Immunotherapy for the treatment of PRAME-expressing cancers

Abstract

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

Description

[Technical Field]

[0001] Field of the Invention The present invention relates to the treatment of PRAME-expressing cancer, particularly by administration of a plurality of PRAME-based synthetic long peptides (SLPs) or nucleic acids encoding such SLPs. Furthermore, the present invention relates to immunogenic compositions suitable for use in the treatment method of the present invention. Moreover, the present invention relates to a method for selecting antigens suitable for use in immunization by determining their ability to increase CCR7 and / or CD40 levels in antigen-presenting cells. [Background Art]

[0002] Background of the Invention Tumor-associated PRAME-specific T cells were first described in 1997 (Ikeda et al. 1997 Immunity 6:199 (Non-patent Literature 1)) and reported as a single CTL clone derived from a patient with melanoma. These T cells recognized peptides presented by HLA-A24. The antigen PRAME had a tissue distribution very similar to that of cancer-testicular antigens, being highly expressed in testicular cells lacking HLA class I antigens, highly expressed in melanoma cancer cells, but low or absent in healthy cells. Several other PRAME CTL epitopes were presented by HLA-A*02:01, and it was quickly discovered that such A2-restricted T cells could be generated from PBMCs of patients with melanoma or healthy donors (Kessler et al. 2001 J. Exp. Med. 193:73 (Non-Patent Literature 2); Griffioen et al. 2006 Clin Cancer Res 12(10):3130 (Non-Patent Literature 3)). These CD8+ CTLs were shown to kill HLA-compatible melanoma cells but not PRAME+ HLA-incompatible melanoma cells or PRAME-negative cells. Other immunogenic PRAME CTL epitopes have been discovered by other groups (Quintarelli et al. 2011 Blood 117(12):3353 (Non-Patent Literature 4); Stanojevic et al. 2021 Cytotherapy 23(8):694 (Non-Patent Literature 5)). In each case, CTLs generated in vitro against these epitopes were able to lyse HLA-matched PRAME+ tumor cells. Attempts to translate these findings into clinical utility envisioned two strategies: (1) ex vivo generation of T cells by peptide stimulation, followed by expansion, proliferation, and reinjection of these T cells, or (2) vaccination with either adjuvant-added PRAME peptide (WO2008118017 (Patent Document 1)) or adjuvant-added PRAME recombinant protein. While reinjection of ex vivo expanding and proliferating T cells was envisioned as a strategy, this had not yet been performed with T cells exclusively stimulated by PRAME peptide.The positive in vivo immunogenic effect of a combination of PRAME and PSMA peptides in cancer patients was demonstrated by Weber et al. 2011 (J Immunther 34(7):556) (Non-patent Literature 6), which in some patients was associated with stable disease for longer than 6 months, but without objective tumor reduction. PRAME peptides, mixed with peptides derived from three other tumor-associated antigen peptides and delivered together in Montanide ISA-51 adjuvant + azacitidine, did not show immunogenicity in patients with myelodysplastic syndrome. This suggests a possible failure in the selection of immunogenic peptides (Holmberg-Thyden et al. 2022 Cancer Immunol Immunother. 71(2):433 (Non-patent Literature 7)). Three papers provide rationale for PRAME recombinant protein vaccination in cancer patients. In all three papers describing these vaccination studies, only vaccines that induced CD4+ T helper cells were reported, and anti-PRAME CD8+ T cells were not demonstrable (Pujol et al. 2016 J. Thoracic Oncol 11(12):2208 (Non-patent Literature 8); Gutzmer et al. 2016 ESMO Open 1(4):e000068 (Non-patent Literature 9)). This explains the lack of clinical utility observed in these studies. Inefficient processing of recombinant PRAME protein for HLA class I presentation is considered the underlying reason why recombinant PRAME vaccines failed to generate CD8+ CTLs in vaccinated patients (discussed in Melssen et al. 2022 J Immunother. Cancer 10(9):e004709 (Non-patent Literature 10)).

[0003] Thus, while significant progress has been made, there is a clear need for improved PRAME-based antigens that are efficiently processed and presented to induce a potent antitumor response.

[0004] In 1998, three reports described the CD8+ CTL response as critically dependent on the delivery of CD4+ helper cells. These helper cells were apparently delivered to antigen-presenting cells such as dendritic cells (DCs) and were involved in the interaction between CD40 ligand (CD40L) on CD4+ T helper cells and CD40 on dendritic cells (DCs) (Ridge et al. 1998 Nature 393:4747 (Non-Patent Literature 11); Bennett et al. 2998 Nature 393:478 (Non-Patent Literature 12); Schoenberger et al. 1998 Nature 393:480 (Non-Patent Literature 13)). In a related article, the interaction of three types of cells—CD4+ T helper cells, DCs, and CD8+ T cells—was referred to as a "license to kill" model for CD8+ CTL generation (Lanzavecchia 1998 Nature 393:413 (Non-Patent Literature 14)). In a recent review, it was found that these crucial findings have been universally confirmed and developed in countless papers in both experimental animal studies such as mice and human immunology research, and that this constitutes standard knowledge regarding the interaction of CD4+ T cells, DCs, and CD8+ T cells (Borst et al. 2018 Nature Rev. Immunol. 18:635 (Non-Patent Literature 15)). The interaction of CD40L on activated CD4+ T cells with the CD40 master switch molecule on DCs was confirmed to be an essential DC activation interaction leading to critical upregulation of costimulatory molecules, including CD80 / CD86 and CD70, on DCs, for the supply of appropriate signaling to CD8+ T cells for effector CD8+ CTL induction and CD8+ memory introduction. CD8+ T cells assisted in this manner differed from CD8+ T cells not assisted by CD4+ T cells in the expression of over 800 genes (Borst et al. 2018 Nature Rev. Immunol. 18:635 (Non-patent Literature 15)). This demonstrates the great importance of such assistance.

[0005] Immature dendritic cells (iDCs) circulate in the blood and are present in many tissues, including the skin. When these iDCs pick up antigens and upregulate CC chemokine receptor 7 (CCR7), they travel to inflow area lymph nodes (dLNs) where they can activate T cells. CCR7 mediates not only the movement of common DCs (cDCs) but also the transport of plasmacytoid DCs (pDCs) to dLNs (W. Hong et al. 2022 Frontiers in Pharmacol. 13:841687 (Non-patent Literature 16)). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2008118017 [Non-patent literature]

[0007] [Non-Patent Document 1] Ikeda et al. 1997 Immunity 6:199 [Non-Patent Document 2] Kessler et al. 2001 J. Exp. Med. 193:73 [Non-Patent Document 3] Griffioen et al. 2006 Clin Cancer Res 12(10): 3130 [Non-Patent Document 4] Quintarelli et al. 2011 Blood 117(12):3353 [Non-Patent Document 5] Stanojevic et al. 2021 Cytotherapy 23(8):694 [Non-Patent Document 6] Weber et al. 2011(J Immunther 34(7):556) [Non-Patent Document 7] Holmberg-Thyden et al. 2022 Cancer Immunol Immunother. 71(2):433 [Non-Patent Document 8] Pujol et al. 2016 J. Thoracic Oncol 11(12):2208 [Non-Patent Document 9] Gutzmer et al. 2016 ESMO Open 1(4):e000068 [Non-Patent Document 10] Melssen et al. 2022 J Immunother. Cancer 10(9):e004709 [Non-Patent Document 11] Ridge et al. 1998 Nature 393:4747 [Non-Patent Document 12] Bennett et al. 2998 Nature 393:478 [Non-Patent Document 13] Schoenberger et al. 1998 Nature 393:480 [Non-Patent Document 14] Lanzavecchia 1998 Nature 393:413 [Non-Patent Document 15] Borst et al. 2018 Nature Rev. Immunol. 18:635 [Non-Patent Document 16] W. Hong et al. 2022 Frontiers in Pharmacol. 13:841687 [Overview of the Initiative]

[0008] Surprisingly, it has now been discovered that certain PRAME-based SLPs have the ability to increase CCR7 and / or CD40 levels in antigen-presenting cells, making them interesting candidates for use in immunotherapy.

[0009] Accordingly, in a first aspect, the present invention provides a method for treating or preventing cancer, comprising the step of administering a plurality of immunogenic peptides to a human subject, wherein the plurality of immunogenic peptides is (a) a peptide 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 1, and (b) a peptide 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 2 The present invention relates to a method comprising the above.

[0010] Similarly, the present invention provides (a) a peptide 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 1, and (b) a peptide 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 2 The present invention relates to a plurality of immunogenic peptides for use in the treatment or prevention of cancer, comprising the above.

[0011] In a further aspect, the present invention provides an immunogenic composition comprising a plurality of peptides, wherein the plurality of peptides is (a) a peptide 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 1, and (b) a peptide 33 to 40 amino acids in length comprising or consisting of the sequence set forth in SEQ ID NO: 2 The present invention relates to an immunogenic composition comprising the above.

[0012] In a further aspect, the present invention provides (i) determining the level of CCR7 and / or CD40 when incubated with a candidate antigen in an antigen-presenting cell, such as a dendritic cell or a CD14+CD11c+ culture monocyte-derived cell, and (ii) selecting a candidate antigen capable of increasing the level of CCR7 and / or CD40 in said antigen-presenting cell The present invention relates to a method for selecting an antigen suitable for use in immunization, comprising the above steps, The antigen is preferably a peptide. Regarding the method.

[0013] In a further aspect, the present invention relates to a method for treating or preventing cancer, comprising the step of administering to a human subject one or more polynucleotides encoding an immunogenic peptide as defined herein.

[0014] In a further aspect, the present invention relates to one or more polynucleotides encoding immunogenic peptides as defined herein for use in the treatment or prevention of cancer.

[0015] In a further aspect, the present invention relates to an immunogenic composition comprising one or more polynucleotides encoding an immunogenic peptide as defined herein. [Brief explanation of the drawing]

[0016] [Figure 1] Part of the peptide structure containing the 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 strategies for flow cytometry data of CD14+CD11c+ cells. (A) DC gates were 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+CD11c+ cells were gated with intact single cells. These cells are depicted in the upper right quadrant. [Figure 3] CCR7 expression on CD14+CD11c+ cells. CCR7 expression is depicted as a change in the geometric mean fluorescence of CCR7 on cells loaded with the indicated SLP divided by the geometric mean fluorescence of cells loaded with an unrelated SLP. Cells were gated as shown in Figure 2. *Cysteine-containing SLP, **Cystine-containing SLP. [Figure 4]CD40 expression on CD14+CD11c+ cells. CD40 expression is depicted as a change in the geometric mean fluorescence of CD40 on cells loaded with the indicated SLP divided by the geometric mean fluorescence of cells loaded with an unrelated SLP. Cells were gated as shown in Figure 2. *Cysteine-containing SLP, **Cystine-containing SLP. [Modes for carrying out the invention]

[0017] Detailed description of the invention definition As used herein, the term "PRAME" refers to the human PRAME protein (UniProt A0A024R1E6).

[0018] The term “immunogenic peptide” means a peptide that can induce or boost an immune response, such as a local and / or systemic CD4+ and / or CD8+ T cell response and / or antibody response. The immunogenic peptides used in the present invention may be unconjugated or unmodified, i.e., simple chains of amino acids linked by peptide bonds, or they may be further modified, such as by covalent bonding to another molecule (e.g., an adjuvant). Similarly, the term “immunogenic composition” means a composition that can induce or boost an immune response, such as a local and / or systemic CD4+ and / or CD8+ T cell response and / or antibody response. The immunogenic peptides described herein, also referred to herein as long-chain peptides, exceed the length of human leukocyte antigen (HLA) class I and class II displaying ligands. Preferably, the long-chain peptides of the present invention are synthetic peptides, referred herein as synthetic long-chain peptides (SLPs).

[0019] In the context of this invention, "peptide of XX-YY amino acid length" means that the number of amino acid residues is XX-YY. For example, a peptide of 33-40 amino acid length means 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues. The peptides used in this invention exceed the length of human leukocyte antigen (HLA) class I and class II presenting epitope peptide sequences. Preferably, the peptides used in this invention are synthetic peptides, also referred to herein as synthetic long-chain peptides (SLPs).

[0020] In the context of this invention, the term “PRAME fragment” means an amino acid sequence that corresponds to, i.e., is identical to, a partial sequence of the PRAME protein. Therefore, it refers to a continuous sequence of the native PRAME protein without any insertions, deletions, or substitutions. When a peptide is identified as containing a PRAME protein fragment of a certain length, it means that the fragment is neither shorter nor longer. For example, if a PRAME fragment is identified as being 33–40 amino acids long, this means that the fragment is neither less than 33 amino acids long nor more than 40 amino acids long. Therefore, such a peptide does not contain, for example, a continuous sequence of PRAME longer than 41 amino acids. However, to avoid ambiguity, “contains” has its usual meaning in the art, i.e., a peptide containing a PRAME fragment may contain additional sequences beyond the identified fragment, such as sequences not derived from PRAME or other partial sequences of PRAME that are not contiguous with the aforementioned fragment.

[0021] The term "antigen-presenting cell" refers to a cell capable of presenting an antigen or fragment thereof to T cells. The term "professional antigen-presenting cell" is used herein to refer to a cell in an intact mammalian organism that processes an antigen or fragment thereof and presents it to T cells, thereby initiating and amplifying the T cell response. Dendritic cells are the primary professional antigen-presenting cells in an intact mammalian organism. Dendritic cells may be derived from isolated CD14+ monocyte culture differentiation. Fully differentiated monocyte-derived dendritic cells no longer express CD14. As used herein, the term CD14+CD11c+ cultured monocyte-derived cell refers to a dendritic cell that is monocyte-derived but not fully differentiated.

[0022] "Treatment" or "to treat" refers to the administration of an effective dose of an immunogenic peptide or immunogenic composition for the purpose of alleviating, improving, preventing, or eradicating (curing) a symptom, disorder, or disease condition. "Prevention" or "to prevent" refers to the administration of an effective dose of an immunogenic peptide or immunogenic composition for the purpose of preventing a symptom, disorder, or disease condition. "Effective dose" refers to an effective dose in the amount and duration required to achieve the desired therapeutic outcome.

[0023] The term "multiple" refers to more than one; for example, "multiple immunogenic peptides" refers to more than one, i.e., two or more immunogenic peptides.

[0024] The indefinite article "a" or "an" refers to an element, and unless the context clearly indicates that there is one element and only one, it does not rule out the possibility of more than one element existing. Therefore, the indefinite article "a" or "an" usually means "at least one."

[0025] Further aspects and embodiments of the present invention As described above, in the first phase, the present invention provides a method for treating or preventing cancer, comprising the step of administering a plurality of immunogenic peptides to a human subject, wherein the plurality of immunogenic peptides are (a) A peptide of 33-40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:1, and (b) A peptide of 33-40 amino acids in length containing or consisting of the sequence shown in SEQ ID NO:2 This includes methods.

[0026] Similarly, the present invention is (a) A peptide of 33-40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:1, and (b) A peptide of 33-40 amino acids in length containing or consisting of the sequence shown in SEQ ID NO:2 This relates to multiple immunogenic peptides, including, for use in the treatment or prevention of cancer.

[0027] The methods and uses of the present invention involve administering an immunogenic peptide containing or comprising a sequence corresponding to a fragment of the human protein PRAME. The immunogenic peptide contains or comprises a contiguous sequence of PRAME having a specific, specified minimum and maximum length.

[0028] In one embodiment, the immunogenic peptide for use in the present invention consists of a specified contiguous sequence of PRAME, i.e., a fragment of PRAME. In another embodiment, the immunogenic peptide for use in the present invention comprises a specified contiguous sequence of PRAME and further comprises an additional flanking region corresponding to a flanking region present in PRAME up to the maximum length of the peptide. In yet another embodiment, the immunogenic peptide for use in the present invention comprises a specified contiguous sequence of PRAME and an additional flanking region up to the maximum length of the peptide, where the flanking region does not correspond to a flanking region in PRAME and / or does not correspond to the PRAME sequence at all. Thus, the immunogenic peptide used in the present invention may or may not contain a non-natural sequence as a result of containing additional amino acids (N-terminus or C-terminus of the PRAME fragment) that are not of PRAME origin. As further described herein, the cysteine ​​in the immunogenic peptide of the present invention may or may not be cysteinylated, i.e., it may or may not exist in the form of cystine. Furthermore, the immunogenic peptide used in the present invention may or may not be conjugated to a non-amino acid portion.

[0029] Preferably, the immunogenic peptides used in the present invention are isolated peptides, where “isolated” means that the peptide has been removed from its natural environment (i.e., subjected to artificial manipulation), and not reflecting the degree to which the peptide has been purified. The peptides may be, for example, recombinantly produced peptides or synthetically produced peptides. Peptides are typically produced synthetically. This may be done by solid-phase peptide synthesis or by any other suitable method.

[0030] The method or use of the present invention may include the administration of more than two immunogenic peptides. That is, the multiple immunogenic peptides may include two or more peptides, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more immunogenic peptides. Typically, each of the multiple immunogenic peptides contains or consists of a sequential fragment of PRAME.

[0031] In one embodiment, the immunogenic peptides used in the present invention are: (a) A peptide consisting of the sequence shown in SEQ ID NO:1, and (b) Peptide consisting of the sequence shown in SEQ ID NO:2 Includes.

[0032] In some embodiments, one or more cysteine ​​residues in an immunogenic peptide may exist in the form of cystine residues (L-cystine or D-cystine). Thus, in such embodiments, one or more cysteine ​​residues in an immunogenic peptide exist in a form in which the thiol group of cysteine ​​in the peptide is oxidized (cysteinylated) and forms a disulfide bond with a cysteine ​​molecule (the latter not being part of the immunogenic peptide). An example of such a cysteine-modified peptide is shown in Figure 1. Cysteine ​​modification prevents the formation of intermolecular and / or intramolecular disulfide bonds, resulting in a more stable peptide-based immunogenic composition.

[0033] In one embodiment, position 12 in the immunogenic peptide shown in SEQ ID NO:2 is cystine. In another embodiment, position 12 in the immunogenic peptide shown in SEQ ID NO:2 is cysteine.

[0034] As described above, the multiple immunogenic peptides may include two or more immunogenic peptides. In one embodiment, the multiple may include the following immunogenic peptides: (a) A peptide of 25-40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:3, (b) A peptide of 35-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:4, and (c) A peptide of 32-40 amino acids in length containing or consisting of the sequence shown in SEQ ID NO:5 This further includes one, two, or all three of them.

[0035] In one embodiment, position 5 in the immunogenic peptide shown in SEQ ID NO:3 is cystine. In another embodiment, position 5 in the immunogenic peptide shown in SEQ ID NO:3 is cysteine.

[0036] In one embodiment, position 33 in the immunogenic peptide shown in SEQ ID NO:4 is cystine. In another embodiment, position 33 in the immunogenic peptide shown in SEQ ID NO:4 is cysteine.

[0037] In one embodiment, multiple peptides are immunogenic peptides, including the following: (a) A peptide of 35-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:6, (b) A peptide of 32-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:7, and (c) A peptide of 35-40 amino acids in length containing or consisting of the sequence shown in SEQ ID NO:8. This further includes one, two, or all three of them.

[0038] In one embodiment, position 7 in the immunogenic peptide shown in SEQ ID NO:8 is cystine. In another embodiment, position 7 in the immunogenic peptide shown in SEQ ID NO:8 is cysteine.

[0039] In one embodiment, the plurality of immunogenic peptides used in the present invention comprises or consists of eight peptides represented by SEQ ID NO:1 to SEQ ID NO:8.

[0040] In one embodiment, the plurality of immunogenic peptides used in the present invention comprises or consists of eight peptides indicated by SEQ ID NO:1 to SEQ ID NO:8, where position 12 in the immunogenic peptide indicated by SEQ ID NO:2 is cystine, position 5 in the immunogenic peptide indicated by SEQ ID NO:3 is cystine, position 33 in the immunogenic peptide indicated by SEQ ID NO:4 is cystine, and position 7 in the immunogenic peptide indicated by SEQ ID NO:8 is cystine.

[0041] In one embodiment, the plurality of immunogenic peptides used in the present invention comprises or consists of eight peptides shown as SEQ ID NO:1 to SEQ ID NO:8, where position 12 in the immunogenic peptide shown as SEQ ID NO:2 is cysteine, position 5 in the immunogenic peptide shown as SEQ ID NO:3 is cysteine, position 33 in the immunogenic peptide shown as SEQ ID NO:4 is cysteine, and position 7 in the immunogenic peptide shown as SEQ ID NO:8 is cysteine.

[0042] In some embodiments, all of the multiple immunogenic peptides to be administered are contained within a single immunogenic composition. In other embodiments, the immunogenic peptides of the present invention are distributed into two or more compositions, for example, into 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 key aspect, the present invention relates to a vaccine (i.e., a vaccine product) comprising two or more compositions containing together multiple immunogenic peptides as defined herein.

[0043] In one embodiment, the method or use of the present invention does not involve the administration of a peptide (WO2008118017) comprising one or more sequences from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0044] In one embodiment, the method or use of the present invention does not involve the administration of a peptide comprising any of the sequences from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0045] In one embodiment, the method or use of the present invention does not involve the administration of peptides containing or consisting of any one of the sequences shown in Table 2, Table 3A or Table 3B, Table 4, or Table 6 of WO2008118017, other than peptides containing or consisting of the sequences shown in SEQ ID NO: 1 to 8 of this disclosure.

[0046] In one embodiment, the method or use of the present invention involves the administration of a peptide containing or comprising the sequences shown in SEQ ID NO: 1-8, but does not involve the administration of a further peptide containing or comprising any one of the sequences shown in Table 2, Table 3A or Table 3B, Table 4, or Table 6 of WO2008118017.

[0047] The methods or uses of the present invention are typically for the treatment or prevention of PRAME-expressing cancers.

[0048] In one embodiment, cancer is selected from the group consisting of neuroblastoma, lymphoma, papilloma, breast or cervical cancer, acute and chronic leukemia, medulloblastoma, non-small cell lung cancer, head and neck cancer, kidney cancer, pancreatic cancer, prostate cancer, small cell lung cancer, multiple myeloma, melanoma, uveal melanoma, sarcoma, and hematological malignancies such as chronic myeloid leukemia and acute myeloid leukemia.

[0049] In some embodiments, the methods or uses of the present invention further include the administration of an adjuvant. The term “adjuvant” is used herein to mean a substance that has an immunoenhancing effect and is co-administered with, added to, or co-formulated with an antigenic substance in order to enhance, induce, evoke, and / or modulate an immunological response to an antigenic substance when administered to a subject. In one embodiment, the adjuvant is physically linked to the peptide to be reconstituted, for example, by a covalent bond.

[0050] In one embodiment, the adjuvant is an emulsifying adjuvant. In one embodiment, the adjuvant is an oily adjuvant. Oily adjuvants can be used to form emulsions (e.g., water-in-oil or oil-in-water emulsions) and are recognized in the art to enhance and induce immune responses. Preferably, the oily adjuvant is a mineral oil-based adjuvant.Non-exclusive examples of oily adjuvants include bio-based oil adjuvants (based on vegetable oils / fish oils, etc.), squalene-based adjuvants (e.g., MF59), Syntex adjuvant formulations (SAF; Vaccine Adjuvants, Volume 42 of the series Methods in Molecular Medicine (trademark) pp. 229-237, ISSN 1543-1894, Lidgate, Deborah M, Preparation of the Syntex Adjuvant Formulation (SAF, SAF-m, and SAF-1)), Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), peanut oil-based adjuvants (e.g., Adjuvant 65), and Lipovant (Zola, H. (Ed.), Laboratory Methods in Immunology. pp. 39-51, Byars, NE, Allison, AC, 1990. Immunologic adjuvants: These include general properties, advantages, and limitations, ASO4 (A. Tagliabue, R. Rappuoli Vaccine adjuvants: the dream becomes real Hum. Vaccine, 4 (5), 2008, pp. 347-349), and Montanide adjuvants based on squalene emulsified with purified squalene and highly purified monooleic acid mannide (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 oily adjuvant is Montanide ISA 51VG(Seppic), which is a mixture of Drakeol VR and mannide monooleate.

[0051] Other suitable adjuvants are those known to act via Toll-like receptors and / or via RIG-I (Retinoic acid-Inducible Gene-1) protein and / or via endothelin receptors. Immunomodulatory compounds capable of activating the innate immune system can be particularly well activated via TLRs, including Toll-like receptors (TLRs) 1-10. Compounds capable of activating TLR receptors, as well as their variants and derivatives, have been well studied in the art. TLR1 may be activated by bacterial lipoproteins and their acetylated derivatives, and TLR2 may be further activated by Gram-positive bacterial glycolipids, LPS, LPA, LTA, pili, outer membrane proteins, bacterial or host-derived heat shock proteins, and mycobacteria lipoarabinomannan. TLR3 may be activated in particular by viral dsRNA or the compound poly(I:C). TLR4 may be activated by Gram-negative LPS, LTA, heat shock proteins of host or bacterial origin, viral coat or envelope proteins, taxol or its derivatives, hyaluronic acid-containing oligosaccharides, and fibronectin. TLR5 may be activated by bacterial flagella or flagellin. TLR6 may be activated by mycobacterial lipoproteins and heat-unstable soluble factor (GBS-F) of Group B Streptococcus or modulin of Staphylococcus. TLR7 may be activated by imidazoquinolines such as imiquimod, reximod, and derivatives such as imiquimod or reximod (e.g., 3M-052). TLR9 may be activated by unmethylated CpG DNA or chromatin-IgG complexes.Particularly preferred adjuvants include, but are not limited to, synthetically produced compounds containing dsRNA, poly(I:C), polyICLC, unmethylated CpG DNA that induces TLR3 and TLR9 receptors, the TLR9 agonist IC31, the TLR4 agonist IMSAVAC, and water-in-oil emulsions containing mineral oil and surfactants derived from the mannide monooleate family (e.g., Montanide ISA-51 and Montanide ISA 720, adjuvants produced by Seppic, France). RIG-I proteins are known to be activated by ds-RNA, similar to TLR3 (Kato et al, (2005) Immunity, 1: 19-28).

[0052] Particularly preferred TLR ligands are Pam3cys and / or its derivatives, preferably Pam3cys lipopeptides or their variants or derivatives, preferably those described in WO2013051936A1 (incorporated herein by reference), and more preferably U-Pam12 or U-Pam14, also known as AMPLIVANT®. TIFF2026530444000001.tif78128

[0053] Pam3cys and / or its derivatives may optionally be covalently linked to the peptide antigen.

[0054] In another embodiment, the method or use of the present invention comprises administration of a TLR2 agonist of the U-Pam-14 variant (as described above), wherein the U-Pam-14 compound is chiral pure and consists solely of a compound containing the Cys((R)-2,3-di(palmitoyloxy)-propyl) moiety (R-diastereoisomer).

[0055] In another embodiment, the method or use of the present invention comprises the administration of a TLR2 agonist selected from the group consisting of: Pam3CysSer, Pam3CysSerLys, Pam3CysSer(Lys)4 (also known as Pam3CSK4), Pam2CysSer(Lys)4 (also known as Pam2CSK4), and Pam1CysSer(Lys)4 (also known as Pam1CSK4).

[0056] Further preferred adjuvants are cyclic dinucleotides (CDNs), muramyl dipeptides (MDPs), and poly-ICLCs. In a preferred embodiment, the adjuvant of the present invention includes, for example, a non-natural adjuvant, such as a Pam3cys lipopeptide derivative as described in WO2013051936A1, an imidazoquinoline such as Poly-ICLC, an imidazoquinoline such as imiquimod, a reximod or its derivative, a CpG oligodeoxynucleotide (CpG-ODN), such as a class A-ODN (or K-type), a class B-ODN (or D-type), a class C-ODN as described in Sheiermann and Klinman, 2014 Vaccine 32(48): 6377-6389, more preferably a class B-ODN (such as CpG7909 or 1018ISS) or a class C-ODN (such as DV-281) having a non-natural sequence, and a peptide-based adjuvant, such as a muramyl dipeptide (MDP) or a tetanus toxoid peptide containing a non-natural amino acid.

[0057] More preferably, the adjuvant is selected from the group consisting of: aluminum salt, 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, β-glucan, Aquila QS21 stimulon, vadimezan, AsA404 (DMXAA), STING (IFN gene stimulant) agonists (e.g., c-di-GMP VacciGrade®), PCI, NKT (natural killer T cell) agonists (e.g., α-galactosylceramide or α-GalCer), RNAdjuvant® (Curevac), retinoic acid-inducible protein I ligands (e.g., 3pRNA or 5'-triphosphate RNA).

[0058] In preferred embodiments of the method or use of the present invention, the adjuvant is AMPLIVANT® or Montanide ISA-51.

[0059] The adjuvant may be mixed with the immunogenic peptide before administration to the patient, or it may be administered separately.

[0060] In a further aspect, the present invention relates to an immunogenic composition comprising a plurality of peptides, wherein the plurality of peptides are (a) A peptide of 33-40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:1, and (b) A peptide of 33-40 amino acids in length containing or consisting of the sequence shown in SEQ ID NO:2 This relates to immunogenic compositions, including [specific components].

[0061] In one embodiment, a plurality of immunogenic peptides in an immunogenic composition are (a) A peptide consisting of the sequence shown in SEQ ID NO:1, and (b) Peptide consisting of the sequence shown in SEQ ID NO:2 Includes.

[0062] In one embodiment, position 12 in the immunogenic peptide indicated by SEQ ID NO:2 is cystine. In another embodiment, position 12 in the immunogenic peptide indicated by SEQ ID NO:2 is cysteine.

[0063] The immunogenic composition may contain multiple immunogenic peptides, including two or more immunogenic peptides. In one embodiment, the immunogenic composition contains the following immunogenic peptides: (a) A peptide of 25-40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:3, (b) A peptide of 35-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:4, and (c) A peptide of 32-40 amino acids in length containing or consisting of the sequence shown in SEQ ID NO:5 This further includes one, two, or all three of them.

[0064] In one embodiment, position 5 in the immunogenic peptide indicated by SEQ ID NO:3 is cystine. In another embodiment, position 5 in the immunogenic peptide indicated by SEQ ID NO:3 is cysteine.

[0065] In one embodiment, position 33 in the immunogenic peptide indicated by SEQ ID NO:4 is cystine. In another embodiment, position 33 in the immunogenic peptide indicated by SEQ ID NO:4 is cysteine.

[0066] In one embodiment, the immunogenic composition comprising the following immunogenic peptides: (a) A peptide of 35-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:6, (b) A peptide of 32-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:7, and (c) A peptide of 35-40 amino acids in length containing or consisting of the sequence shown in SEQ ID NO:8. This further includes one, two, or all three of them.

[0067] In one embodiment, position 7 in the immunogenic peptide indicated by SEQ ID NO:8 is cystine. In another embodiment, position 7 in the immunogenic peptide indicated by SEQ ID NO:8 is cysteine.

[0068] In one embodiment, the immunogenic composition comprises or consists of eight peptides represented by SEQ ID NO:1 to SEQ ID NO:8.

[0069] In one embodiment, the immunogenic composition comprises or consists of eight peptides represented by SEQ ID NO:1 to SEQ ID NO:8, where position 12 in the immunogenic peptide represented by SEQ ID NO:2 is cystine, position 5 in the immunogenic peptide represented by SEQ ID NO:3 is cystine, position 33 in the immunogenic peptide represented by SEQ ID NO:4 is cystine, and position 7 in the immunogenic peptide represented by SEQ ID NO:8 is cystine.

[0070] In one embodiment, the immunogenic composition comprises or consists of eight peptides represented by SEQ ID NO:1 to SEQ ID NO:8, where position 12 in the immunogenic peptide represented by SEQ ID NO:2 is cysteine, position 5 in the immunogenic peptide represented by SEQ ID NO:3 is cysteine, position 33 in the immunogenic peptide represented by SEQ ID NO:4 is cysteine, and position 7 in the immunogenic peptide represented by SEQ ID NO:8 is cysteine.

[0071] The immunogenic compositions according to the present invention may contain pharmaceutically acceptable carriers, which are well known in the art. The immunogenic compositions of the present invention are preferably for administration to human subjects and are therefore formulated to be suitable for administration to human subjects. Preferably, administration is parenteral, such as intravenous, subcutaneous, intramuscular, dermal, intradermal, and / or intratumoral, i.e., by injection.

[0072] The immunogenic composition is preferably chemically stable, i.e., the peptides in the composition do not chemically degrade or decompose. Therefore, preferably, after storage of the solution or liquid composition at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the amount of undegraded, undegraded, and / or unreacted peptides in the solution and / or composition is at least 90% by weight, 91% by weight, 92% by weight, 93% by weight, 94% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight, or even 100% by weight, compared to the original amount. Chemical stability can be evaluated using any suitable technique known in the art, for example, by ULC / MS as illustrated herein. When using UPLC / MS, a solution / composition is defined as chemically stable if, in the UV spectrum after storage at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the total area % of peaks that do not represent the desired peptide product is at most 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% compared to the original.

[0073] The immunogenic composition is preferably also physically stable, i.e., the peptides in the composition do not precipitate or redisperse. Physical stability can be evaluated using any suitable technique known in the art, for example, by visual inspection or by particle distribution using a Malvern Mastersizer as exemplified herein, where the average particle size is denoted as D(0.5). When using a Malvern Mastersizer to evaluate physical stability as exemplified herein, the solution / composition is defined as physically stable if the average D(0.5) after storage at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours is at most 50%, 40%, 30%, 20%, 10%, or 5% compared to its original (i.e., a freshly prepared, immediately prepared solution). Preferably, a solution / composition is defined as physically stable if its average D(0.5) after 3 hours of storage at room temperature is an increase of at most 50%, 40%, 30%, 20%, 10%, or 5%, preferably at most 20%, compared to its original value.

[0074] In one embodiment, the immunogenic composition comprises or consists of a mixture of dried or lyophilized peptides administered together.

[0075] In one embodiment, the immunogenic composition does not contain peptides comprising one or more sequences from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0076] In one embodiment, the immunogenic composition does not contain any peptides comprising any of the sequences from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 among SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0077] As described herein, it has been surprisingly discovered that certain antigens have the ability to increase CCR7 and / or CD40 levels in antigen-presenting cells, making them interesting candidates for use in immunotherapy.

[0078] Therefore, in further aspects, the present invention is (i) A step of determining the CCR7 and / or CD40 levels when incubated with a candidate antigen in professional antigen-presenting cells, such as dendritic cells or CD14+CD11c+ cultured monocyte-derived cells, and (ii) The step of selecting candidate antigens that can increase CCR7 and / or CD40 levels in the antigen-presenting cells. Methods, such as in vitro methods, for selecting antigens suitable for use in immunization, including, The antigen is preferably a peptide. Regarding the method.

[0079] In one embodiment, the method is for selecting an antigen, such as a peptide antigen, that is suitable for use in immunization against PRAME-expressing cancer.

[0080] In a further aspect, the present invention relates to a method for treating or preventing cancer, comprising the step of administering to a human subject one or more polynucleotides encoding an immunogenic peptide as defined herein.

[0081] In a further aspect, the present invention relates to an immunogenic composition comprising one or more plurality of polynucleotides encoding an immunogenic peptide as defined herein.

[0082] The polynucleotide may be any polynucleotide, including, for example, RNA, DNA, and / or cDNA, and may also include nucleotide analogs and / or nucleotide equivalents, such as peptide nucleic acids or morpholino nucleotide analogs. The polynucleotide may be codon-optimized for a selected host to promote the expression of the encoded peptide or polypeptide.

[0083] The polynucleotides used in this aspect of the present invention do not encode full-length PRAME, but rather encode immunogenic peptides, as described herein, either by themselves or adjacent to amino acid sequences not contiguous with PRAME. Therefore, in the polynucleotides of the present invention, the sequence encoding the immunogenic peptide may be part of a larger open reading frame that also contains flanking amino acids, however such flanking amino acids are not contiguous with the immunogenic peptide sequence in PRAME. Such flanking amino acids may originate from proteins other than PRAME, and / or from other locations within the PRAME protein where they are not contiguous with adjacent peptides.

[0084] In one embodiment, a polynucleotide encodes two or more immunogenic peptides as defined herein, arranged as “beads-on-a-string,” thereby linking the peptides according to the present invention (“beads”) directly together and / or via linker sequences derived from proteins other than PRAME and / or from other locations within PRAME that are not contiguous with adjacent peptides. The amino acid sequences adjacent to or linking with the peptides may contain proteolytic cleavage sites.

[0085] The polynucleotides according to the present invention may be applied to deliver the peptides according to the present invention in various ways. The polynucleotides according to the present invention may be used, for example, to produce recombinant proteins or peptides in suitable host cells (e.g., bacterial host cells such as Escherichia coli (E. coli), suitable yeast host cells such as S. cerevisiae, suitable filamentous fungi such as Aspergillus, or mammalian host cells), from which the recombinant proteins or peptides may be purified. Alternatively, the polynucleotides may be functionally linked to an expression regulatory sequence (such as a promoter) and incorporated into an expression construct for human cells. Such (autologous) cells may be transfected or transduced ex vivo for (re)administration to a target in need. Alternatively, such expression constructs according to the present invention may be incorporated into a suitable gene therapy vector. Suitable viral expression constructs include, for example, vectors based on adenovirus, adeno-associated virus (AAV), retrovirus, or modified vaccinia ankara (MVA). The polynucleotide according to the present invention may be functionally ligated to a sequence encoding an adjuvant such as a Toll-like receptor (TLR) ligand, a NOD ligand, or a RIG-I ligand.

[0086] Sequence List TIFF2026530444000002.tif166150 [Examples]

[0087] Example 1. Synthesis of SLP All reagents and solvents for solid-phase peptide synthesis were purchased from Merck, Sigma Aldrich, Actu-All, Bachem and Biosolve, and GL Biochem, and used as received.

[0088] Solid-phase peptide synthesis (SPPS) Peptide synthesis is performed according to established methods using a Tetras peptide synthesizer (Advanced ChemTech) with solid-phase Fmoc / t The reaction was carried out by the Bu chemical reaction. Generally, peptide synthesis was performed using pre-loaded Wang-, HMPB ChemMatrix®, 2-chlorotrityl, or 4-(1',1'-dimethyl-1'-hydroxypropyl)phenoxyacetyl-alanyl-aminomethyl resin. The reaction was typically carried out on a 30–60 mmol scale. Peptides were synthesized by single, double, or triple coupling cycles, or combinations of single, double, and triple coupling cycles.

[0089] Initial swelling procedure (1) Resin swelling: 2 cycles with NMP (2) NMP cleaning (3) iPrOH cleaning

[0090] The single coupling cycle was performed using the following sequence of steps: (1) Deprotection of the Fmoc group: 3 cycles with piperidine in N-methyl-2-pyrrolidone (NMP). (2) NMP cleaning (3) iPrOH cleaning (4) Appropriate amino acid coupling. Fmoc-amino acids in NMP and coupling reagents in NMP (3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one (DEPBT), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium tetrafluoroborate (TBTU), benzotriazol-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), 7-aza-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate (PyAOP), ethylcyano(hydroxyimino)aceto-O2-tri-(1-pyrrolidinyl)-phosphonium Uronium hexafluorophosphate (PyOxim), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 1-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU), 2-(1-oxypyridine-2-yl)-1,1,3,3-tetramethylisothiouronium tetrafluoroborate (TOTT), O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N'-diisopropylcarbodiimide (DIC), or ethyl 2-cyano-2-(hydroxymino)acetate (Oxyma After adding Pure(registered trademark), the reaction mixture was shaken for 1 minute. Optionally, a base such as N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIPEA) was added. The reaction mixture was shaken for at least 30 minutes. (5) NMP cleaning (6) Capping with acetic anhydride or benzoyl chloride in the presence of a base (NMM or pyridine). (7) NMP cleaning (8) iPrOH cleaning

[0091] The double coupling cycle was performed using the following sequence of steps: (1) Deprotection of the Fmoc group: 3 cycles with piperidine in NMP. (2) NMP cleaning (3) iPrOH cleaning (4) First coupling cycle: After adding the Fmoc-amino acid in NMP to the coupling reagent in NMP (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim HATU, COMU, TOTT, HCTU, DIC, or Oxyma Pure®), the reaction mixture was shaken for 1 minute. A base (NMM or DIPEA) was optionally added. The reaction mixture was shaken for at least 15 minutes. (5) Purge the reaction vessel. (6) Second coupling cycle: After adding the Fmoc-amino acid in NMP to the coupling reagent in NMP (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim, HATU, COMU, TOTT, HCTU, DIC, or Oxyma Pure®), the reaction mixture was shaken for 1 minute. A base (NMM or DIPEA) was optionally added. The reaction mixture was shaken for at least 15 minutes. (7) NMP cleaning (8) Capping with acetic anhydride or benzoyl chloride in the presence of a base (NMM or pyridine). (9) NMP cleaning (10) iPrOH cleaning

[0092] The triple coupling cycle was performed using the following sequence of steps: (1) Deprotection of the Fmoc group: 3 cycles with piperidine in NMP. (2) NMP cleaning (3) iPrOH cleaning (4) First coupling cycle: After adding the Fmoc-amino acid in NMP to the coupling reagent in NMP (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim, HATU, COMU, TOTT, HCTU, DIC, or Oxyma Pure®), the reaction mixture was shaken for 1 minute. A base (NMM or DIPEA) was optionally added. The reaction mixture was shaken for at least 15 minutes. (5) Purge the reaction vessel. (6) Second coupling cycle: After adding the Fmoc-amino acid in NMP to the coupling reagent in NMP (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim, HATU, COMU, TOTT, HCTU, DIC, or Oxyma Pure®), the reaction mixture was shaken for 1 minute. A base (NMM or DIPEA) was optionally added. The reaction mixture was shaken for at least 15 minutes. (7) Purge the reaction vessel. (8) Third coupling cycle: After adding the Fmoc amino acid in NMP to the coupling reagent in NMP (DEPBT, HBTU, TBTU, PyBOP, PyAOP, PyOxim, HATU, COMU, TOTT, HCTU, DIC, or Oxyma Pure®), the reaction mixture was shaken for 1 minute. A base (NMM or DIPEA) was optionally added. The reaction mixture was shaken for at least 15 minutes. (9) NMP cleaning (10) Capping with acetic anhydride or benzoyl chloride in the presence of a base (NMM or pyridine). (11) NMP cleaning (12) iPrOH cleaning

[0093] After the final coupling cycle, the following final Fmoc-deprotection and washing steps were performed: (1) Deprotection of the Fmoc group: 3 cycles with piperidine in NMP. (2) NMP cleaning (3) iPrOH cleaning

[0094] Cutting and purification procedure involving cysteine ​​modification during cutting After the final washing step, the resin was dried and cooled. A cleavage cocktail based on Milli-Q water, thioanisole, and TFA was added to the resin, followed immediately by 50 equivalents of 2,2'-dithiobis(5-nitropyridine) (DTNP) per cysteine ​​(Trt) residue present in the peptide. The mixture was allowed to stand 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, which was resuspended by vortexing. After centrifugation, the pellet was isolated and dried under vacuum.

[0095] 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 SPPS synthesis on a 60 μmol scale) was added to the supernatant. After 1 hour, the reaction mixture was diluted with Milli-Q water and filtered. This peptide was purified under acidic conditions (ACN, water, and TFA) using a Waters AutoPurification HPLC / MS system, and then lyophilized overnight to obtain the cystine-containing peptide as a white to off-white powder.

[0096] Cutting and purification procedure using cysteine-modified offresin After the final washing step 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 3 hours. Then, cold diethyl ether was added and the mixture was centrifuged. The supernatant was removed and the pellet was isolated. The following steps were the same as in the "Cleavage and Purification Procedure with Cysteine ​​Modification During Cleavage" described above. 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 and centrifuged, and the obtained pellet was washed twice. 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 and purified by an HPLC / MS system under acidic conditions, followed by lyophilization overnight to obtain the cystine-containing peptide as a white to off-white powder.

[0097] Peptide analysis The identity and purity of the purified peptides were determined by UPLC-UV-MS using a Waters Acquity UPLC / TQD system with a C18 Waters Acquity BEH130 analytical column (particle size 1.7 μm, 2.1 x 150 mm, flow 0.4 mL / min) and a linear gradient (5%B to 95%B, linear gradient over 10 minutes). 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 That decision confirmed the identity of the synthesized peptide.

[0098] Example 2. T cell induction The ability of SLPs to activate T cells was studied. The biological activity of synthesized and purified SLPs was tested using PBMCs derived from healthy donors. Monocytes were isolated by magnetically activated cell sorting (MACS) using anti-CD14 beads, according to the supplier's (Miltenyi Biotec) protocol. Briefly, PBMCs were isolated by centrifugation on a Ficol gradient and cryopreserved. Approximately 50*10 thawed PBMCs were used for the isolation of CD14-positive cells to produce dendritic cells (DCs). Cells were cultured at 37°C for 3 days in 2 mL / well IMDM 4% human serum (HS) containing 800 U / ml GM-CSF and 500 U / ml IL-4 (Peprotech). Three days later, 1 mL / well of IMDM 4% HS containing GM-CSF (2400 U / mL) and IL-4 (1500 U / mL) was added to monocyte-derived dendritic cells (DCs), and these adherent cells were cultured for a further three days. On day six, the long-chain peptides, distributed into two pools, were added to naive donor monocyte-derived DCs at a concentration of 13 μM and incubated overnight at 37°C. The following day (day seven), the peptide-loaded DCs were harvested, irradiated (1000 rad), washed, and mixed with autologous PBMCs in IMDM 8% human serum in the presence of IL-7 (10 ng / mL) and IL-12p70 (100 pg / mL) in a 1:10 ratio. Since these conditions are suitable for T cell culture, they will hereafter be referred to as cultured T cells.

[0099] New dendritic cells (DCs) were generated on day 10, and six days later, on day 16, they were loaded with SLP as described above. The following day, these peptide-loaded DCs were harvested, irradiated (1000 rad), and washed. Cultured T cells were also harvested. Both were counted and mixed in a 1:10 ratio (DC:T) in 8% IMDM human serum in the presence of IL-7 (10 ng / mL) and IL-12p70 (100 pg / mL) for the first restimulation.

[0100] On the same day, new DCs were prepared as described above, and on day 23, they were loaded with SLP as described above. The following day, the peptide-loaded DCs were collected, irradiated (1000 rad), and washed. Cultured and restimulated T cells were also collected. Both were counted and mixed in a 1:10 ratio (DC:T) in 8% IMDM human serum in the presence of IL-7 (10 ng / mL) and IL-12p70 (100 pg / mL) for a second restimulation.

[0101] On day 24, we also started Experiment 1, which involved culturing individual SLP-loaded DCs with T cells recovered from restimulation 1. In Experiment 1, the DCs and recovered T cells were cultured in a 1:10 ratio for 2 days, after which the cells were transferred to coated ELISpot plates (see description below).

[0102] On day 24, new DCs were prepared as described above, and on day 30, they were loaded with SLP as described above. The following day, the peptide-loaded DCs were collected, irradiated (1000 rad), and washed. T cells that had been cultured and restimulated twice were also collected. Both were counted and mixed in a 1:10 ratio (DC:T) in 8% IMDM human serum in the presence of IL-2 (30 IU / mL) and IL-12p70 (100 pg / mL) for a third restimulation.

[0103] On day 31, we also started Experiment 2, in which we cultured individual SLP-loaded DCs and T cells recovered from restimulation 2. In Experiment 2, the DCs and recovered T cells were cultured in a 1:10 ratio for 2 days, and then the cells were transferred to coated ELISpot plates (see explanation below).

[0104] T cell cultures were restimulated three times in 1-week cycles using peptide-loaded auto-moDCs. After the second and third restimulations, responsiveness to a single SLP was tested (Test 1 and Test 2, respectively). Responsiveness was determined by measuring IFNγ production using ELISpot.

[0105] For ELISpot analysis, IFNγ-coated antibody was coated onto multiscreen plates overnight at 4°C. The following day, the plates were washed four times with PBS and blocked at 37°C for at least 1 hour using IMDM 8% HS. Each sample was tested in triplicates. As a positive control, phytoheamagglutinin (PHA, 1 μg / mL) was added to cells that had not been stimulated with SLP after thawing. The plates were incubated overnight at 37°C. The cells were then discarded, and the plates were washed with PBS / 0.05% Tween-20. Diluted IFNγ detection antibody was added to each well and incubated at room temperature for 2 hours. Next, the plates were washed with PBS / 0.05% Tween-20 and then incubated with streptavidin-ALP at room temperature for 1 hour. The plates were washed with PBS / 0.05% Tween-20. BCIP / NPT ALP substrate was filtered and added to each well at room temperature for 10-20 minutes.

[0106] SLPs having the sequences shown 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 cysteine-containing SLPs, cysteinylated SLPs were tested. Each SLP was tested minimally in seven donors. As controls, cultures were used with medium only, an albumin-derived peptide control, and Candida albicans antigen, with PHA used as a positive control on the ELISpot plate. When stimulated with two peptide pools, a positive IFNγ response was detected for 7 out of 10 peptides (Table 1). Next, T cell cultures were prepared in an additional 6 donors by stimulating them with a peptide pool consisting only of the 3 peptides that had not yet tested positive. When PBMCs were stimulated with only these remaining three peptides, SEQ ID NO:3 also showed a positive IFNγ response (Table 2). SLPs with the sequences shown in SEQ ID NO:9 and SEQ ID NO:14 did not show a conclusive response in any of the 13 donors tested.

[0107] In conclusion, eight of the ten SLPs tested showed positive results in these in vitro human T cell cultures, demonstrating their immunogenicity. The peptides may test positive after a second restimation (restim2 / test1), but will not test positive after a third restimation (restim3 / test2), likely because other specific T cells proliferate excessively in these cultures thereafter.

[0108] (Table 1) IFNγ ELISpot results for individual SLPs in Trial 1 and Trial 2. Results are expressed as - (negative) or + (positive). A response was defined as positive if the mean number of spots was higher than the mean number of spots for the unrelated SLP control + 2 * standard deviations. The last row represents a summary of the results in the table. If at least one donor had a positive response, it is denoted as POS. BCxxx = buffy coat code for healthy donors, nt = untested. TIFF2026530444000003.tif146148

[0109] (Table 2) IFNγ ELISpot results from Test 1 and Test 2 for three individual SLPs that did not test positive in the experiments shown in Table 1. These three SLPs were tested in six additional donors. Results are expressed as - (negative) or + (positive). A positive response was defined as the mean number of spots being higher than the mean number of spots for the unrelated SLP control + 2 * standard deviations. The last row represents a summary of the results in the table. If at least one donor had a positive response, it is denoted as POS. BCxxx = buffy coat code of a healthy donor, nt = not tested. TIFF2026530444000004.tif80148

[0110] Example 3. Derivation of CCR7 and CD40 When studying the immunogenicity of SLPs in moDC cultures, an abnormal effect on the morphology of moDCs cultured with SEQ ID NO:1 was observed (not shown). Based on this, we decided to test a complete set of PRAME-derived SLPs and further investigate and characterize this effect using APC-related cell surface markers.

[0111] As described in Zom et al Oncotarget, 2016 7 (41): 67087 and above, to prepare monocyte-derived DC cell cultures, magnetically activated cell sorting (Miltenyi Biotec) was used to obtain CD14 cells from buffy coat obtained from healthy donors using the manufacturer's instructions. +Cells were isolated. Briefly, these cells were cultured in IMDM supplemented with 4% human serum, penicillin / streptomycin, and L-glutamine. On days 0 and 3, 500 IU / mL of IL-4 and 800 IU / mL of GM-CSF were added to these cultures.

[0112] SLPs having the sequences shown 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 each SLP (SEQ ID NO:2), both non-cysteinylated and cysteinylated SLPs were tested. On day 6, cells were loaded with 13 μM of each individual SLP as shown, and on day 7, they were harvested after overnight stimulation, and the expression of surface markers was determined by flow cytometry.

[0113] Cells were stained with appropriate antibodies from Miltenyi Biotec, covering a common set of cell surface markers and costimulatory molecules that identify DCs, using CD11c (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 using an LSR-II instrument (BD Biosciences). Live cells were gated by FSCs and SSCs (using Flow Jo software), and doublets were excluded based on SSC-H and SSC-A (Figures 2A and B).

[0114] The expression of CCR7 and CD40 was compared to CD14 on SLP-loaded samples and control samples. + CD11c + Evaluation was performed on living single cells (Figure 2C). Unrelated albumin-derived SLPs were used as a negative control and to correct for background. CD14 was measured in cells loaded with each test SLP. + CD11c +The change factor was calculated by dividing the geometric mean of CCR7 or CD40 on living single cells by the geometric mean of the same marker on cells loaded with an unrelated albumin-derived SLP.

[0115] Notably, cells loaded with SEQ ID NO:2 (cysteine(*)), SEQ ID NO:2 (cystine(Cys**)), or SEQ ID NO:1 were observed to show higher increased expression of CCR7 (Figure 3) and CD40 (Figure 4) on their cell surface compared to the background, whereas other SLPs did not induce this effect. The effect is similar to that of the expected positive control, the lipopeptide TLR2 agonist, AMPLIVANT (U-Pam-14) R-diastereoisomer (see above), 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 the step of administering multiple immunogenic peptides to a human subject, wherein the multiple immunogenic peptides are (a) A peptide of 33 to 40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:1, and (b) Peptides with a length of 33 to 40 amino acids, containing or consisting of the sequence shown in SEQ ID NO:2 The method, including the method described above.

2. The aforementioned plurality of immunogenic peptides (a) A peptide consisting of the sequence shown in SEQ ID NO:1, and (b) Peptide consisting of the sequence shown in SEQ ID NO:2 Includes, Preferably, position 12 in SEQ ID NO:2 is cystine. The method according to claim 1.

3. The aforementioned multiple immunogenic peptides are the following immunogenic peptides: (a) A peptide of 25 to 40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:3 (b) A peptide of 35-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:4, and (c) A peptide with a length of 32 to 40 amino acids, containing or consisting of the sequence shown in SEQ ID NO:

5. The method according to any one of the claims, further comprising one, two, or all three of the above.

4. The aforementioned plurality of peptides are the following immunogenic peptides: (a) A peptide of 35-40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:6 (b) A peptide of 32 to 40 amino acids in length, comprising or consisting of the sequence shown in SEQ ID NO:7, and (c) A peptide of 35-40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:

8. The method according to any one of the claims, further comprising one, two, or all three of the above.

5. The method according to any one of the claims, wherein the plurality of immunogenic peptides include or consist of eight peptides indicated by SEQ ID NO:1 to SEQ ID NO:

8.

6. The above treatment (i) Not administering a peptide containing one of the sequences from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, or (ii) The administration of a peptide containing any of the sequences from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 among SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, The method according to any one of the above claims.

7. The method according to any one of the claims, wherein the cancer is a PRAME-expressing cancer, preferably selected from the group consisting of neuroblastoma, lymphoma, papilloma, breast or cervical cancer, acute and chronic leukemia, medulloblastoma, non-small cell lung cancer, head and neck cancer, kidney cancer, pancreatic cancer, prostate cancer, small cell lung cancer, multiple myeloma, melanoma, uveal melanoma, sarcoma, and hematological malignancies such as chronic myeloid leukemia and acute myeloid leukemia.

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

9. (a) A peptide of 33 to 40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:1, and (b) Peptides with a length of 33 to 40 amino acids, containing or consisting of the sequence shown in SEQ ID NO:2 Multiple immunogenic peptides, including, for use in the treatment or prevention of cancer.

10. A 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 multiple peptides, wherein the multiple peptides are (a) A peptide of 33 to 40 amino acids in length, containing or consisting of the sequence shown in SEQ ID NO:1, and (b) Peptides with a length of 33 to 40 amino acids, containing or consisting of the sequence shown in SEQ ID NO:2 The immunogenic composition comprising the above.

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

13. (i) Determining the CCR7 and / or CD40 levels in antigen-presenting cells such as dendritic cells or CD14+CD11c+ cultured monocyte-derived cells when incubated with a candidate antigen, and (ii) The step of selecting candidate antigens that can increase CCR7 and / or CD40 levels in the antigen-presenting cells. A method for selecting antigens suitable for use in immunization, including, The antigen is preferably a peptide. The aforementioned method.

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

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

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