Vaccine for TERT-positive tumors

Abstract

The present invention relates to novel peptides containing the TERT epitope that exhibit good immunogenicity and are useful for cancer immunotherapy.

Description

[Technical Field]

[0001] The present invention relates to the field of cancer treatment and to the identification, modification, and formulation of specific peptides useful for cancer immunotherapy. [Background technology]

[0002] Telomeres are specialized structures at the ends of eukaryotic chromosomes. Because canonical DNA polymerases cannot completely replicate linear DNA, 50–100 nucleotides are deleted with each cell division, leaving unreplicated DNA at the 3' end (Zhao 2009). After a certain number of cell doublings, telomere shortening reaches a critical length threshold, leading to cell growth arrest (Colebatch 2019). In cells that require multiple cell divisions, such as stem cells, this so-called "end-replication" problem can be resolved by the expression of a catalytic protein called telomerase, which prevents telomeres from shortening (Hayflick 1998). Telomerase is a macromolecular complex system, in which telomerase reverse transcriptase (TERT) plays a key role (Huang et al., 2013). In addition to its telomere function, which occurs within the nucleus, extratelomeric (also known as non-canonical) functions of TERT have been reported, primarily performed by mitochondrial TERT. Mitochondrial TERT protects mitochondrial DNA by binding to a subunit of complex I of the respiratory chain and reduces reactive oxygen species (ROS) levels (Rosen 2020). Noncanonical extratelomeric functions of telomerase can be exploited by cancer cells to promote carcinogenesis. For example, TERT can stimulate cell proliferation by inducing the expression of several growth-promoting genes (e.g., EGFR) or increase cell migration (Liu 2016), or it can increase mitochondrial membrane potential, inducing resistance to chemotherapeutic agents and proapoptotic stimuli (Chiodi and Mondello 2012). TERT can also stimulate angiogenesis, influence the tumor microenvironment by disrupting the Wnt / β-catenin signaling pathway, and positively regulate the NF-κB pathway, a key transcription factor that activates many inflammatory genes (Ghosh 2012; Li & Tergaonkar, 2014).

[0003] TERT expression is tightly regulated in normal cells (Armstrong 2000). Multiple mechanisms contribute to this regulation, including epigenetic, transcriptional, and post-transcriptional processes. Epigenetic regulation is mediated by the methylation status of the TERT promoter (Lee 2018). The TERT promoter contains binding motifs for several transcription factors, most notably the MAX / MAD1 complex, which acts as a repressor of TERT expression under physiological conditions (Dogan 2021). Post-transcriptional regulation is primarily mediated by alternative splicing of the hTERT pre-mRNA (Slusher 2020). To date, 22 alternative splice variants of hTERT have been reported; however, only full-length hTERT mRNA without any deletions or insertions exhibits telomerase activity (Wong 2014).

[0004] Under physiological conditions, TERT is undetectable or present at low levels in most healthy tissues, but remains constitutively expressed in stem cells, in early progenitor cells in the bone marrow, in spermatocytes, and in specific subsets of lymphocytes in the thymus, spleen, and lymph nodes (Hiyama 2007).

[0005] In cancer, telomerase reactivation has been reported in approximately 85–95% of human primary tumors (Low 2013). Pan-cancer studies have revealed that somatic TERT promoter mutations are particularly frequent in glioma (85%), cutaneous melanoma (85%), urothelial carcinoma (73%), poorly differentiated thyroid cancer (21–60%) and anaplastic thyroid cancer (13–73%), and hepatocellular carcinoma (44%) (Gupta 2021).

[0006] Aberrant upregulation of TERT in cancer cells can be caused by both genetic and epigenetic mechanisms, including alternative RNA splicing (increased full-length hTERT mRNA, allowing telomerase activity), TERT promoter hypermethylation, and, primarily, TERT promoter mutations. The two most frequent promoter mutations, C228T and C250T, are found at two hotspot positions, corresponding to positions -124 bp (C>T) and -146 bp (C>T) upstream from the ATG start, respectively. These C228T and C250T mutations accounted for 77% and 21% of TERT alterations, respectively (Killela 2013). TERT promoter mutations are one of the most common genetic alterations in adult gliomas, occurring in over 95% of oligodendrogliomas and 80% of IDH wild-type glioblastomas (Pierini et al. 2020).

[0007] Because of its crucial biological role in cancer, TERT is therefore a promising target for immunotherapy.

[0008] More than 25 immunogenic TERT epitopes have been identified, some of which are involved in enhancing CD8+ T cell responses via MHC class I, and others in inducing CD4+ T cell responses via MHC class II (see in particular Vonderheide, Biochimie 90 (2008) 173e180, table 1, page 175; Ellingsen 2021; WO2018206462; Dosset et al (Cancers 2020, 12(6), 1687)). Several MHC class II peptides have been identified that are capable of binding not only to the most commonly expressed HLA DR molecules, but also to most HLA class II molecules (Godet et al. Clin Can Res 2012; EP2639299). The "universal" TERT peptides UCP2 (SEQ ID NO: 1) and UCP4 (SEQ ID NO: 2) is immunogenic in a number of patients (Dosset et al Clin Can Res 2012, and Immunocology 2013), including patients with lung cancer (Adotevi et al 2023), and is currently being evaluated in newly diagnosed glioblastoma (NCT04280848).

[0009] Table 1. List of identified immunogenic TERT epitopes (adapted from Dosset et al. and Vonderheide, supra) TIFF2026507024000001.tif179141TIFF2026507024000002.tif83141

[0010] Several other clinical trials using various TERT peptide vaccines are also underway in various solid tumors and myeloma. TERT-specific immune responses were detected in over 50% of immunized patients, and tumor responses were observed in some cases, without any significant side effects (Ellingsen 2021). One TERT vaccine advanced to Phase III in patients with advanced pancreatic cancer but failed to demonstrate a survival advantage over chemotherapy (Middleton 2014). Collectively, these clinical trials demonstrate that therapeutic TERT-based vaccination can induce T cell responses against TERT, but its anti-cancer efficacy when used alone is currently limited. The efficacy of TERT vaccination may be significantly improved by improving vaccine technology and / or by combining it with immune checkpoint inhibitors, such as anti-PD1 antibodies, to enhance T cell responses in cancer patients.

[0011] Priming of lymphocytes or antibodies against a specific target (a process termed immunization) results from the presentation of an antigen to T cells by antigen-presenting cells. While this process can be achieved in vitro, it is more easily accomplished in vivo by administering the antigen to live animals or humans (a procedure termed vaccination). Despite all advances, vaccines still face several limitations. Most antigens are weakly immunogenic. The dose of peptide antigen required to induce immunity (typically in the range of 10–300 μg) can be a limiting factor, especially when antigen production is difficult or demand exceeds production capacity. Furthermore, because extracellularly injected antigens are typically presented by MHC class II and not MHC class I (thus preferentially inducing CD4+ lymphocytes and antibodies), inducing CD8+ lymphocytes remains a challenging task. Finally, vaccine technologies such as emulsions, liposomes, nanoparticles, fusion molecules, and DNA and RNA vaccines can be either unstable or difficult to synthesize, resulting in high production costs.

[0012] Therefore, adjuvants are generally used to increase the immunogenicity of administered antigens. WO2017089529 discloses that melanin can be used as an adjuvant to enhance the immune response to an epitope-bearing antigen.

[0013] WO2021165306 discloses that the addition of amino acids with nucleophilic residues to peptides is useful for improving immunogenicity when complexed with melanin.

[0014] Carpentier et al. PLoS One. 2017 Jul 17;12(7) disclose that synthetic melanin conjugated to a subunit vaccine antigen (obtained by oxidative polymerization of L-dopa mixed with a peptide) significantly enhances CD8+ T cell responses. Summary of the Invention

[0015] The inventors have modified the epitope UCP2 disclosed in Godet et al. Clin Can Res 2012 and Dosset et al Clin Can Res 2012 by adding a serine to its N-terminus and shown that this modified peptide complexed with melanin increases the immune response generated when administered in vivo compared to the preferred amino acid additions disclosed in WO 2021165306 (adding cysteine, lysine, or methionine to its terminus).

[0016] Thus, in a first aspect, the present invention relates to a polypeptide or peptide comprising SEQ ID NO: 7 or a peptide consisting of SEQ ID NO: 7 for generating an immune response against TERT useful for the treatment of cancer.

[0017] As shown below, a peptide preferably contains at most 100 amino acids, more preferably at most 50 amino acids. If the peptide is larger than SEQ ID NO: 7, SEQ ID NO: 7 may be located at the N-terminus of the peptide or elsewhere within the peptide. However, a peptide may contain more than 100 amino acids. It may also be a protein with biological activity.

[0018] In some embodiments, the polypeptide or peptide comprising SEQ ID NO: 7 further comprises another immunogenic epitope, particularly a CD4 or CD8 epitope of an antigen, particularly a cancer antigen, notably a cancer targeted by the immunogenic compositions disclosed herein. The other epitope may be linked to SEQ ID NO: 7 by a stretch of amino acids (which is the preferred form) or by any other acceptable linker, such as a polyether compound or other linker used in dendrimer constructs. When melanin is used as an adjuvant, it is preferred if SEQ ID NO: 7 is at the N-terminus of the polypeptide or peptide.

[0019] The other epitope may be another epitope from TERT, or may be an epitope from a protein other than TERT, the expression of which is associated with the same cancer as TERT.

[0020] Other epitopes include the pan-DR epitope (PADRE) and Pol 711 The epitope may be a universal T-helper epitope, such as an epitope. Other universal T-helper epitopes are widely disclosed in the literature, which can improve the immune response to cells expressing TERT via a response to SEQ ID NO: 7.

[0021] The present invention also relates to nucleic acid molecules encoding the polypeptides or peptides disclosed above. In some embodiments, the nucleic acid molecule is DNA. In other embodiments, the nucleic acid molecule is RNA. In some embodiments, the nucleic acid molecule is a chimeric DNA-RNA. When administered to a subject, nucleic acids can be protected from degradation by methods known in the art, particularly by liposome encapsulation.

[0022] The polypeptides or peptides disclosed herein can be used to treat or prevent cancers with high TERT expression, particularly glioblastoma, glioma, melanoma, hepatocellular carcinoma, lung cancer, urothelial cancer, or thyroid cancer.

[0023] The present invention therefore relates to a polypeptide or peptide as disclosed herein, a nucleic acid molecule, or a vaccine composition as disclosed below, for use as a medicament, in particular as a vaccine (whether prophylactic or therapeutic).

[0024] Therefore, the present invention also relates to a vaccine composition (or immunogenic composition) comprising the polypeptide, peptide, or nucleic acid molecule disclosed herein. In the vaccine composition, the polypeptide, peptide, or nucleic acid molecule is formulated with a suitable excipient and optionally some adjuvants, and is to be injected into a mammal, particularly a human. In particular, administration can be intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, or direct injection into a tumor. In other embodiments, the vaccine composition can be in the form of an oral composition, an inhalable composition, or an intradermal composition, particularly in the form of a patch. Subcutaneous administration is of particular interest.

[0025] It is preferred that the vaccine composition comprises a polypeptide or peptide disclosed herein and an adjuvant, particularly when the adjuvant is melanin. Preferably, synthetic melanin (i.e., melanin obtained in vitro by oxidative polymerization of a precursor) is used. In particular, the melanin is soluble melanin. In this embodiment, the peptide is complexed with or bound to the melanin. The vaccine composition can be obtained by oxidative polymerization of a melanin precursor in the presence of the peptide, as disclosed in WO2017089529, or by adding the peptide to already synthesized melanin, as disclosed in WO2021165306.

[0026] The present invention also relates to a polypeptide, peptide, nucleic acid, or vaccine composition disclosed herein for use in the treatment of cancer.

[0027] The present invention also relates to the use of a polypeptide, peptide, nucleic acid, or immunogenic composition disclosed herein for the preparation of a medicament for preventing or treating cancer in a patient, in this embodiment, the medicament comprises the polypeptide, peptide, nucleic acid, or immunogenic composition and a suitable excipient or adjuvant.

[0028] Vaccines can be prophylactic (i.e., intended to protect the recipient from developing disease) or therapeutic (i.e., intended to help the recipient combat disease that is already present). The disease is linked to a target antigen (TERT) that is expressed or presented by cells during the course of the disease.

[0029] The present invention also relates to a method for treating a patient in need thereof (particularly a patient with cancer), comprising administering to the patient an effective amount of a polypeptide, peptide, nucleic acid, or immunogenic composition disclosed herein. Such administration results in the generation of an immune response directed against TERT, which then attacks and eliminates tumor cells, thereby providing a therapeutic effect.

[0030] The present invention also relates to a method for protecting a patient from cancer, comprising administering to the patient a therapeutic or effective amount of a polypeptide, peptide, nucleic acid, or immunostimulatory composition disclosed herein to induce an immune response against TERT associated with the cancer, wherein the immune response has a protective (prophylactic) effect against the cancer.

[0031] In particular, the cancer is a low-grade or high-grade glial tumor.

[0032] In another embodiment, the cancer is melanoma.

[0033] In another aspect, the cancer is urothelial carcinoma.

[0034] In another embodiment, the cancer is lung cancer.

[0035] In another embodiment, the cancer is small cell lung cancer.

[0036] In another embodiment, the cancer is thyroid cancer.

[0037] In another embodiment, the cancer is hepatocellular carcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0038] As used herein, an "effective amount" or "therapeutic amount" of an agent refers to an amount sufficient to induce a beneficial or desired result, such as a clinical outcome or the generation of an immune response, particularly a T-cell-mediated immune response. In this context, a therapeutic amount of an agent is an amount sufficient to achieve, for example, the generation of an immune response to an antigen and a reduction in the severity of symptoms of a disease associated with the antigen, compared to the situation observed in the absence of administration of the composition. An effective amount is an amount that provides therapeutic improvement while minimizing side effects or adverse effects. An effective amount may be 10 μg to 5 mg of antigen, preferably 20 μg to 500 μg. When melanin is used in a vaccine formulation, the amount of melanin that may be used may be comprised between 40 μg and 10 mg, particularly between 40 μg and 1 mg.

[0039] The resulting vaccine formulation can be used to protect animals from diseases that are associated with (i.e., involve and / or involve) cells that internally express, surface express, or secrete TERT.

[0040] Alternatively, the polypeptides, peptides, nucleic acids, or immunostimulatory compositions can be used in vitro in the presence of live cells (e.g., macrophages, dendritic cells, antigen-presenting cells, or lymphocytes) to prime them against an antigen, for example, before administration (preferably injection) in humans. The resulting composition thus induces an immune response in the recipient against the antigen TERT. In particular, US 6,210,662 discloses such a principle of forming a therapeutic or immunogenic composition consisting of antigen-presenting cells activated by contact with an antigen complex.

[0041] Therefore, the present invention relates to an in vitro method for priming or stimulating CD4 or CD8 lymphocytes against TERT, comprising contacting and incubating the polypeptide, peptide, or nucleic acid molecule or immunogenic composition disclosed herein, optionally with an adjuvant, with antigen-presenting cells and T lymphocytes.Preferably, the antigen-presenting cells and T lymphocytes are isolated from patients with cancer.The resulting lymphocytes can then be collected and administered to patients for the treatment of TERT-related cancer.

[0042] The present invention also relates to methods for detecting an immune response to TERT (particularly to the epitope set forth in SEQ NO: 7) by co-incubating an immunostimulatory composition as disclosed herein with a patient's tissue containing immune cells (such as blood or isolated lymphocytes) and detecting a specific immune response of the patient's lymphocytes to the TERT antigen present in the immunostimulatory composition. Such a response can be detected by measuring molecules secreted by lymphocytes, such as cytokines, particularly gamma-interferon.

[0043] The present invention also relates to methods for detecting an immune response to TERT (particularly to the epitope set forth in SEQ ID NO: 7) by co-incubating a population of antigen-presenting cells (such as monocytes or dendritic cells) with patient tissue containing immune cells (such as blood or isolated lymphocytes) and detecting a specific immune response of the patient's lymphocytes to the TERT antigen present in the immunostimulatory composition. Such a response can be detected by measuring molecules secreted by lymphocytes, such as cytokines, particularly gamma-interferon. The antigen-presenting cells have been pre-incubated in the presence of the immunogenic composition disclosed herein and thus present the epitome set forth in SEQ ID NO: 7 on their surface via MHC I molecules.

[0044] These methods are carried out in vitro.

[0045] melanin " melanin It should be noted that "eumelanin" refers to a pigment that is a macromolecule obtained from the oxidative polymerization of precursors related to indole or catechol, generally beginning with the oxidation of the amino acid tyrosine (or another precursor), followed by polymerization. This oxidation is a key step and is generally mediated by the enzyme tyrosinase, which converts tyrosine to DOPA. WO2017089529 and WO2021165306 disclose a synthetic process for eumelanin. Melanins that can be used as adjuvants with the epitopes disclosed herein can be "natural" melanins such as those found in nature, such as eumelanin, MAP-like polymers (containing a high proportion of melanin precursors), or synthetic melanin molecules obtained by in vitro oxidative polymerization of precursor derivatives, such as those described below. Synthetic melanins are prepared, in particular, by oxidizing tyrosine or L-dopa with hydrogen peroxide and are thus commercially available, for example, from Sigma-Aldrich.

[0046] In the context of the present application, the preferred melanin is eumelanin.

[0047] Process for obtaining synthetic melanin Synthetic melanin is obtained in vitro after oxidative polymerization of melanin precursors.

[0048] Polymerization of melanin precursors can be carried out by methods known in the art. In particular, melanin precursors can be incubated with enzymes such as phenylalanine hydroxylase, tyrosinase, mushroom tyrosinase, tyrosine hydroxylase, peroxidase, phenoloxidase, dopachrome tautomerase, DHICA oxidase, and DHI oxidase, with or without buffer. The choice of enzyme will depend on the nature of the precursors present in the solution before polymerization, and will be left to those skilled in the art. Oxidative polymerization is preferably carried out in the presence of tyrosinase.

[0049] The mixture is also exposed to an oxidizing agent (oxidizer or oxidizing agent) as disclosed below to promote polymerization and yield synthetic melanin.

[0050] In particular, the skilled artisan can optimize various parameters such as the ratio of melanin precursors when a mixture is used, the type of oxidizing agent, the pH, the buffer, the length of incubation, or the temperature of the reaction.

[0051] In particular, melanin synthesis depends on pH (alkaline pH favors the autoxidation of catechol) and on the metal ions present in the incubation solution (e.g., Cu). 2+ , Ni 2+ , Fe 3+ , Fe 2+ , Co 2+ , Zn 2+ , Mn 2+ , Mg 2+ ...) (Palumbo et al, Biochim Biophys Acta. 1987; 13; 925(2):203-9; Palumbo et al, Biochim Biophys Acta. 1991; 1115(1):1-5; WO95009629). Therefore, working at pH 8.5 + / - 0.5 is suitable. To increase the reaction kinetics, the physicochemical conditions can be modified: for example, by increasing the temperature above 20°C (e.g., 60-80°C), bubbling air through the reaction mixture, or increasing the atmospheric pressure.

[0052] Such synthetic melanins can be distinguished from natural melanins because they can be more homogeneous than natural melanins. In the context of the present invention, it is preferred to use synthetic eumelanin-like forms, especially those obtained by in vitro oxidative polymerization of L-dopa.

[0053] In one embodiment, the synthetic melanin (after polymerization) is purified by filtration over a 5 kDa to 100 kDa filter, preferably a 10 kDa filter.

[0054] In a preferred embodiment, the synthetic melanin is a soluble melanin, ie, in the form of particles less than 500 nm.

[0055] When synthesized, the melanin is washed by ultrafiltration or by filtration on an approximately 10 kDa filter (the melanin remains in the retentate) and then resuspended in water or a buffer (such as phosphate buffer). The melanin may be filtered through a 0.2 μm filter for sterilization. Thus, in some embodiments, the melanin is resuspended in water, with or without a buffer (such as phosphate buffer), before being combined with the peptide to obtain the immunogenic composition.

[0056] Melanin precursors "Melanin precursors" refer to molecules used or synthesized in vitro to synthesize melanin, particularly eumelanin, including L-phenylalanine, L-tyrosine, L-dopa, dopaquinone, cyclodopa, dopachrome, dihydroxyindolecarboxylic acid or 5,6-dihydroxyindole-2-carboxylic acid (DHICA), indole-5,6 quinone, 5,6-dihydroxyindole (DHI), dopamine-o-quinone, dopamine-leucodopaminochrome, leucodopachrome (cyclodopa), dopaminochrome, norepinephrine, noradequinone, noradenochrome, epinephrine, epinephrine-o-quinone, adenochrome, 3-aminotyrosine, 6-hydroxydopa, dihydrocaffeic acid, caffeic acid, methides, benzothiazoles, benzothiazines, and dihydroesculetin.

[0057] Indeed, the term "melanin precursor" further includes derivatives of such precursors and / or polymers containing a high proportion of such precursors (e.g., mussel adhesive proteins, etc.) Such melanin precursors and derivatives are described in WO2017089529 (incorporated by reference for the teachings thereof) and can be used as equivalent melanin precursors in the context of the present invention.

[0058] The melanin precursor is preferably selected from the group consisting of DHICA, DHI, L-dopa, L-tyrosine, D-dopa, 6-hydroxydopa, dopaquinone, cyclodopa, dopachrome, dopamine-o-quinone, dopamine, leucodopaminochrome, and dopaminochrome.

[0059] A preferred melanin precursor is L-dopa. Another preferred melanin precursor is DHICA. Another preferred melanin precursor is DHI. Another preferred melanin precursor is L-tyrosine. In certain embodiments, the melanin precursor is a mixture of DHICA and DHI. In other embodiments, the melanin precursor is dopachrome.

[0060] Other melanin precursors or derivatives thereof are described in the art, such as the products described in WO2017089529.

[0061] oxidizing agent " oxidizing agent "or" Oxidized molecules " is a compound capable of supplying oxygen to a solution containing melanin precursors and promoting their polymerization and the formation of melanin macromolecules.

[0062] Oxidizing agents that can accomplish this purpose include oxygen, hydrogen peroxide, ammonium persulfate, ferric ion, sodium iodide with hydrogen peroxide, and treatment with salts of transition metal cations such as copper sulfate as a catalyst for air oxidation.

[0063] Therefore, it is preferred if the oxidizing agent is selected from the group consisting of oxygen, hydrogen peroxide, ammonium persulfate, and ferric ions.

[0064] Vaccines, immunogenic or immunostimulatory compositions " Immunogenic or immunostimulatory compositions" refers to a composition that, when administered to an animal, is capable of generating an immune response in that animal. Preferably, the animal is a mammal, but may also be an avian (chicken, duck, goose, turkey, quail, etc.), particularly if the composition is to be used in avian livestock. The animal may also be a fish, so that the immunogenic composition may be used in fish farming.

[0065] The immunogenic composition is preferably used in mammals. Such mammals are preferably humans, but may also be other mammals when the composition is used in the veterinary field, particularly to induce immunity in livestock animals such as cows (cows), sheep, goats, or horses, but also in pets such as dogs or cats.

[0066] Thus, an immunogenic composition is a composition that contains an antigen, particularly a peptide containing an epitope from an antigen, and is capable of generating an immune response against such an antigen. The generated immune response can be a cellular (T cell-mediated) immune response or a humoral (B cell-mediated, antibody production) immune response. An immunogenic composition can also induce both a cellular and a humoral immune response.

[0067] The cellular immune response can be a CD8 T lymphocyte-mediated response (i.e., a cytotoxic response) or a CD4 T lymphocyte-mediated response (a helper response). A cytotoxic immune response can also be combined with a helper cellular immune response. A helper response can involve Th1, Th2, or Th17 lymphocytes (such lymphocytes can induce various cytokine responses, as is known in the art).

[0068] The immunogenic composition may allow for better presentation of antigens present therein through the MHC1 or MHC2 pathway.

[0069] The immunogenic composition may comprise a polypeptide or peptide comprising SEQ ID NO: 7. It may also be a mixture of polypeptides or peptides.

[0070] In some embodiments, the immunogenic composition comprises a nucleic acid encoding a polypeptide or peptide comprising SEQ ID NO: 7. Administration of this immunogenic composition allows for expression of the polypeptide or peptide in cells in vivo, resulting in an immune response against these polypeptides or peptides. Cells can also be transfected in vitro with a nucleic acid encoding a polypeptide or peptide comprising SEQ ID NO: 7, and the resulting cell composition can be used as the immunogenic composition.

[0071] Adjuvants " Adjuvants " refers to a substance that has the ability to modify or enhance the immune response to an antigen. In other words, the immune response to an antigen may be higher or different in the presence of an adjuvant than in the absence of the adjuvant (including when the response is modified, e.g., when the subset of T cells activated in the presence of an adjuvant is different from the subset activated in the absence of an adjuvant). Many adjuvants are known in the art and are widely used in the vaccine field.

[0072] These include alum, emulsions (either oil-in-water or water-in-oil, such as Freund's Incomplete Adjuvant (IFA) and MF59®), PRR (pattern recognition receptor) ligands, TLR3 (Toll-like receptor 3) and RLR (RIG-I-like receptor) ligands, such as double-stranded RNA (dsRNA), or synthetic analogs of dsRNA, such as poly(I:C), TLR4 ligands, such as bacterial lipopolysaccharide (LPS), MPLA (monophosphoryl lipid A), especially when formulated with alum, TLR5 ligands, such as bacterial flagellin, TLR7 / 8 ligands, such as imidazoquinolines (i.e., imiquimod, gardiquimod, and R848), TLR9 ligands, such as oligodeoxynucleotides containing specific CpG motifs (CpG ODN), or NOD2 (nucleotide-binding oligomerization domain-containing protein 2) ligands. The term ligand above preferably describes an agonist of a receptor, ie a substance that binds to and activates the receptor, in particular the TLR3 and TLR9 receptors.

[0073] The melanin described in WO2017089529 or WO2021165306 acts as an adjuvant, and it is also possible to use it in combination with another adjuvant. When melanin is used and another adjuvant is added, it is preferably selected from the group consisting of TLR3 agonists and TLR9 agonists, particularly when this further adjuvant is selected from polyinosinic:polycytidylic acid (poly I:C) and CpG oligonucleotides.

[0074] peptide A peptide is a chain of amino acids linked by peptide bonds. In the context of the present invention, a peptide comprises at least 9 amino acids, more preferably at least 10 amino acids, more preferably at least 11 amino acids, or at least 12 amino acids. In some embodiments, a peptide comprises at most 100 amino acids, more preferably at most 50 amino acids, more preferably at most 30 amino acids, or more preferably at most 25 amino acids. Peptides of 10 to 25 amino acids are well suited. However, in other embodiments, a peptide (sometimes called a polypeptide) may comprise more than 100 amino acids. It may also be a protein.

[0075] One or more amino acids of the peptide may be artificial (different from one of the 20 amino acids found in natural proteins). Such artificial amino acids may be D-amino acids or unnatural amino acids (such as citrulline, hydroxyproline, norleucine 3-nitrotyrosine, nitroarginine, ornithine, naphthylalanine, etc.).

[0076] The peptides may be capped or modified at their N- and / or C-termini, in particular, acetylation or capping of the N-terminus helps to minimize degradation of the peptide by aminopeptidases, and amidation of the C-terminus helps to stabilize the peptide from degradation by carboxypeptidases.

[0077] Thus, in the context of the present invention, a biologically active peptide should comprise SEQ ID NO: 7. As indicated, this sequence may be comprised in a larger protein or longer peptide, and may be further modified with glycosylation or terminal protection.

[0078] vaccine In the context of the present invention, a vaccine is a composition that, when administered to an animal, produces or artificially increases immunity to a particular antigen. It is therefore understood that the terms "immunogenic composition," "immunostimulatory composition," and "vaccine" can be used interchangeably.

[0079] Obtaining an immunogenic composition An immunogenic composition can be obtained by combining a polypeptide or peptide comprising a modified epitope disclosed herein with an adjuvant.

[0080] In particular, the polypeptide or peptide bearing the modified epitope is combined with a melanin, particularly a synthetic melanin as described herein.

[0081] The polypeptide or peptide can be added to a synthetic melanin solution as disclosed above (weight ratio of polypeptide or peptide / melanin: 1 / 1 to 1 / 10) and incubated for various periods of time, which may depend on the temperature of incubation, before use. The resulting solution can be washed and resuspended in water or any suitable buffer.

[0082] The binding of polypeptides or peptides to melanin can be verified by Tricine-SDS-PAGE analysis, as described by Carpentier (2017). Briefly, samples (peptide-melanin or peptide alone) are loaded onto an acrylamide gel. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R-250, allowing for quantification of free peptide in the gel. The binding of peptides to melanin is determined by the ratio: [Amount of unbound peptide in peptide-melanin sample / Amount of peptide in control sample containing only peptide] It can be expressed as:

[0083] The immunostimulatory composition may also include another adjuvant, as disclosed above. In a preferred embodiment, the adjuvant is added to the resulting composition immediately prior to administration, i.e., less than one hour before administration. [Brief explanation of the drawings]

[0084] [Figure 1] Cross-reactivity of T cell responses obtained after immunization with SEQ 7 in transgenic SURE mice. Mice were immunized against SEQ 7 as described in Table 1 and sacrificed on day 8. Spleen cells (5.10 cells / well) were restimulated in vitro for 18 hours with either SEQ ID NO: 7 or the native epitope SEQ ID NO: 1 (neither conjugated to melanin) at various concentrations (5 μg / ml to 5 ng / ml), and the number of IFNγ-SFCs (spot-forming cells) was measured after 18 hours of culture. [Example]

[0085] With the aim of selecting an optimized TERT antigen for use in human patients, several peptides containing one of the known TERT immune epitopes (SEQ ID NO: 1) were screened in the context of a melanin-based vaccine, as described in WO2021165306.

[0086] These peptides (SEQ ID NO: 4 to SEQ ID NO: 7; Table 2) all contain the UCP2 epitope (SEQ ID NO: 1) and have an additional amino acid at the NH2-terminus, as WO2021165306 has disclosed that such a modification improves the efficacy of melanin-based vaccines. This application teaches that cysteine, acetylcysteine, methionine, proline, hydroxyproline, histidine, and lysine are preferred amino acids that can increase peptide binding, with cysteine ​​being the preferred amino acid for optimal binding.

[0087] Table 2: List of sequences used and corresponding T cell responses obtained after subcutaneous immunization in transgenic Surel1 mice. TIFF2026507024000003.tif62136* Described in Godet et al. Clin Can Res 2012 & Dosset et al Clin Can Res 2012

[0088] Example 1. Binding of peptides to melanin L-Dopa alone (0.8 mg / ml) was polymerized to melanin at pH 8.5 for 2 hours at 60°C under stirring. The reaction mixture was then filtered on a 10 kDa filter, and the retentate containing the synthetic melanin was resuspended in phosphate buffer at pH 7.5. The peptide was then added (at a melanin / peptide weight ratio of 2), and the mixture was incubated at room temperature for 18 hours.

[0089] Tricine-SDS-PAGE analysis was performed as described in Carpentier; 2017 (ibid.). Briefly, samples (peptide-melanin or peptide alone) were loaded onto an acrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and imaged using a ChemiDoc XRS+ system (Bio-Rad Laboratory), allowing for quantification of free peptide in the gel. Binding of peptide to melanin was determined by the ratio: [Amount of unbound peptide in peptide-melanin sample / Amount of peptide in control sample containing only peptide] As shown in Table 3, binding was observed with SEQ ID NO: 7, which was unexpectedly much higher than that observed with the unmodified epitope peptide SEQ ID NO: 1 and higher than that observed with peptides modified according to preferred embodiments of WO2021165306 (e.g., SEQ ID NO: 5 and SEQ ID NO: 6).

[0090] Table 3: Peptide conjugation to synthetic melanin. L-Dopa (0.8 mg / ml) was subjected to oxidative polymerization under aerobic conditions at pH 8.5 and 60°C for 2 hours. The reaction mixture was then filtered on a 10 kDa filter, and the retentate containing the synthetic melanin was resuspended at pH 7.5. The peptide was then added (at a melanin / peptide weight ratio of 2), and the mixture was incubated at room temperature or 60°C for various times. The percentage of peptide conjugated to melanin was then quantified by SPS-page analysis. TIFF2026507024000004.tif49128

[0091] Example 2. Immunogenicity of various peptides The immunogenicity of these various peptides was screened by vaccination in HLA-A2 / DR1 mice (also known as the Surel1 model), which are transgenic mice that mimic the human immune system.

[0092] For immunization, L-dopa (0.8 mg / ml) was subjected to oxidative polymerization under aerobic conditions at pH 8.5 and 60°C for 2 hours. The reaction mixture was then filtered through a 10 kDa filter, and the retentate containing the synthetic melanin was resuspended in water. Peptide (10 μg / mouse) was then added (peptide / L-dopa = 1 / 4 weight ratio), and after an 18-hour incubation period, the mixture was used for subcutaneous immunization in mice. The phosphorothioate oligonucleotide CpG-28 (5'-TAAACGTTATAACGTTATGACGTCAT, SEQ ID NO: 3) was added to the vaccine formulation (10 μg / mouse) immediately before immunization. Mice were sacrificed on day 8, and T cell responses were measured as described in Carpentier (2017). Briefly, splenocytes were restimulated in vitro with the corresponding peptides (not conjugated to melanin) and the number of IFNg-SFCs (spot-forming cells) was determined and expressed as mean + / - SEM.

[0093] Surprisingly and unexpectedly, peptide SEQ ID NO: 7 was observed to be highly effective in eliciting an immune response after immunization of mice (Table 2), i.e., SEQ ID NO: 7 was more efficient than the most preferred modifications recommended in WO2021165306 (NH2-terminal methionine or lysine described as SEQ ID NO: 5 and SEQ ID NO: 6) and almost as effective as SEQ ID NO: 4, which has a cysteine ​​at the NH2-terminus.

[0094] The immunogenicity of SEQ ID NO: 7 was also demonstrated in other conditions. After immunization on days 1 and 14 with 10 μg antigen / mouse in combination with either the phosphorothioate oligonucleotide CpG-28 (SEQ ID NO: 3) or Montanide, specific immune responses were observed on day 28: IFNg-SFC of 22 + / - 5 and 15 + / - 5 for CpG-28 and Montanide, respectively, compared with 2 + / - 1 for controls using an irrelevant epitope (mean + / - SEM, n = 8 / group, compiled from two separate experiments).

[0095] These experiments therefore demonstrate that SEQ ID NO: 7 has an epitope that can be recognized by the immune system and is therefore immunogenic.

[0096] Example 3. Stability and filterability of peptides in solution Developing a valid vaccine requires adequate stability of the drug substance (>95% over time), as even minor degradation products of the peptide can cause unwanted toxicity when administered to patients and can compromise the purity of the drug required for development.

[0097] To assess stability, peptides were diluted to 0.5 mg / mL in water, adjusted to pH 7.4, frozen at -20°C, and analyzed by HPLC (Hypersil GOLD C8 15 cm x 4.6 mm x 5 μm (ThermoFisher)) on the indicated days using an elution buffer of 15% CH3CN and 0.1% TFA. Pick areas were compared with reference standards.

[0098] As shown in Table 4, SEQ ID NO: 5 and SEQ ID NO: 4 were very unstable in solution at physiological pH (pH 7.4) after oxidation, and SEQ ID NO: 6 showed unexpected significant degradation under non-frozen conditions, while SEQ ID NO: 7 was very stable. In fact, after 7 days at -20°C, the pic of SEQ ID NO: 4 decreased by 53%, whereas SEQ ID NO: 7 was stable (>95%) at pH 7.4 for at least 3 months at -20°C and at room temperature for at least 7 days.

[0099] Table 4: Stability of peptides after a 7-day incubation period in a solution at pH 7.4 at various temperatures (ratio of pick area on the indicated day / pic area of ​​a fresh solution of the same peptide) (mean data + / - SD of two or three different experiments). TIFF2026507024000005.tif42133

[0100] Filterability of pharmaceutical formulations on 0.2μ filters is important to achieve a sterile solution. Surprisingly, SEQ ID NO: 7 appears to be particularly relevant for further pharmaceutical development because, as described in Example 2, the mixed peptide plus melanin does not precipitate, can be filtered on a 0.2μ filter, and remains suspended in isotonic solution for more than 18 hours, whereas SEQ ID NO: 6 precipitates and is not filterable under such conditions.

[0101] In preferred conditions, the weight ratio of melanin to peptide mixture of SEQ ID NO: 7 is greater than 2.5:1, preferably 2.7:1, to obtain optimal filterability on a 0.2μ filter.

[0102] In fact, lower ratios may result in some precipitation, along with a slight decrease in the absorbance of the supernatant (measured by optical density at 220 nm).

[0103] Example 4. Verification of cross-reactivity of selected peptides Thus, SEQ ID NO: 7 exhibited good binding, immunogenicity, stability, and filterability.

[0104] Because SEQ ID NO: 7 differs from the natural epitope SEQ ID NO: 1, it can be expected that this modification may result in an entirely new epitope that does not have cross-reactivity with the native epitope. Therefore, mice were immunized with SEQ ID NO: 7 to determine whether the T cell response elicited by SEQ ID NO: 7 could similarly recognize the native epitope SEQ ID NO: 1.

[0105] This was shown to be the case (Figure 1), thus demonstrating cross-reactivity of the immune response to both epitopes.

[0106] References TIFF2026507024000006.tif225144TIFF2026507024000007.tif91143

Claims

1. A peptide comprising SEQ ID NO:

7.

2. 2. The peptide of claim 1, consisting of SEQ ID NO:

7.

3. 2. The peptide of claim 1, comprising at most 50 amino acids.

4. 4. The peptide of claim 1 or 3, further comprising other immunogenic epitopes.

5. A nucleic acid molecule encoding the peptide according to any one of claims 1 to 4.

6. A vaccine composition comprising a peptide according to any one of claims 1 to 4 or a nucleic acid molecule according to claim 5.

7. 7. The vaccine composition according to claim 6, comprising a peptide according to any one of claims 1 to 4 and an adjuvant.

8. 8. The vaccine composition of claim 7, wherein the adjuvant is melanin.

9. A peptide according to any one of claims 1 to 4, a nucleic acid according to claim 5 or a vaccine composition according to any one of claims 6 to 8 for use as a medicament or as a vaccine.

10. A peptide according to any one of claims 1 to 7, a nucleic acid according to claim 5, or a vaccine composition according to any one of claims 6 to 8 for use in the treatment of cancer.

11. 11. The peptide of any one of claims 1 to 4, the nucleic acid of claim 5, or the vaccine composition of any one of claims 6 to 8, for use according to claim 10, wherein the cancer is selected from the group consisting of glioblastoma, glioma, melanoma, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, urothelial cancer, and thyroid cancer.

12. 11. The peptide of any one of claims 1 to 4, the nucleic acid of claim 5, or the vaccine composition of any one of claims 6 to 8, for use according to claim 10, wherein the cancer is a low-grade or high-grade glial tumor.

13. 13. The peptide of any one of claims 1 to 4, the nucleic acid of claim 5 or the vaccine composition of any one of claims 6 to 8 in a form suitable for intramuscular, intravenous, subcutaneous, intraperitoneal, intratumoral, buccal, inhalable or intradermal administration for the use according to any one of claims 9 to 12.

14. An in vitro method for detecting, priming or stimulating lymphocytes directed against TERT, comprising contacting antigen-presenting cells and T lymphocytes with a peptide described in any one of claims 1 to 4 or a nucleic acid molecule described in claim 5, optionally together with an adjuvant.

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