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EP-4735030-A1 - CHIMERIC VLP FORMING POLYPEPTIDES COMPRISING BETA-RETROVIRAL GAG

EP4735030A1EP 4735030 A1EP4735030 A1EP 4735030A1EP-4735030-A1

Abstract

The present invention relates to a platform concept for presenting antigenic polypeptides, for instance HERV envelope proteins (Env), as part of a virus like particle (VLP) construct, which comprises a Gag (group-specific antigen) protein of a beta-retrovirus, for instance of a human endogenous retrovirus K (HERV-K). Surprisingly it was found that antigenic polypeptide expression in a VLP comprising a Gag protein of HERV-K promotes antigenic polypeptide display and immunogenicity.

Inventors

  • HOLST, PETER JOHANNES
  • SKANDORFF PEDERSEN, Isabella
  • ANDERSSON, Anne-Marie Carola

Assignees

  • InProTher ApS

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. CLAIMS 1. At least one nucleic acid molecule encoding (1) a first polypeptide A, wherein the polypeptide A is a group-specific antigen (Gag) protein of a beta-retrovirus; and (2) a second polypeptide, wherein the second polypeptide comprises the polypeptides B, C and D in the order B-C-D or D-C-B; wherein polypeptide B is an antigenic polypeptide, polypeptide C is a transmembrane domain (TMD), and polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof, wherein said first polypeptide and second polypeptide is encoded by the same nucleic acid molecule or by two different nucleic acid molecules.
  2. 2. VLP comprising the polypeptides A, B, C and D as defined in claim 1.
  3. 3. The at least one nucleic acid according to claim 1 or the VLP according to claim 2, wherein polypeptide D is a cytoplasmic tail (CT) of a viral envelope protein or fragment thereof capable of binding to the first polypeptide A so that when the first and second polypeptides are expressed in the same cell, virus like particles (VLPs) are formed that comprise said first and second polypeptide.
  4. 4. The at least one nucleic acid or the VLP according to any of the preceding claims, wherein (i) (a) polypeptides C, D and A are from the same beta-retrovirus and preferably of HERV-K; and (b) polypeptide B is any antigen but not an antigen from the beta-retrovirus mentioned in (a) above and preferably polypeptide B is an antigenic part of an Env protein, preferably of HERV-W; or wherein (ii) (c) polypeptides B, C, D are from the same virus and preferably of HERV-W; and (d) polypeptide A is of a beta-retrovirus, preferably of HERV-K.
  5. 5. The at least one nucleic acid or the VLP according to claim 4, wherein polypeptide D (CT) is of a cytoplasmic tail of an envelope (Env) protein of said beta-retrovirus or a fragment thereof.
  6. 6. The at least one nucleic acid molecule or the VLP according to any of the preceding claims, wherein the polypeptide D comprises at least 6 continuous amino acid residues of the C- terminus of a beta-retroviral envelope protein and wherein the C-terminal amino acid of said continuous amino acid residues is the C-terminal amino acid of said envelope protein; and preferably wherein polypeptide D comprises the amino acid residues IVTVSV (SEQ ID NO: 9), or comprises the amino acid residues the amino acid residues QIVTVSV (SEQ ID NO: 10), or comprises the amino acid residues KRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 11), or comprises the amino acid residues AVLSKRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 12), or comprises the amino acid residues RAMMTMAVLSKRKGGNVGKSKRDQIVTVSV (SEQ ID NO: 27), or wherein polypeptide D comprises a polypeptide according to any of SEQ ID NOs: 9, 10, 11, 12 or 27 in which the polypeptide comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 9, 10, 11, 12 or 27.
  7. 7. The at least one nucleic acid molecule or the VLP according to any one of claims 1-6, wherein (a) in polypeptide D at least the 6 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, preferably wherein at least the 20 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, more preferably wherein at least the 30 continuous terminal amino acid residues of the C-terminus of polypeptide D are deleted, or wherein polypeptide D is deleted; or (b) wherein polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQ (SEQ ID NO: 29), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQ (SEQ ID NO: 29) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 29; or (c) wherein polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLS (SEQ ID NO: 30) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 30; or (d) wherein polypeptides C and D together comprise the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRE (SEQ ID NO: 31), or comprises the polypeptide sequence TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRE (SEQ ID NO: 31) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 31; or (e) polypeptide A is HERV-K Gag and polypeptide C and D are as defined in (a), (b), (c) or (d), or (f) polypeptide A is HERV-K Gag, polypeptide B is an antigenic part of an Env protein, preferably of HERV-W, and polypeptide C and D are as defined in (a), (b), (c) or (d).
  8. 8. The at least one nucleic acid molecule or the VLP according to any of the preceding claims, wherein at the C-terminus of polypeptide D, 6 continuous amino acid residues of the C- terminus of a beta-retroviral envelope protein are attached, and preferably wherein the 6 continuous amino acid residues of the C-terminus of a beta-retroviral envelope protein comprise the amino acid sequence IVTVSV (SEQ ID NO: 9) or an amino acid sequence according to SEQ ID NO: 9 in which the amino acid sequence comprises at least one conservative amino acid substitution compared with the respective reference sequence SEQ ID NO: 9.
  9. 9. The at least one nucleic acid molecule or the VLP according to any of the preceding claims, wherein polypeptide C comprises at least on alpha helical polypeptide structure, more preferably wherein polypeptide C comprises from about 15 to 55 amino acid residues, even more preferably wherein polypeptide C is a transmembrane domain (TMD) of a bitopic type I integral transmembrane protein, even more preferably wherein polypeptide C is a TMD of a virus, preferably of a beta-retrovirus, even more preferably wherein polypeptide C is of the same beta-retrovirus as the first polypeptide A (Gag), most preferably wherein polypeptide C is of an envelope (Env) protein of said same beta-retrovirus; or wherein polypeptide C and D together comprise the polypeptide TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQIVTVSV SEQ ID (NO: 8) or comprise the polypeptide TIGSTTIINLILILVCLFCLLLVCRCTQQLRRDSDHRERAMMTMAVLSKRKGGNVGKS KRDQIVTVSV SEQ ID (NO: 8) in which the polypeptide comprises at least one conservative amino acid substitution compared with the reference sequence SEQ ID NO: 8.
  10. 10. The at least one nucleic acid molecule or the VLP according to any of the preceding claims, wherein polypeptide B is a viral protein or an antigenic part of a viral protein, even more preferably an endogenous retrovirus (ERV) protein, even more preferably a gamma-ERV protein, and even more preferably wherein polypeptide B is an antigenic part of a viral Env protein, preferably an antigenic part of the Env protein of a human endogenous retrovirus (HERV) and most preferably an antigenic part of the Env protein of a gamma-ERV.
  11. 11. The at least one nucleic acid molecule or the VLP according to claim 10, wherein said viral protein is selected from the group consisting of HERV-H Env, HERV-W Env, HERV-R Env, HERV-FRD Env, HERV-MSRV Env, HERV-MER34 Env, HERV-W-1 Env (= Syncytin-1), HERV-H19 Env (=HERV-H_2q24.3), HERV-H_2q24.1 Env, ERV3-1 Env and ERVFRD-1 Env (=Syncytin-2) and/or wherein said antigenic part is or comprises the surface unit (SU) and/or ectodomain (Ecto) of said Env protein.
  12. 12. The at least one nucleic acid molecule or the VLP according to any of the preceding claims, wherein the first polypeptide A is a Gag protein of an endogenous beta-retrovirus (beta-ERV) or of a foreign beta-retrovirus, preferably wherein the beta-ERV is selected from the group consisting of human beta-ERV and non-human beta-ERV, more preferably wherein the human beta-ERV is HERV-K and wherein the non-human beta-ERV is selected from IAPE (Intracisternal A-type Particles elements with an Envelope) murine endogenous retrovirus and simian retrovirus 2 (SRV2), even more preferably wherein the HERV-K is selected from the group consisting of HERV- K108 (=ERVK-6), ERVK-19, HERV-K115 (=ERVK-8), ERVK-9, HERV-K113, ERVK-21, ERVK-25, HERV-K102 (=ERVK-7), HERV-K101 (=ERVK-24), and HERV-K110 (=ERVK- 18).
  13. 13. The at least one nucleic acid molecule according to any one of claims 1 or 3 to 12, wherein the at least one nucleic acid is an RNA, preferably an RNA selected from the group consisting of mRNA, circular RNA and self-amplifying RNA.
  14. 14. The at least one nucleic acid molecule according to any one of claims 1 or 3 to 13, wherein the first polypeptide A in (1) and the second polypeptide in (2) are encoded on the same nucleic acid molecule, wherein the first polypeptide A in (1) is encoded by a first open reading frame (ORF) and wherein the second polypeptide c in (2) is encoded by a separate second ORF.
  15. 15. The at least one nucleic acid molecule according to claim 14, wherein the sequence of the first ORF encoding the first polypeptide in (1) and the sequence of the separate second ORF encoding the second polypeptide in (2) are connected by a sequence enabling separate translation of the first and second polypeptide, preferably wherein said sequence enabling separate translation encodes a p2A self-cleaving peptide or an internal ribosomal entry site (IRES).
  16. 16. The at least one nucleic acid molecule according to any one of claims 1 or 3 to 13, wherein the first polypeptide A in (1) is encoded on a first nucleic acid molecule and wherein the second polypeptide comprising polypeptides B, C and D in (2) is encoded on a separate second nucleic acid molecule.
  17. 17. The at least one nucleic acid molecule according to any one of claims 1 or 3 to 16, wherein the at least one nucleic acid molecule comprises at least 60 adenosine nucleotides at the 3’-UTR.
  18. 18. The at least one nucleic acid molecule according to any one of claims 1 or 3 to 17, wherein the at least one nucleic acid molecule is codon optimized for expression in a human.
  19. 19. The at least one nucleic acid molecule according to any one of claims 1 or 3 to 18, wherein the at least one nucleic acid molecule comprises at least 300 nucleotides.
  20. 20. The VLP according to any of the preceding claims, wherein the polypeptide B is displayed on the surface of the VLP.

Description

InProTher ApS (Hervolution ApS) ZSP ref.: 1195-5 PCT2 Chimeric VLP forming polypeptides comprising beta-retroviral Gag Although immune cells are able to detect and kill diseased cells including tumor cells, this system is not always functional, as evident from the almost 9 million annual deaths worldwide due to cancer. To boost and/or activate the body’s own defence mechanisms against harmful disease triggers that enter from outside of or even originate in the body itself, vaccination and or immune therapy can be used to stimulate the immune system’s response to certain target antigens which are otherwise overlooked by the bodies surveillance mechanisms. One elegant vaccination strategy is the presentation of antigens (e.g. viral proteins such as viral envelope proteins) to the immune system on virus-like particles (VLPs), which are encoded in a nucleotide comprised by a vaccine. These particles do not contain viral nucleic acids and are therefore non-infectious. Nevertheless, VLPs are highly immunogenic and displayed proteins are presented in a natural context. For example, a viral envelope (Env) protein integrated in VLPs is presented on a virus-like surface, which promotes correct folding and conformation. In addition to the advantage of a strong immunogenicity, the vaccination strategy with VLPs includes also practical benefits. Thus, VLPs are relatively easy to produce as they are built from few proteins and production can be performed in cell cultures. Bayer et al. (2010) showed that only the combination of encoded and capsid presented antigens was able to increase the level of functional antibodies. This observation was assigned to the fact that while the presentation on the adenoviral capsid helped to cross-link B cell receptors, encoded antigens were required for an essential CD4+ T cell responses promoting affinity maturation of B cells. With this vaccination strategy Bayer et al. were able to reduce viral load of F- MLV after challenge. However, no indication of increased CD8+ T cell responses against the target antigen could be observed (Bayer W, Tenbusch M, Lietz R, Johrden L, Schimmer S, Uberla K, Dittmer U, Wildner O, J. Virol. 2010;84:1967–1976). Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection (J Virol. 2010 Feb;84(4):1967-76). Shoji et al. primarily focused on the optimization of an adenovirus-based HIV vaccine and investigated the in-situ formation of group specific antigen (Gag) protein based VLPs. In their study such a setting showed the highest immune responses compared to other display strategies that did not promote in situ formation of VLPs (Shoji M, Yoshizaki S, Mizuguchi H, Okuda K, Shimada M. Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters. PLoS One.2012;7(1):e30302). One particularly interesting field of application for vaccinations sensitizing the body to fight otherwise undetected diseases is to vaccinate against the formation of tumor pathologies in the body and to target malignancy developing body cells. Vaccination approaches to induce specific immune responses against tumor cells is a relatively old topic in cancer immunotherapies but is still under development. One vaccination strategy involves the vaccination with attenuated tumor cells, e.g., irradiated autologous tumors or allogeneic tumor cell lines, often secreting the granulocyte- macrophage colony-stimulating factor (GM-CSF). In both cases the injected material encompasses cancer-antigens that are likely present in the actual tumor. Other vaccination strategies include the administration of peptides or proteins to induce specific immune responses. These antigens are either injected directly in combination with an adjuvant, or are encoded by DNA plasmids or viral vectors. An example of suitable antigenic polypeptide targets for anti-cancer vaccinations present in the body can be proteins or peptides of endogenous retroviruses (ERVs) and especially of human endogenous retroviruses (HERVs). ERVs are the evidence of ancient infections with retroviruses in distant ancestors. Upon infection, viral RNA was reverse transcribed into parvoviral DNA, which was integrated into the host genome. Eventually, the provirus was integrated into cells of the germ line and became inheritable, giving rise to endogenous retroviruses. Over millions of years the viral DNA was passed down generations and became fixed in the populations. Today, every human genome consists of about 8% endogenous retroviral DNA, but these are just relics of the former retrovirus. Due to mutations, deletions and insertions most of the retroviral genes became inactivated or got completely lost from the genome. Today, no functional, full-length endogenous retrovirus is present in humans anymore. However, ERVs underwent duplication process