector functions and antibody production. Nucleic acid vaccines have many advantages over traditional peptide/protein vaccines with vaccine design being straightforward, thereby reducing cost and production time. Moreover nucleic acid vaccines allow easy delivery of multiple antigens with one immunization and can induce both humoral and cellular immune responses, which makes tumor/pathogen escape less likely. Additionally, unlike peptide-based vaccines, nucleic acid-based vaccines are not restricted by the patient's HLA type. Furthermore, the in vivo expression of an antigen and endogenous post-translational modification results in native protein structures ensuring appropriate processing and immune presentation. From a safety aspect, cloning or synth