WO2025008085A1

WO2025008085A1 - Immunotherapy for the treatment of prame-expressing cancers

Info

Publication number: WO2025008085A1
Application number: PCT/EP2024/059307
Authority: WO
Inventors: Anna-Sophia WIEKMEIJER; Esmé Teunisje Ida VAN DER GRACHT; Cornelis Joseph Maria Melief
Original assignee: ISA PHARMACEUTICALS BV
Current assignee: ISA PHARMACEUTICALS BV
Priority date: 2023-07-05
Filing date: 2024-04-05
Publication date: 2025-01-09
Anticipated expiration: 2026-01-05

Abstract

The present invention relates to the treatment of PRAME-expressing cancers, in particular by administration of a plurality of PRAME-based synthetic long peptides or nucleic acids encoding such SLPs. Furthermore, the invention relates to immunogenic compositions suitable for use in the method of treatment of the invention. Moreover, the invention relates to methods for selecting antigens suitable for use in immunization by determining their ability to increase CCR7 and/or CD40 levels in antigen-presenting cells.

Description

IMMUNOTHERAPY FOR THE TREATMENT OF PRAME-EXPRESSING CANCERS

FIELD OF THE INVENTION

The present invention relates to the treatment of PRAME-expressing cancers, in particular by administration of a plurality of PRAME-based synthetic long peptides (SLPs) or nucleic acids encoding such SLPs. Furthermore, the invention relates to immunogenic compositions suitable for use in the method of treatment of the invention. Moreover, the invention relates to methods for selecting antigens suitable for use in immunization by determining their ability to increase CCR7 and/or CD40 levels in antigen-presenting cells.

BACKGROUND OF THE INVENTION

Tumor associated PRAME specific T cells were first described in 1997 (Ikeda et al. 1997 Immunity 6: 199) and were reported as a single CTL clone, derived from a patient with melanoma. These T cells recognized a peptide presented by HLA-A24. The antigen PRAME had a tissue distribution highly similar to cancer testis antigens with high expression on testis cells in the absence of HLA class I antigens and high expression in melanoma cancer cells, but low or absent expression on healthy cells. It was soon discovered that several other PRAME CTL epitopes were presented by HLA-A*02:01 and that such A2-restricted T cells could be generated from patients with melanoma or from healthy donor PBMC (Kessler et al. 2001 J. Exp. Med. 193:73; Griffioen et al. 2006 Clin Cancer Res 12(10): 3130). These CD8+ CTL were shown to kill HLA-matched melanoma cells, but not PRAME+ HLA- mismatched melanoma cells or PRAME-negative cells. Other immunogenic PRAME CTL epitopes were discovered by other groups (Quintarelli et al. 2011 Blood 117(12):3353; Stanojevic et al. 2021 Cytotherapy 23(8):694). In each case CTLs generated in vitro against these epitopes were capable of lysis of HLA-matched PRAME+ tumor cells. Attempts to generate clinical benefit from these insights has envisaged two strategies: 1) Generation of T cells ex vivo by stimulation with peptides, followed by T-cell expansion and reinfusion or 2) Vaccination with either adjuvanted PRAME peptides (W02008118017) or adjuvanted PRAME recombinant protein. Although reinfusion of ex vivo expanded T cells has been envisaged as a strategy, this has not been implemented yet with T cells exclusively stimulated with PRAME peptides. Positive direct in vivo immunogenicity of a combination of PRAME and PSMA peptides in patients with cancer was shown by Weber et al. 2011(J Immunther 34(7): 556), in some patients this was associated with stable disease > 6 months, but not with objective tumor regression. A PRAME peptide mixed with peptides from three other tumor-associated antigens, delivered together in Montanide ISA-51 adjuvant + azacytidine did not show immunogenicity in patients with myelodysplastic syndrome, possibly indicating failure of selection of immunogenic peptides (Holmberg-Thyden et al. 2022 Cancer Immunol Immunother. 71(2) :433). In three papers an account is given of PRAME recombinant protein vaccination of patients with cancer. In all three papers describing these vaccination studies, only vaccine induced CD4+ T helper cells were reported and no anti-PRAME CD8+ T cells were demonstrable (Pujol et al. 2016 J. Thoracic Oncol ll(12):2208; Gutzmer et al. 2016 ESMO Open l(4):e000068), explaining the lack of clinical benefit observed in these studies. The inefficient processing of recombinant PRAME protein for HLA class I presentation is the likely root cause of the failure of the recombinant PRAME vaccines to generate CD8+ CTL in the vaccinated patients (discussed in Meissen et al. 2022 J Immunother. Cancer 10(9):e004709).

Thus, while important progress has been made, there is a clear need for improved PRAME-based antigens that are processed and presented efficiently and induce strong anti-tumor responses.

In 1998 three reports described that CD8+ CTL responses crucially depend on delivery of CD4+ help. This help was clearly delivered to antigen presenting cells such as dendritic cells (DC) and involved interaction between CD40 ligand (CD40L) on CD4+ T helper cells and CD40 on dendritic cells (DC) (Ridge et al. 1998 Nature 393:4747; Bennett et al. 2998 Nature 393:478; Schoenberger et al. 1998 Nature 393:480). In an accompanying editorial, the three cell interaction between CD4+ T helper cells, DC and CD8+ T cells was dubbed the "license to kill" model for CD8+ CTL generation (Lanzavecchia 1998 Nature 393:413). In a recent review these pivotal findings were found to have been universally confirmed and expanded by countless papers in both experimental animals such as mice and in human immunology studies, constituting canonical knowledge regarding the interaction of CD4+ T cells, DC and CD8+ T cells (Borst et al. 2018 Nature Rev. Immunol. 18: 635). Interaction of CD40L on activated CD4+ T cells with the CD40 master switch molecule on DC was confirmed to be the crucial DC activating interaction that led to important upregulation of co-stimulatory molecules including CD80/CD86 and CD70 on DC for provision of the appropriate signals to CD8+ T cells for effector CD8+ CTL induction and CD8+ memory installment. CD8+ T cells helped in this manner differed in more than 800 gene expressions from CD8+ T cells not helped by CD4+ T cells (Borst et al. 2018 Nature Rev. Immunol. 18:635), indicating the major importance of such help.

Immature dendritic cells (iDC) circulate in the blood and exist in many tissues including the skin. When such iDC have picked up antigens and upregulate the CC chemokine receptor 7 (CCR7), they will home to draining lymph nodes (dLN) where they can activate T cells. CCR7 not only mediates the migration of common DCs (eDCs), but also trafficking of plasmacytoid DCs (pDCs) to dLN (W. Hong et al. 2022 Frontiers in Pharmacol. 13:841687).

SUMMARY OF THE INVENTION

It has now surprisingly been found that certain PRAME-based SLPs have the ability to increase CCR7 and/or CD40 levels in antigen-presenting cells, rendering them interesting candidates for use in immunotherapy.

Thus, in a first aspect, the invention relates to a method for treating or preventing cancer, comprising administering to a human subject a plurality of immunogenic peptides, wherein the plurality of immunogenic peptides comprises:

(a) a peptide of 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 1, and

(b) a peptide of 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 2.

Similarly, the invention relates to a plurality of immunogenic peptides for use in the treatment or prevention of cancer, wherein the plurality of immunogenic peptides comprises:

(b) a peptide of 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO:2.

In a further aspect, the invention relates to an immunogenic composition comprising a plurality of peptides, wherein the plurality of peptides comprises:

In a further aspect, the invention relates to a method for selecting an antigen suitable for use in immunization comprising:

(i) determining CCR7 and/or CD40 levels in antigen-presenting cells, such as dendritic cells or CD14+CD11C+ cultured monocyte-derived cells, upon incubation with a candidate antigen, and

(ii) selecting a candidate antigen that is able to increase CCR7 and/or CD40 levels in said antigen-presenting cells, wherein the antigen preferably is a peptide. In a further aspect, the invention relates to a method for treating or preventing cancer, comprising administering to a human subject one or more polynucleotides encoding the immunogenic peptides as defined herein.

In a further aspect, the invention relates to one or more polynucleotides encoding the immunogenic peptides as defined herein for use in the treatment or prevention of cancer.

In a further aspect, the invention relates to an immunogenic composition comprising one or more polynucleotides encoding the immunogenic peptides as defined herein.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1: Part of a peptide structure containing a cystine moiety (FKDC(C)LFK) (SEQ ID NO: 16). The cysteine is cysteinylated and forms a disulfide bond with another cysteine molecule.

Figure 2: Gating strategy of flow cytometry data for CD14⁺CDllc⁺ cells. A) A DC gate was set based on SSC-A and FSC-A containing intact cells. B) within this population single cells were gated based on SSC-H and SSC-A. C) CD14⁺CDllc⁺ cells were gated on the intact single cells. These cells are depicted in the upper right quadrant.

Figure 3: Expression of CCR7 on CD14⁺CDllc⁺ cells. The expression of CCR7 is depicted as a fold change by dividing the geometric mean fluorescence of CCR7 on cells loaded with the indicated SLP by the geometric mean fluorescence of cells loaded with irrelevant SLP. Cells were gated as depicted in Figure 2. *SLP containing a cysteine, ** SLP containing a cystine

Figure 4: Expression of CD40 on CD14⁺CDllc⁺ cells. The expression of CD40 is depicted as a fold change by dividing the geometric mean fluorescence of CD40 on cells loaded with the indicated SLP by the geometric mean fluorescence of cells loaded with irrelevant SLP. Cells were gated as depicted in Figure 2. *SLP containing a cysteine, ** SLP containing a cystine

DETAILED DESCRIPTION OF THE INVENTION

Definitions

When used herein, the term "PRAME" refers to the human PRAME protein (UniProt A0A024R1E6).

The term "immunogenic peptide" means a peptide capable of triggering or boosting an immune response, such as a local and/or systemic CD4+ and/or CD8+ T cell response and/or an antibody response. An immunogenic peptide used in the invention may be unconjugated or unmodified, i.e. be a simple chain of amino acids linked by peptide bonds, or it may be further modified, e.g. conjugated, such as covalently bound to another molecule, e.g. an adjuvant. Likewise, the term "immunogenic composition" means a composition capable of triggering or boosting an immune response, such as a local and/or systemic CD4+ and/or CD8+ T cell response and/or an antibody response. Immunogenic peptides described herein, also denominated herein as long peptides, exceed the length of human leukocyte antigen (HLA) class I and class II presented ligands. Preferably, the long peptides of the invention are synthetic peptides, denominated herein as synthetic long peptides (SLPs).

Within the context of the present invention "a peptide of XX to YY amino acids in length" means that the number of amino acid residues is from XX to YY, for example 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues for a peptide of 33 to 40 amino acids in length. Peptides used in the invention exceed the length of human leukocyte antigen (HLA) class I and class II presented epitope peptide sequences. Preferably, the peptides used in the invention are synthetic peptides, also denominated herein as synthetic long peptides (SLPs).

Within the context of the present invention, the term "fragment of PRAME" means an amino acid sequence that corresponds to, i.e. is identical to, a partial sequence of a PRAME protein. Thus, it refers to a consecutive sequence of a natural PRAME protein without insertions, deletions or substitutions. If it is specified that a peptide comprises a fragment of a PRAME protein of a certain length, it means that the fragment is not shorter or longer. For example, if it is specified that the PRAME fragment is 33-40 amino acids in length, this means that said fragment is not less than 33 amino acids or more than 40 amino acids in length. Thus, such a peptide does e.g. not comprise a consecutive sequence of PRAME of 41 amino acids in length or more. However, for the avoidance of doubt, "comprising" has its usual meaning in the art, i.e. a peptide comprising a fragment of PRAME can comprise additional sequences beyond the specified fragment, e.g. sequences not derived from PRAME or other partial sequences of PRAME which are not contiguous with said fragment in PRAME.

The term "antigen-presenting cells" refers to cells that can present an antigen, or fragment thereof, to T cells. The term "professional antigen-presenting cells" is used herein for cells that in an intact mammalian organism process and present an antigen, or fragment thereof, to T cells to initiate and expand T cell responses. Dendritic cells are the main professional antigen-presenting cells in an intact mammalian organism. Dendritic cells may be derived from isolated CD14+ monocyte culture differentiation methods. Monocyte derived and fully differentiated dendritic cells no longer express CD14. When used herein, the term CD14+CD11C+ cultured monocyte-derived cells refers to monocyte-derived, but not fully differentiated dendritic cells.

"Treatment" or "treating" refers to the administration of an effective amount of an immunogenic peptide or immunogenic composition with the purpose of easing, ameliorating, arresting, eradicating (curing) symptoms, disorders or disease states. "Prevention" or "preventing" refers to the administration of an effective amount of an immunogenic peptide or immunogenic composition with the purpose of preventing symptoms, disorders or disease states. An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.

The term "plurality" refers to more than one, e.g. a plurality of immunogenic peptides refers to more than one, i.e. two or more, immunogenic peptides.

Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Further aspects and embodiments of the invention

As described above, in a first aspect, the invention relates to a method for treating or preventing cancer, comprising administering to a human subject a plurality of immunogenic peptides, wherein the plurality of immunogenic peptides comprises:

The method and use of the invention comprise administration of immunogenic peptides that comprise or consist of sequences corresponding to fragments of the human protein PRAME. The immunogenic peptides have a certain specified minimum and maximum length and comprise or consist of a consecutive sequence of PRAME. In one embodiment, an immunogenic peptide for use in the invention consists of a specified consecutive sequence of PRAME, i.e. a fragment of PRAME. In another embodiment, an immunogenic peptide for use in the invention comprises a specified consecutive sequence of PRAME and further comprises additional flanking regions corresponding to the flanking regions present in PRAME, up to the maximum length of the peptide. In another embodiment, an immunogenic peptide for use in the invention comprises a specified consecutive sequence of PRAME and additional flanking regions up to the maximum length of the peptide, wherein the flanking regions do not correspond to the flanking regions in PRAME and/or do not correspond to PRAME sequences at all. Thus, the immunogenic peptide used in the invention may not or may comprise a non- naturally occurring sequence as a result of comprising additional amino acids (N- terminal or C-terminal of the PRAME fragment) not originating from PRAME. As explained further herein, cysteines in the immunogenic peptides of the invention may or may not be cysteinylated, i.e. present in the form of a cystine. Furthermore, immunogenic peptides used in the invention may or may not be conjugated to non-amino acid moieties.

Preferably, an immunogenic peptide used in the invention is an isolated peptide, wherein "isolated" does not reflect the extent to which the peptide is purified, but indicates that the peptide has been removed from its natural milieu (/.e., that has been subject to human manipulation). The peptide may, e.g., be a recombinantly-produced peptide or a synthetically-produced peptide. Peptides are typically produced synthetically. This may be done by solid phase peptide synthesis or by any other suitable method.

The method or use of the invention may comprise administration of more than two immunogenic peptides, i.e. the plurality of immunogenic peptides may comprise two or more peptides, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more immunogenic peptides. Typically each of the immunogenic peptides of the plurality comprises or consists of a consecutive fragment of PRAME.

In one embodiment, the plurality of immunogenic peptides used in the invention comprises:

(a) a peptide consisting of the sequence set forth in SEQ ID NO: 1, and

(b) a peptide consisting of the sequence set forth in SEQ ID NO:2.

In some embodiments, cysteine residues in one or more immunogenic peptides may be present in the form of a cystine residue (L-cystine or D-cystine). Thus, in such an embodiment, the one or more cysteine residue in the immunogenic peptide is present in a form wherein the thiol group of the cysteine in the peptide has been oxidised (cysteinylated) and forms a disulfide bond with a cysteine molecule (the latter not being part of the immunogenic peptide). An example of such a peptide wherein the cysteine has been modified is shown in Figure 1. The cysteine modification prevents the formation of intermolecular and/or intramolecular disulfide bonds, resulting in a more stable peptide-based immunogenic composition.

In one embodiment, position 12 in the immunogenic peptide set forth in SEQ ID NO:2 is a cystine. In another embodiment, position 12 in the immunogenic peptide set forth in SEQ ID NO: 2 is a cysteine.

As mentioned above, the plurality of immunogenic peptides may comprise more than two immunogenic peptides. In one embodiment, the plurality further comprises one, two or all three of the following immunogenic peptides:

(a) a peptide of 25 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 3,

(b) a peptide of 35 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO:4, and

(c) a peptide of 32 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 5.

In one embodiment, position 5 in the immunogenic peptide set forth in SEQ ID NO:3 is a cystine. In another embodiment, position 5 in the immunogenic peptide set forth in SEQ ID NO: 3 is a cysteine.

In one embodiment, position 33 in the immunogenic peptide set forth in SEQ ID NO:4 is a cystine. In another embodiment, position 33 in the immunogenic peptide set forth in SEQ ID NO: 4 is a cysteine.

In one embodiment, the plurality of peptides further comprises one, two or all three of the following immunogenic peptides:

(a) a peptide of 35 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 6,

(b) a peptide of 32 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO:7, and

(c) a peptide of 35 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 8.

In one embodiment, position 7 in the immunogenic peptide set forth in SEQ ID NO:8 is a cystine. In another embodiment, position 7 in the immunogenic peptide set forth in SEQ ID NO: 8 is a cysteine.

In one embodiment, the plurality of immunogenic peptides used in the invention comprises, or consists of, the eight peptides set forth in SEQ ID NO: 1 to SEQ ID NO:8. In one embodiment, the plurality of immunogenic peptides used in the invention comprises, or consists of, the eight peptides set forth in SEQ ID NO: 1 to SEQ ID NO:8, wherein: position 12 in the immunogenic peptide set forth in SEQ ID NO:2 is a cystine, position 5 in the immunogenic peptide set forth in SEQ ID NO:3 is a cystine, position 33 in the immunogenic peptide set forth in SEQ ID NO:4 is a cystine, and position 7 in the immunogenic peptide set forth in SEQ ID NO:8 is a cystine.

In one embodiment, the plurality of immunogenic peptides used in the invention comprises, or consists of, the eight peptides set forth in SEQ ID NO: 1 to SEQ ID NO:8, wherein: position 12 in the immunogenic peptide set forth in SEQ ID NO:2 is a cysteine, position 5 in the immunogenic peptide set forth in SEQ ID NO:3 is a cysteine, position 33 in the immunogenic peptide set forth in SEQ ID NO:4 is a cysteine, and position 7 in the immunogenic peptide set forth in SEQ ID NO:8 is a cysteine.

In some embodiments, all immunogenic peptides of the plurality to be administered are comprised within one immunogenic composition. In other embodiments, the immunogenic peptides of the invention are distributed over two or more compositions, e.g. distributed over two or more vials. In such embodiments, the compositions may be mixed before administration to the patient or the compositions may be administered separately. Thus, in a further main aspect, the invention relates to a vaccine (i.e. a vaccine product) comprising two or more compositions which together comprise the plurality of immunogenic peptides as defined herein.

In one embodiment, the method or use of the invention does not comprise administration of peptides comprising, or consisting of, one or more of the sequences of the group consisting of: SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 (W02008118017).

In one embodiment, the method or use of the invention does not comprise administration of peptides comprising, or consisting of any of the sequences of the group consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

In one embodiment, the method or use of the invention does not comprise administration of peptides comprising or consisting of any one of the sequence set forth in Table 2, Table 3A or Table 3B, Table 4 or Table 6 of W02008118017, other than peptides comprising or consisting of the sequences set forth in SEQ ID NO: 1-

In one embodiment, the method or use of the invention comprises administration of peptides comprising or consisting of the sequences set forth in SEQ ID NO: 1-8, but does not comprise administration of further peptides comprising or consisting of any one of the sequence set forth in Table 2, Table 3A or Table 3B, Table 4 or Table 6 of W02008118017.

The method or use of the invention is typically for the treatment or prevention of a PRAME-expressing cancer.

In one embodiment, the cancer is selected from the group consisting of: neuroblastoma, lymphoma, papillomas, breast or cervical carcinomas, acute and chronic leukemias, medulloblastoma, non-small cell lung carcinoma, head and neck cancer, renal carcinoma, pancreatic carcinoma, prostate cancer, small cell lung cancer, multiple myeloma, melanoma, uveal melanoma, sarcomas and hematological malignancies like chronic myeloid leukemia and acute myeloid leukemia.

In some embodiments, the method or use of the invention further comprises administration of an adjuvant. The term "adjuvant" is used herein to refer to substances that have immune-potentiating effects and are co-administered, or added to, or co-formulated with an antigenic agent in order to enhance, induce, elicit, and/or modulate the immunological response against the antigenic agent when administered to a subject. In one embodiment, the adjuvant is physically linked, such as covalently linked, to the peptide(s) to be reconstituted.

In one embodiment, the adjuvant is an emulsifying adjuvant. In one embodiment, the adjuvant is an oil-based adjuvant. Oil-based adjuvants can be used to form emulsions (e.g. water-in-oil or oil-in-water emulsions) and are appreciated in the art to enhance and direct the immune response. Preferably the oil-based adjuvant is a mineral oil-based adjuvant. Non-limiting examples of oilbased adjuvants are bio-based oil adjuvants (based on vegetable oil I fish oil, etc.), squalene-based adjuvant (e.g. MF59), Syntex Adjuvant Formulation (SAF; Lidgate, Deborah M, Preparation of the Syntex Adjuvant Formulation (SAF, SAF-m, and SAF-1), In: Vaccine Adjuvants, Volume 42 of the series Methods in Molecular Medicine™ p229-237, ISSN1543-1894), Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvant (FIA), adjuvants based on peanut oil (e.g. Adjuvant 65) , Lipovant {Byars, N.E., Allison, A.C., 1990. Immunologic adjuvants: general properties, advantages, and limitations. In: Zola, H. (Ed.), Laboratory Methods in Immunology. p39-51), ASO4 A. Tagliabue, R. Rappuoli Vaccine adjuvants: the dream becomes real Hum. Vaccine, 4 (5), 2008, p347-349), Montanide adjuvants, which are based on purified squalene and squalene emulsified with highly purified mannide mono-oleate (e.g. Montanide ISA 25 VG, 28 VG, 35 VG, 50 V, 50 V2, 51 VG, 61 VG, 70 VG, 70 M VG, 71 VG, 720 VG, 760 VG, 763 A VG, 775 VG, 780 VG, 201 VG, 206 VG, 207 VG). More preferably, the oil-based adjuvant is Montanide ISA 51VG (Seppic), which is a mixture of Drakeol VR and mannide monooleate.

Other suitable adjuvants are adjuvants that are known to act via the Tolllike receptors and/or via a RIG-I (Retinoic acid- Inducible Gene-1) protein and/or via an endothelin receptor. Immune modifying compounds that are capable of activation of the innate immune system can be activated particularly well via Toll like receptors (TLRs), including TLRs 1 - 10. Compounds capable of activating TLR receptors and modifications and derivatives thereof are well documented in the art. TLR1 may be activated by bacterial lipoproteins and acetylated forms thereof, TLR2 may in addition be activated by Gram positive bacterial glycolipids, LPS, LPA, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or from the host, and Mycobacterial lipoarabinomannans. TLR3 may be activated by dsRNA, in particular of viral origin, or by the chemical compound poly(I:C). TLR4 may be activated by Gram negative LPS, LTA, Heat shock proteins from the host or from bacterial origin, viral coat or envelope proteins, taxol or derivatives thereof, hyaluronan containing oligosaccharides and fibronectins. TLR5 may be activated with bacterial flagellae or flagellin. TLR6 may be activated by mycobacterial lipoproteins and group B Streptococcus heat labile soluble factor (GBS-F) or Staphylococcus modulins. TLR7 may be activated by imidazoquinolines, such as imiquimod, resiquimod and derivatives imiquimod or resiquimod (e.g. 3M-052). TLR9 may be activated by unmethylated CpG DNA or chromatin - IgG complexes. Particularly preferred adjuvants comprise, but are not limited to, synthetically produced compounds comprising dsRNA, poly(I:C), poly ICLC, unmethylated CpG DNA which trigger TLR3 and TLR9 receptors, IC31, a TLR 9 agonist, IMSAVAC, a TLR4 agonist, a water-in-oil emulsion comprising a mineral oil and a surfactant from the mannide monooleate family (e.g. Montanide ISA-51, Montanide ISA 720 an adjuvant produced by Seppic, France). RIG-I protein is known to be activated by ds-RNA just like TLR3 Kato et al, (2005) Immunity, 1: 19-28).

A further particularly preferred TLR ligand is a Pam3cys and/or derivative thereof, preferably a Pam3cys lipopeptide or variant or derivative thereof, preferably such as described in WO2013051936A1 (incorporated herein by reference), more preferably U-Paml2 or U-Paml4 a.k.a. AMPLIVANT®.

Pam3cys and/or derivatives thereof may optionally be covalently linked to the peptide antigen(s).

In another embodiment, the method or use of the invention comprises administration of a TLR2 agonist of the U-Pam-14 variant (above), wherein the U- Pam-14 compound is chirally pure and only consists of compounds comprising a Cys((R)-2,3-di(palmitoyloxy)-propyl) moiety (R-diastereoisomer).

In another embodiment, the method or use of the invention comprises administration of a TLR2 agonist selected from the group consisting of: Pam3CysSer, Pam3CysSerLys, Pam3CysSer(Lys)4 (also termed Pam3CSK4), Pam2CysSer(Lys)4 (also termed Pam2CSK4), PamlCysSer(Lys)4 (also termed PamlCSK4).

Further preferred adjuvants are Cyclic dinucleotides (CDNs), Muramyl dipeptide (MDP) and Poly-ICLC. In a preferred embodiment, the adjuvants of the invention are non-naturally occurring adjuvants such as the Pam3cys lipopeptide derivative as described in WO2013051936A1, Poly-ICLC, imidazoquinoline such as imiquimod, resiquimod or derivatives thereof, CpG oligodeoxynucleotides (CpG- ODNs), such as class A-ODN (or K-type), class B-ODN (or D-type), class C-ODN as described in Sheiermann and Klinman, 2014 Vaccine 32(48): 6377-6389, more preferably class B-ODN (such as CpG7909 or 1018ISS) or class C-ODN (such as DV-281), having a non-naturally occurring sequence, and peptide-based adjuvants, such as muramyl dipeptide (MDP) or tetanus toxoid peptide, comprising non- naturally occurring amino acids.

Further preferred are adjuvants selected from the group consisting of: aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CyaA, dSLIM, GM-CSF, IC30, IC31, ImuFact EV1P321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGA microparticles, SRL172, Pam3Cys- GDPKHPKSF, YF-17D, VEGF trap, R848, beta-glucan, Aquila's QS21 stimulon, vadimezan, AsA404 (DMXAA), STING (stimulator of IFN genes) agonist (e.g. c-di- GMP VacciGrade™), PCI, NKT (natural killer T cell) agonist (e.g. alphagalactosylceramide or alpha-GalCer, RNAdjuvant® (Curevac), retinoic acid inducible protein I ligands (e.g. 3pRNA or 5'-triphosphate RNA).

In a preferred embodiment of the method or use of the invention, the adjuvant is AMPLIVANT® or Montanide ISA-51.

The adjuvant may be mixed with the immunogenic peptides prior to administration to the patient or be administered separately.

In one embodiment, the plurality of immunogenic peptides in the immunogenic composition comprises:

(a) a peptide consisting of the sequence set forth in SEQ ID NO: 1, and

(b) a peptide consisting of the sequence set forth in SEQ ID NO:2.

The plurality of immunogenic peptides comprised within the immunogenic composition may comprise more than two immunogenic peptides. In one embodiment, the immunogenic composition further comprises one, two or all three of the following immunogenic peptides:

In one embodiment, position 5 in the immunogenic peptide set forth in SEQ ID NO:3 is a cystine. In another embodiment, position 5 in the immunogenic peptide set forth in SEQ ID NO: 3 is a cysteine. In one embodiment, position 33 in the immunogenic peptide set forth in SEQ ID NO:4 is a cystine. In another embodiment, position 33 in the immunogenic peptide set forth in SEQ ID NO:4 is a cysteine.

In one embodiment, the immunogenic composition of peptides further comprises one, two or all three of the following immunogenic peptides:

In one embodiment, the immunogenic composition comprises, or consists of, the eight peptides set forth in SEQ ID NO: 1 to SEQ ID NO:8.

In one embodiment, the immunogenic composition comprises, or consists of, the eight peptides set forth in SEQ ID NO: 1 to SEQ ID NO:8, wherein: position 12 in the immunogenic peptide set forth in SEQ ID NO:2 is a cystine, position 5 in the immunogenic peptide set forth in SEQ ID NO:3 is a cystine, position 33 in the immunogenic peptide set forth in SEQ ID NO:4 is a cystine, and position 7 in the immunogenic peptide set forth in SEQ ID NO:8 is a cystine.

In one embodiment, the immunogenic composition comprises, or consists of, the eight peptides set forth in SEQ ID NO: 1 to SEQ ID NO:8, wherein: position 12 in the immunogenic peptide set forth in SEQ ID NO:2 is a cysteine, position 5 in the immunogenic peptide set forth in SEQ ID NO:3 is a cysteine, position 33 in the immunogenic peptide set forth in SEQ ID NO:4 is a cysteine, and position 7 in the immunogenic peptide set forth in SEQ ID NO:8 is a cysteine.

Immunogenic compositions according to the invention may comprise a pharmaceutically-acceptable carrier. Pharmaceutically-acceptable carriers are well- known in the art. Immunogenic compositions of the invention are preferably for, and therefore formulated to be suitable for, administration to a human subject. Preferably, the administration is parenteral, e.g. intravenous, subcutaneous, intramuscular, intradermal intracutaneous and/or intratumoral administration, i.e. by injection.

The immunogenic compositions are preferably chemically stable, i.e. the peptides in the composition do not chemically degrade or decompose. Thus, preferably, the amount of un-degraded, un-decomposed and/or unreacted peptides within the solution and/or composition is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% by weight as compared to its original, after storage of the solution or liquid composition for at least about 0.5, 1, 1.5, 2 or at least 3 hours at room temperature. Chemical stability can be assessed using any suitable technique known in the art, for instance using UPLC/MS as exemplified herein. When using UPLC/MS, a solution/composition is defined as chemically stable if the total %area of peaks that do not represent the desired peptide product in the UV spectrum after storage of at least about 0.5, 1, 1.5, 2 or at least 3 hours at room temperature is at most 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% as compared to its original.

The immunogenic compositions are preferably also physically stable, i.e. the peptides in the composition do not precipitate or re-disperse. Physical stability can be assessed using any suitable technique known in the art, for instance by visual inspection or by particle distribution using a Malvern Mastersizer as exemplified herein, wherein average particle size is expressed in D(0.5). When using Malvern Mastersizer for assessing physical stability as exemplified herein, a solution/composition is defined as physically stable if the average D (0.5) after storage of at least about 0.5, 1, 1.5, 2 or at least 3 hours at room temperature is increased at most 50%, 40%, 30%, 20%, 10% or 5% as compared to its original (/.e. the freshly prepared solution directly after preparation). Preferably, a solution/composition is defined as physically stable if the average D(0.5) after storage of 3 hours at room temperature is increased at most 50%, 40%, 30%, 20%, 10% or 5%, preferably at most 20%, as compared to its original.

In one embodiment, the immunogenic composition comprises or consists of a mixture of dry or lyophilized peptides that are to be administered together.

In one embodiment, the immunogenic composition does not comprise peptides comprising, or consisting of, one or more of the sequences of the group consisting of: SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

In one embodiment, the immunogenic composition does not comprise peptides comprising, or consisting of any of the sequences of the group consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

As shown herein, it has surprisingly been found that certain antigens have the ability to increase CCR7 and/or CD40 levels in antigen-presenting cells, rendering them interesting candidates for use in immunotherapy. Accordingly, in a further aspect, the invention relates to a method, such as an in vitro method, for selecting an antigen suitable for use in immunization comprising:

(i) determining CCR7 and/or CD40 levels in antigen-presenting cells, for example professional antigen-presenting cells, such as dendritic cells or CD14+CDllc+ cultured monocyte-derived cells, upon incubation with a candidate antigen, and

(ii) selecting a candidate antigen that is able to increase CCR7 and/or CD40 levels in said antigen-presenting cells, wherein the antigen preferably is a peptide.

In one embodiment, the method is for selecting an antigen, such as a peptide antigen, suitable for use in immunization against PRAME-expressing cancer.

In a further aspect, the invention relates to a method for treating or preventing cancer, comprising administering to a human subject one or more polynucleotides encoding the immunogenic peptides as defined herein.

In an even further aspect, the invention relates to an immunogenic composition comprising one or more plurality of polynucleotides encoding the immunogenic peptides as defined herein.

A polynucleotide may be any polynucleotide comprising e.g. RNA, DNA and/or cDNA and may comprise nucleotide analogues and/or nucleotide equivalents such as a peptide nucleic acid or a morpholino nucleotide analogue. A polynucleotide may be codon-optimized for a host of choice to facilitate expression of the encoded peptide or polypeptide.

The polynucleotide used in this aspect of the invention does not encode full- length PRAME, but rather encodes an immunogenic peptide as described herein, as such, or flanked by amino acid sequences that are not contiguous with PRAME. Thus, in the polynucleotide of the invention, the sequence encoding the immunogenic peptide may be part of a larger open reading frame also containing flanking amino acids, provided that such flanking amino acids are not contiguous with the immunogenic peptide sequence in PRAME. Such flanking amino acids may for example be from proteins other than PRAME and/or they may be from other locations within a PRAME protein that are not contiguous with the peptide they flank.

In one embodiment, the polynucleotide encodes two or more immunogenic peptides as defined herein arranged as "beads-on-a-string", whereby the peptides according to the invention (the "beads") are linked directly together and/or are linked through linker sequences that are from proteins other than PRAME and/or from other locations within PRAME that are not contiguous with the peptide they flank. The amino acid sequences flanking or linking the peptides may comprise proteolytic cleavage sites.

A polynucleotide according to the invention may be applied to deliver a peptide according to the invention in various ways. A polynucleotide according to the invention may e.g. be used in the production of recombinant protein or peptide in a suitable host cell (e.g. a bacterial host cell such as E. coli, a suitable yeast host cell such as S. cerevisiae, a suitable filamentous fungal such as an Aspergillus or mammalian host cell) from which the recombinant protein or peptide may be purified. Alternatively, the polynucleotide may be operably linked to expression regulatory sequences (promoters and the like) and incorporated in an expression construct for human cells. Such (autologous) cells may be transfected or transduced ex vivo to be (re)-administered to a subject in need thereof. Alternatively, such expression construct according to the invention may be incorporated into a suitable gene therapy vector. Suitable viral expression constructs include e.g. vectors that are based on adenovirus, adeno-associated virus (AAV), retroviruses or modified vaccinia Ankara (MVA). The polynucleotide according to the invention may also be operably linked to a sequence encoding and adjuvant such as a Toll-like receptor (TLR) ligand, a NOD ligand, or a RIG-I ligand.

Sequence listing

EXAMPLES

Example 1. Synthesis of SLPs

All reagents and solvents for solid phase peptide synthesis were purchased from Merck, Sigma Aldrich, Actu-AII, Bachem and Biosolve, GL Biochem and used as received.

Solid phase peptide synthesis (SPPS)

Peptides synthesis was performed on a Tetras peptide synthesizer (Advanced ChemTech) by solid phase Fmoc/^tBu chemistry according to established methods. In general, the peptide synthesis was carried out using pre-loaded Wang-, HMPB ChemMatrix®, 2-chlorotrityl, or 4-(l',l'-dimethyl-l'-hydroxypropyl)phenoxyacetyl- alanyl-aminomethyl resin. Reactions were typically carried out on a 30 to 60 mmol scale. The peptides were synthesized by single, double or triple coupling cycles or a combination of single, double and triple coupling cycles.

The initial swell procedure

1) Swelling of the resin: 2 cycles with NMP

2) NMP wash

3) iPrOH wash

A single coupling cycle was performed by the following consecutive steps:

1) Deprotection of the Fmoc-group: 3 cycles with piperidine in /V-methyl-2- pyrrolidone (NMP).

2) NMP wash

3) iPrOH wash

4) Coupling of the appropriate amino acid. After addition of the Fmoc-amino acid in NMP and the coupling reagent (3-(diethoxy-phosphoryloxy)-l,2,3- benzo[d]triazin-4(3H)-one (DEPBT), 2-(l/-/-benzotriazole-l-yl)-l,l,3,3- tetramethylaminium hexafluorophosphate (HBTU), 2-(lH-benzotriazol-l- yl)-/V,/V,/V',/V'-tetramethylaminium tetrafluoroborate (TBTU), benzotriazole- 1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), 7- aza-benzotriazol-l-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyAOP), Ethyl cyano(hydroxyimino)acetato-O2-tri-(l-pyrrolidinyl)- phosphonium hexafluorophosphate (PyOxim), 1-

[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), l-[l-(Cyano-2-ethoxy-2- oxoethylideneaminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU), 2-(l-Oxy-pyridin-2-yl)-l, 1,3,3- tetramethylisothiouronium tetrafluoroborate (TOTT),O-(lH-6- chlorobenzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HCTU), /V,/V'-diisopropylcarbodiimide (DIC) or ethyl 2-cyano-2- (hydroximino)acetate (Oxyma Pure®)) in NMP, the reaction mixture was shaken for 1 min. Optionally a base such as /V-methylmorpholine (NMM) or /V,/V-diisopropylethylamine (DIPEA) was added. The reaction mixture was shaken for at least 30 min.

5) NMP wash

6) Capping by acetic anhydride or benzoyl chloride in the presence of a base

(NMM or pyridine).

7) NMP wash

8) iPrOH wash

A double coupling cycle was performed by the following consecutive steps:

1) Deprotection of the Fmoc-group: 3 cycles with piperidine in NMP.

2) NMP wash

3) iPrOH wash

4) First coupling cycle: After addition of the Fmoc-amino acid in NMP and the coupling reagent (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim HATU, COMU, TOTT, HCTU, DIC or Oxyma Pure®) in NMP, the reaction mixture was shaken for 1 min. Optionally a base (NMM or DIPEA) was added. The reaction mixture was shaken for at least 15 min.

5) Purge of the reaction vessel.

6) Second coupling cycle: After addition of the Fmoc-amino acid in NMP and the coupling reagent (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim, HATU, COMU, TOTT, HCTU, DIC or Oxyma Pure®)) in NMP, the reaction mixture was shaken for 1 min. Optionally a base (NMM or DIPEA) was added. The reaction mixture was shaken for at least 15 min.

7) NMP wash 8) Capping by acetic anhydride or benzoyl chloride in the presence of a base

(NMM or pyridine).

9) NMP wash

10) iPrOH wash

A triple coupling cycle was performed by the following consecutive steps:

1) Deprotection of the Fmoc-group: 3 cycles with piperidine in NMP.

2) NMP wash

3) iPrOH wash

5) Purge of the reaction vessel.

7) Purge of the reaction vessel.

8) Third coupling cycle: After addition of the Fmoc-amino acid in NMP and the coupling reagent (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim, HATU, COMU, TOTT, HCTU, DIC or Oxyma Pure®)) in NMP, the reaction mixture was shaken for 1 min. Optionally a base (NMM or DIPEA) was added. The reaction mixture was shaken for at least 15 min.

9) NMP wash

10) Capping by acetic anhydride or benzoyl chloride in the presence of a base (NMM or pyridine).

11) NMP wash

12) iPrOH wash

After the last coupling cycle, the following final Fmoc-deprotection step and wash steps were performed:

1) Deprotection of the Fmoc-group: 3 cycles with piperidine in NMP.

2) NMP wash

3) iPrOH wash

Cleavage and purification procedure with cysteine modification during cleavage After the final wash steps, the resin was dried and cooled. A cleavage cocktail based on Milli-Q water, thioanisole, and TFA was added to the resin, immediately followed by the addition of 50 equivalents of 2,2'-dithiobis(5-nitropyridine) (DTNP) per cysteine(Trt) residue present in the peptide. The mixture was left standing at room temperature for 90 minutes. Next, the cleavage mixture was filtered and the residue was washed with diethyl ether. The filtrate was collected and centrifuged. The supernatant was removed and fresh diethyl ether was added to the pellet and resuspended by vortexing. After centrifugation, the pellet was isolated and dried under vacuum.

The crude peptide was dissolved in a mixture based on Milli-Q water, MeCN and acetic acid. After centrifugation, the supernatant was isolated. L-cysteine (145 mg, for a SPPS synthesis at a scale of 60 pmol) was added to the supernatant. After 1 h, the reaction mixture was diluted with Milli-Q water and filtered. The peptide was purified by a Waters AutoPurification HPLC/MS system under acidic conditions (ACN, water and TFA) followed by lyophilization overnight to obtain the cystine-containing peptide as a white to off-white powder.

Cleavage and purification procedure with cysteine modification off-resin

After the final wash steps in the peptide synthesizer, the resin was dried and cooled. A cleavage cocktail based on Milli-Q water, ethanethiol, triisopropylsilane, and TFA was added to the resin and mixed for 3h. Subsequently, cold diethylether was added and the mixture was centrifuged. The supernatant was removed and the pellet was isolated. The following steps were identical as to the above described "cleavage and purification procedure with cysteine modification during cleavage". Briefly, the obtained filtrate was treated with the cleavage cocktail and DTNP. After 90 minutes, the solution was filtered into diethyl ether. The filtrate was collected, centrifuged and the obtained pellet was washed a second time. Next, the pellet was dissolved in Milli-Q water, MeCN and acetic acid and centrifuged, L- Cysteine was added to the supernatant. After 30 minutes, the reaction mixture was filtered, purified by HPLC/MS system under acidic conditions, followed by lyophilization overnight to obtain the cystine-containing peptide as a white to off- white powder.

Analysis of the peptide

The identity and purity of the purified peptides were determined by UPLC-UV-MS on a Waters Acquity UPLC/TQD system using an C18 Waters Acquity BEH130 analytical column (1.7 um particle size, 2.1 x 150 mm, flow 0.4 mL/min) with a linear gradient (5% B to 95% B, linear gradient in 10 min). The absorbance was measured at 220 nm.

Solvent system:

A: 0.05% trifluoroacetic acid (TFA) and 1% ACN in H2O

B: 0.05% TFA in ACN

The determination confirmed the identity of the synthesized peptides.

Example 2. T cell induction

The capacity of the SLPs to activate T cells was studied. The biological activity of the synthesized and purified SLPs was tested using PBMC from healthy donors. Monocytes were isolated using anti-CD14 beads by magnet activated cell sorting (MACS) following the protocol of the supplier (Miltenyi Biotec). In short, PBMCs were isolated by centrifugation over a Ficoll gradient and cryopreserved. To generate dendritic cells (DCs), approximately 50*10 thawed PBMC were used for the isolation of CD14 positive cells. The cells were cultured for three days at 37°C in 2 ml/well of IMDM 4% human serum (HS) containing 800 U/ml GM-CSF and 500 U/ml IL-4 (Peprotech). After 3 days 1 mL/well of IMDM 4% HS with GM-CSF (2400U/mL) and IL-4 (1500U/mL) was added to the monocytes-derived DCs and these adhered cells were cultured for an additional 3 days. On day 6, long peptides distributed over 2 pools were added to monocyte-derived DCs of naive donors at a 13pM concentration and incubated overnight at 37°C. The next day (day 7) peptide-loaded DCs were harvested, irradiated (1000 rad), washed and mixed in a 1: 10 ratio with autologous PBMC in IMDM 8% human serum in the presence of IL-7 (10 ng/mL) and IL-12p70 (100 pg/mL). Conditions in favor of T cell culture, hence from here on referred to as, cultured T cells.

On day 10 new DCs were generated and loaded, after 6 days, with SLPs on day 16 as described above. The next day, these peptide-loaded DCs were harvested, irradiated (1000 rad) and washed. Also the cultured T cells were harvested. Both were counted and mixed in a 1: 10 ratio (DC:T) in IMDM 8% human serum in the presence of IL-7 (10 ng/mL) and IL-12p70 (100 pg/mL) for the first restimulation.

On the same day, new DCs were generated as described above that were loaded with SLPs on day 23 as described above. The next day, peptide-loaded DCs were harvested, irradiated (1000 rad) and washed. Also the cultured and restimulated T cells were harvested. Both were counted and mixed in a 1: 10 ratio (DC:T) in IMDM 8% human serum in the presence of IL-7 (10 ng/mL) and IL- 12p70 (100 pg/mL) for the second restimulation. On this day 24, also test 1 was started in which DCs loaded with individual SLPs were cultured with harvested T cells from restimulation 1. For test 1, DCs and harvested T cells were cultured in a 1: 10 ratio for 2 days after which cells were transferred to a coated ELISpot plate (see description below).

On day 24, new DCs were generated as described above that were loaded with SLPs on day 30 as described above. The next day, peptide-loaded DCs were harvested, irradiated (1000 rad) and washed. Also the cultured twice restimulated T cells were harvested. Both were counted and mixed in a 1 : 10 ratio (DC:T) in IMDM 8% human serum in the presence of IL-2 (30 ZU/mL) and IL-12p70 (100 pg/mL) for the third restimulation.

On day 31, also test 2 was started in which DCs loaded with individual SLPs were cultured with harvested T cells from restimulation 2. For test 2, DCs and harvested T cells were cultured in a 1: 10 ratio for 2 days after which cells were transferred to a coated ELISpot plate (see description below).

The T cell cultures were restimulated three times in 1-week cycles using peptide loaded autologous moDCs. After the 2nd and 3rd restimulation, reactivity towards single SLPs was tested (test 1 and test 2, respectively). Reactivity was determined by measuring IFNy production using ELISpot.

For ELISpot analysis, multiscreen plates were coated with an IFNy coating antibody overnight at 4°C. The next day, the plate was washed 4x with PBS and blocking was done using IMDM 8% HS at 37°C for at least one hour. Each sample was tested in triplicate. As a positive control, phytoheamagglutinin (PHA, 1 pg/mL) was added to cells that were not stimulated with SLP after thawing. The plate was cultured overnight at 37°C. Thereafter, cells were discarded and the plate was washed using PBS/0.05% Tween-20. The IFNy detection antibody was diluted and added to each well and incubated for 2 hours at room temperature. Next, the plate was washed using PBS/0.05% Tween-20 and then incubated with streptavidin-ALP for 1 hour at room temperature. The plate was washed with PBS/0.05% Tween-20. BCIP/NPT ALP substrate was filtered and added per well for 10-20 minutes at room temperature.

SLPs having the sequences set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 14 were synthesized as described in Example 1. For the cysteine containing SLPs, the cysteinylated SLPs were tested. Each SLP was minimally tested on 7 donors. As controls, medium only, an Albumin-derived peptide control and Candida albicans antigen were included in the culture, PHA was used as a positive control on the ELISpot plate. Positive IFNy responses were detected to 7 of the 10 peptides upon stimulation with 2 peptide pools (Table 1). Next, T cell cultures were generated by stimulating with a pool of peptides consisting only of the three peptides that had not tested positive yet in an additional 6 donors. Upon stimulation of PBMCs with these remaining 3 peptides only, also SEQ ID NO:3 showed a positive IFNy response (Table 2). SLPs having the sequences set forth in SEQ ID NO:9 and SEQ ID NO: 14 did not show convincing responses in any of the 13 donors tested.

In conclusion, 8 of the 10 tested SLPs demonstrated a positive result in these in vitro human T cell cultures, demonstrating their immunogenicity. Peptides can test positive after the second restimulation (restim2/test 1) but not after the third (restim3/test 2), as these cultures are then likely overgrown by T cells with other specificities.

Table 1: Results of IFNy ELISpot of test 1 and test 2 for the individual SLPs. Results are depicted as - (negative) or + (positive). A response was defined as positive when the average spot count was higher than the average spot count+2*standard deviation of the irrelevant SLP control. The last row depicts a summary of the results in the table. When at least one donor had a positive response, this is denoted as POS. BCxxx = healthy donor buffy coat codes, nt = not tested.

POS POS POS NEG POS POS NEG NEG POS POS

Table 2: Results of IFNy ELISpot of test 1 and test 2 for 3 individual SLPs that did not test positive in the experiments depicted in Table 1. These 3 SLPs were tested in an additional 6 donors. Results are depicted as - (negative) or + (positive). A response was defined as positive when the average spot count was higher than the average spot count+2*standard deviation of the irrelevant SLP control. The last row depicts a summary of the results in the table. When at least one donor had a positive response, this is denoted as POS. BCxxx = healthy donor buffy coat codes, nt = not tested.

NEG NEG POS

Example 3. Induction of CCR7 and CD40

When studying the immunogenicity of SLPs in moDCs cultures, unusual effects were observed on the morphology of moDCs cultured with SEQ ID NO: 1 (not shown). Based on that we decided to test the complete set of the PRAME-derived SLPs to further investigate and characterize the effect using APC related cell surface markers.

CD14⁺ cells were isolated from buffy coats obtained from healthy donors using magnet activated cell sorting (Miltenyi Biotec) using the manufacturer's instructions, to prepare a monocyte derived DC cell culture as described in Zom et al Oncotarget, 2016 7 (41): 67087 and above. In brief, these cells were cultured in IMDM supplemented with 4% human serum, penicillin/streptomycin and L- glutamine. 500 Ill/mL IL-4 and 800 Ill/mL GM-CSF were added to these cultures on day 0 and day 3.

SLPs having the sequences set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 14 were synthesized as described in Example 1. For one SLP (SEQ ID NO:2) both the non-cysteinylated and cysteinylated SLPs were tested. On day 6 cells were loaded with 13 pM of each individual SLP as indicated and harvested after o/n stimulation on day 7 to determine expression of surface markers with flow cytometry.

Cells were stained with suitable antibodies from Miltenyi Biotec covering a general set of DC identifying cell surface markers and co-stimulatory molecules, using CDllc (REA618), CD14 (REA599), CD40 (REA733) and CCR7 (REA546). Antibodies were used according to the manufacturer's instructions (Miltenyi Biotec) and samples were prepared for flow cytometry analysis on an LSR-II instrument (BD Biosciences). Live cells were gated (using Flow Jo software) on FSC and SSC and doublets were excluded based on SSC-H and SSC-A (Fig 2A and B).

Expression of CCR7 and CD40 was assessed on CD14⁺CDllc⁺ live single cells (Fig. 2C) on the SLP-loaded and control samples. An albumin-derived irrelevant SLP was used as negative control and to correct for background. A fold change was calculated by dividing the geometric mean of CCR7 or CD40 on CD14⁺CDllc⁺ live single cells measured on cells loaded with an individual test SLP with the geometric mean of that same marker on cells loaded with the albumin-derived irrelevant SLP.

Remarkably, it was observed that cells loaded with SEQ ID NO:2 (cysteine (*)), SEQ ID NO:2 (cystine (Cys**)) or SEQ ID NO: 1 show increased expression of CCR7 (Fig. 3) and CD40 (Fig. 4) on their cell surface above background, while other SLPs did not induce this effect. Effects are similar to the expected positive control, a lipopeptide TLR2 agonist, the R-diastereoisomer (see above) of AMPLIVANT (U-Pam-14), as shown here and described in Willems et al. 2014 J Med Chem 57(15):6873, Stegmann et al. 2023 Cancer Immunology Immunotherapy May 24. doi: 10.1007/s00262-023-03462-y. Online ahead of print. PMID: 37222770.

Claims

1. A method for treating or preventing cancer, comprising administering to a human subject a plurality of immunogenic peptides, wherein the plurality of immunogenic peptides comprises:

2. The method according to claim 1, wherein the plurality of immunogenic peptides comprises:

(a) a peptide consisting of the sequence set forth in SEQ ID NO: 1, and

(b) a peptide consisting of the sequence set forth in SEQ ID NO:2, wherein preferably position 12 in SEQ ID NO:2 is a cystine.

3. The method according to any one of the preceding claims, wherein the plurality of immunogenic peptides further comprises one, two or all three of the following immunogenic peptides:

(b) a peptide of 35 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 4, and

4. The method according to any one of the preceding claims, wherein the plurality of peptides further comprises one, two or all three of the following immunogenic peptides:

(b) a peptide of 32 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 7, and

5. The method according to any one of the preceding claims, wherein the plurality of immunogenic peptides comprises, or consists of, the eight peptides set forth in SEQ ID NO: 1 to SEQ ID NO:8.

6. The method according to any one of the preceding claims, wherein the treatment: (i) does not comprise administration of peptides comprising, or consisting of, one of the sequences of the group consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, or

(ii) does not comprise administration of peptides comprising or consisting of any of the sequences of the group consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

7. The method according to any one of the preceding claims, wherein the cancer is a PRAME-expressing cancer, preferably selected from the group consisting of: neuroblastoma, lymphoma, papillomas, breast or cervical carcinomas, acute and chronic leukemias, medulloblastoma, non-small cell lung carcinoma, head and neck cancer, renal carcinoma, pancreatic carcinoma, prostate cancer, small cell lung cancer, multiple myeloma, melanoma, uveal melanoma, sarcomas and hematological malignancies like chronic myeloid leukemia and acute myeloid leukemia.

8. The method according to any one of the preceding claims, further comprising administration of an adjuvant, wherein the adjuvant preferably is AMPLIVANT® or Montanide ISA-51.

9. A plurality of immunogenic peptides for use in the treatment or prevention of cancer, wherein the plurality of immunogenic peptides comprises:

10. The plurality of immunogenic peptides for use according to claim 9, further comprising the features of any one of claims 2 to 8.

11. An immunogenic composition comprising a plurality of peptides, wherein the plurality of peptides comprises: (a) a peptide of 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 1, and

12. The immunogenic composition according to claim 11, further comprising the features of any one of claims 2 to 8.

13. A method for selecting an antigen suitable for use in immunization comprising:

14. A method for treating or preventing cancer, comprising administering to a human subject one or more polynucleotides encoding the immunogenic peptides as defined in any one of claims 1 to 6.

15. An immunogenic composition comprising one or more plurality of polynucleotides encoding the immunogenic peptides as defined in any one of claims 1 to 6.