Vaccines against TERT-positive tumors

Abstract

The present invention relates to a novel peptide containing a TERT epitope, which has good immunogenicity and is useful in the immunotherapy of cancer.

Description

Technical Field

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

[0002] Telomeres are specialized structures at the ends of eukaryotic chromosomes. Because standard DNA polymerases are unable to fully replicate linear DNA, each cell division results in the loss of 50–100 nucleotides, leaving unreplicated DNA at the 3' end (Zhao 2009). After a certain number of cell doublings, telomeres shorten to a critical length threshold, and the cell enters growth arrest (Colebatch 2019). For cells that divide repeatedly, such as stem cells, this so-called "end replication" problem can be addressed by expressing catalytic proteins (called telomerases) that prevent telomere shortening (Hayflick 1998). Telomerase is a complex system composed of macromolecules, in which telomerase reverse transcriptase (TERT) plays a key role (Huang et al. 2013). In addition to its telomere functions within the nucleus, TERT also has extratelomeric (also known as non-canonical) functions, primarily embodied by mitochondrial TERT. Mitochondrial TERT protects mitochondrial DNA by binding to subunits of respiratory chain complex I and reducing reactive oxygen species (ROS) levels (Rosen 2020). The non-canonical extratelomeric functions of telomerase may be misused by cancer cells to promote carcinogenesis. For example, TERT can stimulate cell proliferation by inducing the expression of multiple growth-promoting genes (e.g., EGFR) (Liu 2016), increasing cell migration, or increasing mitochondrial membrane potential, inducing resistance to chemotherapeutic drugs, and inducing resistance to pro-apoptotic stimuli (Chiodi and Mondello 2012). TERT can also stimulate angiogenesis and influence the tumor microenvironment by interfering with the Wnt / β-catenin signaling pathway and positively regulating the NF-κB pathway, a key transcription factor that activates many inflammatory genes (Ghosh 2012; Li & Tergaonkar 2014).

[0003] In normal cells, TERT expression is tightly regulated (Armstrong 2000). Multiple mechanisms are involved in 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, particularly the MAX / MAD1 complex, which acts as a repressor of TERT expression under physiological conditions (Dogan 2021). Post-transcriptional regulation is primarily achieved through alternative splicing of the hTERT precursor mRNA (Slusher 2020). To date, 22 hTERT alternative splicing variants have been reported, but only the full-length hTERT mRNA, devoid of deletions or insertions, exhibits telomerase activity (Wong 2014).

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

[0005] In cancer, telomerase reactivation has been reported in approximately 85% to 95% of human primary tumors (Low 2013). A pan-cancer study showed that somatic TERT promoter mutations were particularly common in gliomas (85%), cutaneous melanomas (85%), urothelial carcinomas (73%), poorly differentiated thyroid cancers (21-60%) and anaplastic thyroid cancers (13-73%), or hepatocellular carcinomas (44%) (Gupta 2021).

[0006] Aberrant upregulation of TERT in cancer cells can be driven by genetic and epigenetic mechanisms, including alternative RNA splicing (increasing full-length hTERT mRNA with telomerase activity), TERT promoter hypermethylation, and (primarily) TERT promoter mutations. The two most common promoter mutations, C228T and C250T, are located in hotspots at positions -124 bp (C>T) and -146 bp (C>T) upstream of the corresponding ATG start site, respectively. These C228T and C250T mutations account for 77% and 21% of TERT alterations, respectively (Killela 2013). TERT promoter mutations are among the most common genetic alterations in adult gliomas, occurring in over 95% of oligodendrogliomas and 80% of IDH-wildtype glioblastomas (Pierini et al., 2020).

[0007] Due to its key biological role in cancer, TERT has become a promising target for immunotherapy.

[0008] More than 25 immunogenic TERT epitopes have been identified, some of which are associated with the enhancement of MHC class I-mediated CD8+ T cell responses, while others are involved in the induction of MHC class II-mediated CD4+ T cell responses (see in particular Vonderheide, Biochimie 90 (2008) 173e180, Table 1, page 175; Ellingsen 2021; WO2018206462; Dosset et al. (Cancers 2020, 12(6), 1687). Some MHC class II peptides have been identified that can bind not only to the most commonly expressed HLA DR molecules, but also to the majority of 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) are immunogenic in a large number of patients (Dosset et al., Clin Can Res 2012). 2012 and Immunooncology 2013), including in patients with lung cancer (Adotevi et al., 2023), and is currently being evaluated in newly diagnosed glioblastoma patients (NCT04280848).

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

[0010]

[0011]

[0012] Several other clinical trials using different TERT peptide vaccines have been conducted in various solid tumors and myeloma. Immune responses against TERT have been detected in ≥50% of immunized patients, and tumor responses have been observed in some cases without any significant side effects (Ellingsen 2021). A TERT vaccine has advanced to Phase III in patients with advanced pancreatic cancer but has failed to demonstrate a survival advantage over chemotherapy (Middleton 2014). Overall, clinical trials have shown that TERT-based therapeutic vaccination can trigger T cell responses against TERT, but to date has had limited anticancer effects when used alone. The effectiveness of TERT vaccination may be significantly improved by improving vaccine technology and / or combining treatment with immune checkpoint inhibitors (such as anti-PD1 antibodies), which enhance T cell responses in cancer patients.

[0013] Antigen-presenting cells present an antigen to T cells, which in turn trigger the production of lymphocytes or antibodies against a specific target (a process known as immunization). This process can be achieved in vitro but is more easily achieved in vivo by administering the antigen to a living animal or human (a process known as vaccination). Despite many advances, vaccines still face several limitations. Most antigens have poor immunogenicity. The dose of peptide antigen required to trigger immunity (typically in the range of 10 to 300 μg) can be a limiting factor, especially when the antigen is difficult to prepare or demand exceeds production capacity. In addition, inducing CD8+ lymphocytes remains a daunting challenge because injected extracellular antigens are typically presented by MHC class II rather than MHC class I (thus, preferentially producing CD4+ lymphocytes and antibodies). Finally, vaccine technologies (such as emulsions, liposomes, nanoparticles, fusion molecules, DNA, and RNA vaccines) can be unstable or difficult to synthesize, resulting in sometimes prohibitive production costs.

[0014] Therefore, adjuvants are often used to enhance the immunogenicity of administered antigens. WO2017089529 discloses that melanin can be used as an adjuvant to enhance the immune response against antigens carrying epitopes.

[0015] WO2021165306 discloses that the addition of amino acids with nucleophilic residues into a peptide helps to improve the immunogenicity of the peptide when complexed with melanin.

[0016] Carpentier et al., PLoS One. 2017 Jul 17, 12(7) reported that synthetic melanin significantly enhanced CD8+ T cell responses when combined with subunit vaccine antigens (obtained by oxidative polymerization of L-dopa and peptides). Summary of the Invention

[0017] The inventors modified the epitope UCP2 disclosed by Godet et al. (Clin Can Res 2012) and Dosset et al. (Clin Can Res 2012) by adding serine to its N-terminus, and demonstrated that this modified peptide, after binding to melanin, produces a higher immune response when administered in vivo than the preferred amino acid addition disclosed in WO 2021165306 (i.e., adding cysteine, lysine or methionine to the terminal).

[0018] Therefore, in a first embodiment, 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 for the treatment of cancer.

[0019] As described below, the peptide preferably contains no more than 100, more preferably no more than 50, amino acids. When the peptide is longer than SEQ ID NO: 7, SEQ ID NO: 7 can be located at the N-terminus of the peptide, or elsewhere within the peptide. However, the peptide can contain more than 100 amino acids. It can also be a biologically active protein.

[0020] In some embodiments, the polypeptide or peptide comprising SEQ ID NO: 7 further comprises other immunogenic epitopes of an antigen (particularly a cancer antigen, especially a cancer targeted by the immunogenic compositions disclosed herein), particularly CD4 or CD8 epitopes. The other epitopes can be linked to SEQ ID NO: 7 via an amino acid extension (which is a preferred embodiment) or any other acceptable linker (e.g., polyether compounds or other linkers used in dendrimer constructs). When melanin is used as an adjuvant, SEQ ID NO: 7 is preferably located at the N-terminus of the polypeptide or peptide.

[0021] The other epitope may be another epitope from TERT, or an epitope from another protein other than TERT, the expression of which is more associated with cancer than TERT.

[0022] Other epitopes may be universal T helper cell epitopes, such as the pan-DR epitope (PADRE) and Pol 711 Epitope. Other universal T helper cell epitopes are widely disclosed in the literature. They can enhance the immune response against cells expressing TERT via a response against SEQ ID NO: 7.

[0023] The present invention also relates to a nucleic acid molecule encoding a polypeptide or peptide 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 DNA-RNA chimera. The nucleic acid molecule can be prevented from degradation when administered to a subject by methods known in the art (particularly by liposome encapsulation).

[0024] 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 carcinoma or thyroid cancer.

[0025] Therefore, the present invention relates to the use of a polypeptide or peptide disclosed herein, a nucleic acid molecule disclosed below or a vaccine composition as a medicament, in particular as a vaccine, whether prophylactic or therapeutic.

[0026] Therefore, the present invention also relates to a vaccine composition (or immunogenic composition) comprising a polypeptide, peptide or nucleic acid molecule disclosed herein. In the vaccine composition, the polypeptide, peptide or nucleic acid molecule is formulated together with an appropriate excipient and optionally some adjuvants for injection into a mammal, especially a human. In particular, administration can be by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection or direct injection into the tumor. In other embodiments, the vaccine composition can be in the form of an oral, inhaled or intradermal composition, especially in the form of a patch. Subcutaneous administration is of particular interest.

[0027] The vaccine composition preferably comprises a polypeptide or peptide disclosed herein and an adjuvant, particularly when the adjuvant is melanin. Synthetic melanin (i.e., melanin obtained in vitro by oxidative polymerization of a precursor) is preferably used. In particular, the melanin is a soluble melanin. In this embodiment, the peptide should be complexed or combined with the melanin. The vaccine composition can be obtained by oxidative polymerization of a melanin precursor in the presence of a peptide, as disclosed in WO2017089529, or by adding the peptide to a synthesized melanin, as disclosed in WO2021165306.

[0028] The present invention also relates to the use of the polypeptide, peptide, nucleic acid or vaccine composition disclosed herein for treating cancer.

[0029] The present invention also relates to the use of the polypeptides, peptides, nucleic acids or immunogenic compositions disclosed herein in the preparation of a medicament for preventing or treating cancer in a patient. In this embodiment, the medicament comprises the polypeptides, peptides, nucleic acids or immunogenic compositions and a suitable excipient or adjuvant.

[0030] Vaccines can be preventative vaccines (ie, intended to protect the vaccinee from developing a disease), or therapeutic vaccines (ie, intended to help the vaccinee fight an existing disease).The disease is associated with a target antigen (TERT) that is expressed or presented by cells during the disease process.

[0031] The present invention also relates to a method for treating a patient in need thereof (particularly a cancer patient), comprising administering to the patient an effective amount of a polypeptide, peptide, nucleic acid, or immunogenic composition disclosed herein. Such administration results in an immune response directed against TERT, which in turn attacks and eliminates tumor cells, thereby exerting a therapeutic effect.

[0032] The present invention also relates to a method for protecting a patient from cancer, comprising administering to the patient a therapeutic amount or an effective amount of a polypeptide, peptide, nucleic acid or immunostimulatory composition disclosed herein to induce an immune response against cancer-associated TERT, wherein the immune response has a protective (preventive) effect against cancer.

[0033] In particular, the cancer is a low-grade or high-grade glial tumor.

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

[0035] In another embodiment, the cancer is urothelial carcinoma.

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

[0037] In another embodiment, the cancer is small cell lung cancer.

[0038] In another embodiment, the cancer is thyroid cancer.

[0039] In another embodiment, the cancer is hepatocellular carcinoma.

[0040] As used herein, an "effective amount" or "therapeutic amount" of an agent refers to an amount sufficient to induce a beneficial or desired result (e.g., a clinical result or an immune response, particularly a T cell-mediated immune response). In this article, the therapeutic amount of an agent refers to an amount that is, for example, sufficient to induce an immune response to an antigen and reduce the severity of the disease symptoms associated with the antigen (compared to the situation observed when the composition is not administered). An effective amount refers to an amount that minimizes side effects or adverse reactions while providing treatment improvement. 10 μg to 5 mg of antigen, preferably 20 μg to 500 μg, can be used as an effective amount. If melanin is used in the vaccine formulation, the amount of melanin that can be used can be contained between 40 μg and 10 mg, particularly between 40 μg and 1 mg.

[0041] The resulting vaccine formulations can be used to protect animals from diseases involving (ie, involving and / or associated with) cells that express, surface, or secrete TERT.

[0042] Alternatively, polypeptides, peptides, nucleic acids or immunostimulatory compositions can be used in vitro in the presence of living cells (e.g., macrophages, dendritic cells, antigen-presenting cells or lymphocytes) to sensitize them to the antigen, for example, before human administration (preferably injection). Thus, the resulting composition will elicit an immune response in the recipient against the antigen TERT. In particular, US 6210662 discloses the principle of forming such therapeutic or immunogenic compositions, including activation of antigen-presenting cells by contact with antigen complexes.

[0043] Therefore, the present invention relates to an in vitro method for eliciting or stimulating CD4 or CD8 lymphocytes against TERT, comprising contacting and incubating a 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 a cancer patient. The resulting lymphocytes can then be recovered and administered to a patient for the treatment of a TERT-associated cancer.

[0044] The present invention also relates to a method for detecting an immune response against TERT (particularly against the epitope set forth in SEQ ID NO: 7), by incubating the immunostimulatory composition disclosed herein with a patient's tissue containing immune cells (e.g., blood or isolated lymphocytes), and detecting a specific immune response of the patient's lymphocytes against the TERT antigen present in the immunostimulatory composition. This response can be detected by measuring molecules secreted by the lymphocytes, such as cytokines, particularly interferon-gamma.

[0045] The present invention also relates to a method for detecting an immune response against TERT (particularly against the epitope shown in SEQ ID NO: 7) by co-incubating a population of antigen-presenting cells (e.g., monocytes or dendritic cells) with a patient's tissue containing immune cells (e.g., blood or isolated lymphocytes), and detecting a specific immune response of the patient's lymphocytes against the TERT antigen present in an immunostimulatory composition. This response can be detected by measuring molecules secreted by the lymphocytes (e.g., cytokines, particularly gamma interferon). The antigen-presenting cells have previously been incubated in the presence of an immunogenic composition disclosed herein, and thus present the epitope shown in SEQ ID NO: 7 on their surface via MHC I molecules.

[0046] These methods are performed in vitro.

[0047] melanin

[0048] It should be noted that " melanin" is a pigment that is a macromolecule obtained by oxidative polymerization of indole- or catechol-related precursors (usually starting from the oxidation of the amino acid tyrosine (or another precursor) and then polymerizing). The oxidation is a key step and is usually mediated by tyrosinase, which converts tyrosine to DOPA. WO2017089529 and WO2021165306 disclose methods for the synthesis of true melanin. As melanins that can be used as adjuvants for the epitopes disclosed herein, "natural" melanins occurring in nature, such as true melanin, MAPs-like polymers (containing a high proportion of melanin precursors) or synthetic melanin molecules obtained by in vitro oxidative polymerization of precursor derivatives (as described below) can be used. Therefore, synthetic melanin (especially prepared by oxidizing tyrosine or L-DOPA with hydrogen peroxide) is sold in the form of synthetic melanin, for example, by SigmaAldrich.

[0049] In the context of the present application, the preferred melanin is eumelanin.

[0050] Obtaining synthetic melanin

[0051] Melanin precursors are oxidatively polymerized in vitro to produce synthetic melanin.

[0052] The polymerization of the melanin precursor can be carried out by methods known in the art. In particular, the melanin precursor can be incubated with an enzyme (e.g., phenylalanine hydroxylase, tyrosinase, mushroom tyrosinase, tyrosine hydroxylase, peroxidase, phenol oxidase, dopachrome tautomerase, DHICA oxidase, DHI oxidase) with or without a buffer. Those skilled in the art will select the enzyme based on the properties of the precursor present in the solution before polymerization. Oxidative polymerization is preferably carried out in the presence of tyrosinase.

[0053] The mixture is also exposed to an oxidizing agent (oxidant or oxidative agent) as described below to promote polymerization and obtain synthetic melanin.

[0054] Among them, those skilled in the art can optimize various parameters, such as the ratio of melanin precursors in the mixture used, the type of oxidant, pH, buffer, incubation time or reaction temperature.

[0055] In particular, melanin synthesis may be affected by pH (alkaline pH promotes catechol autooxidation) and the presence of metal ions (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, it is appropriate to carry out the reaction at a pH of 8.5+ / -0.5. Physicochemical conditions can be adjusted to improve the reaction kinetics, for example, by raising the temperature to above 20°C (e.g., 60 to 80°C), by bubbling air into the reaction mixture, or by increasing the pressure.

[0056] Such synthetic melanins may be more homogeneous than natural melanins and thus distinguishable therefrom. In the context of the present invention, preference is given to using synthetic eumelanins, in particular those obtained by in vitro oxidative polymerization of L-dopa.

[0057] In one embodiment, the synthetic melanin (post-polymerization) is purified by filtration over a 5 kDa-100 kDa filter, preferably a 10 kDa filter.

[0058] In a preferred embodiment, the synthetic melanin is in the form of soluble melanin, ie, in the form of particles smaller than 500 nm.

[0059] When synthesized, the melanin is washed by ultrafiltration or filtration through a filter of about 10 kDa (the melanin remains in the retentate) and then resuspended in water or a buffer (e.g., phosphate buffer). The melanin can be filtered through a 0.2 μm filter to ensure sterility. Therefore, in one embodiment, the melanin is resuspended in water with or without a buffer (e.g., phosphate buffer) and then mixed with the peptide to obtain an immunogenic composition.

[0060] Melanin precursor

[0061] "Melanin precursors" are molecules used or synthesized in the in vitro synthesis of melanin (particularly eumelanin). Examples include 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 leukodopaminochrome, leukodopachrome (cyclodopa), dopamine chrome, norepinephrine, noradequinone, noradenochrome, epinephrine, epinephrine-o-quinone, adenochrome, 3-aminotyrosine, 6-hydroxydopa, dihydrocaffeic acid, caffeic acid, methylated compounds, benzothiazole, benzothiazine, and dihydroescin.

[0062] In fact, 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). Such melanin precursors and their derivatives have been described in WO2017089529 (the teachings of which are incorporated by reference) and can be used as equivalent melanin precursors in the present invention.

[0063] 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, leukocyte dopamine pigment and dopamine pigment.

[0064] 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 one specific embodiment, the melanin precursor is a mixture of DHICA and DHI. In another embodiment, the melanin precursor is dopachrome.

[0065] Other melanin precursors or derivatives thereof are described in the art, for example the products described in WO2017089529.

[0066] oxidants

[0067] “ oxidants "or" Oxidized molecules "It is a compound that can provide oxygen to a solution containing melanin precursors, promoting their polymerization and forming melanin macromolecules.

[0068] Oxidants that can achieve this purpose include oxygen, hydrogen peroxide, ammonium persulfate, iron ions, sodium iodide and hydrogen peroxide, and treatment with salts of transition metal cations (such as copper sulfate) as catalysts for air oxidation.

[0069] Therefore, the oxidizing agent is preferably selected from oxygen, hydrogen peroxide, ammonium persulfate and iron ions.

[0070] Vaccines, immunogenic or immunostimulatory compositions

[0071] “ Immunogenic or immunostimulatory compositions " refers to a composition that is capable of generating an immune response in an animal when administered to the animal. Preferably, the animal is a mammal, but may also be a bird (e.g., chicken, duck, goose, turkey, quail), particularly when the composition is for poultry livestock. The animal may also be a fish, as the immunogenic composition may be used in fish farming.

[0072] The immunogenic composition is preferably used in mammals. Such mammals are preferably humans, but when the composition is used in the veterinary field, it can also be other mammals, especially for inducing immunity in livestock such as cattle (cows), sheep, goats or horses, and can also be used in pets such as dogs or cats.

[0073] Thus, an immunogenic composition is a composition containing an antigen (particularly a peptide containing an antigenic epitope) that is capable of generating an immune response against the antigen. The immune response generated may be a cellular (T cell-mediated) or humoral (B cell-mediated, antibody-producing) immune response. An immunogenic composition may also induce both a cellular and a humoral immune response.

[0074] 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). It can also combine a cytotoxic immune response with a helper cellular immune response. The helper response can involve Th1, Th2, or Th17 lymphocytes (these lymphocytes are capable of eliciting different cytokine responses, as is known in the art).

[0075] An immunogenic composition may better present the antigen present therein via the MHC1 or MHC2 pathway.

[0076] The immunogenic composition should contain a polypeptide or peptide comprising SEQ ID NO: 7. It may also be a mixture of polypeptides or peptides.

[0077] In some embodiments, an immunogenic composition comprises a nucleic acid encoding a polypeptide or peptide comprising SEQ ID NO: 7. Administration of such an immunogenic composition can result in in vivo expression of the polypeptide or peptide within cells and an immune response against the polypeptide or peptide. 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 an immunogenic composition.

[0078] adjuvant

[0079] "Adjuvant" refers to a substance that improves or enhances the immune response to an antigen. In other words, in the presence of an adjuvant, the immune response to the antigen may be higher or different than in its absence (this includes situations where the response is modified, for example, where a different T cell subset is activated in the presence of an adjuvant than in its absence). Various adjuvants are known in the art and are widely used in the field of vaccines.

[0080] Alum, emulsions (oil-in-water or water-in-oil, such as Freund's incomplete adjuvant (IFA) and ), 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 formulated with alum, TLR5 ligands, such as bacterial flagellin, TLR7 / 8 ligands, such as imidazoquinolines (i.e., imiquimod, gadiquimod 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 refers to an agonist of a receptor, i.e., a substance that binds to and activates a receptor, in particular TLR3 and TLR9 receptors.

[0081] As described in WO2017089529 or WO2021165306, melanin can be used as an adjuvant or in combination with other adjuvants. When melanin is used and another adjuvant is added, it is preferably selected from TLR3 agonists and TLR9 agonists, particularly when the further adjuvant is selected from polyinosinic acid: polycytidylic acid (poly I: C) and CpG oligonucleotides.

[0082] peptides

[0083] Peptide is an amino acid chain connected by peptide bonds. In the case of the present invention, the peptide should include 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, the peptide should include up to 100 amino acids, more preferably up to 50 amino acids, more preferably up to 30 amino acids, more preferably up to 25 amino acids. 10 to 25 amino acid peptides are relatively suitable. However, in other embodiments, the peptide (which may be referred to as a polypeptide) may include more than 100 amino acids. It may be a protein.

[0084] One or more amino acids in the peptide may be artificially synthesized (different from each of the 20 amino acids found in natural proteins). Such artificially synthesized amino acids may be D-amino acids, or non-natural amino acids (e.g., citrulline, hydroxyproline, norleucine, 3-nitrotyrosine, nitroarginine, ornithine, naphthylalanine, etc.).

[0085] The N-terminus and / or C-terminus of the peptide may be capped or modified. In particular, N-terminal acetylation or capping helps minimize degradation of the peptide by aminopeptidases, while C-terminal amidation helps stabilize the peptide and prevent degradation by carboxypeptidases.

[0086] Thus, in the context of the present invention, the biologically active peptide would contain SEQ ID NO: 7. As indicated, this sequence may be contained within a large protein or a longer peptide and further modified by glycosylation or terminal protection.

[0087] vaccine

[0088] In the context of the present invention, a vaccine refers to a composition that is administered to an animal to produce or artificially increase immunity against a specific antigen. Thus, it should be understood that the terms "immunogenic composition," "immunostimulatory composition," and "vaccine" can be used interchangeably.

[0089] Obtaining an immunogenic composition

[0090] Immunogenic compositions can be obtained by combining a polypeptide or peptide containing a modified epitope disclosed herein with an adjuvant.

[0091] In particular, the polypeptide or peptide carrying the modified epitope is combined with a melanin, in particular with a synthetic melanin as described herein.

[0092] The polypeptide or peptide can be added to a synthetic melanin solution as described above (polypeptide or peptide / melanin weight ratio between 1 / 1 and 1 / 10) and incubated for various times before use, the duration depending on the incubation temperature. The resulting solution can be washed and resuspended in water or any suitable buffer.

[0093] The binding of polypeptides or peptides to melanin can be verified by Tricine-SDS-PAGE analysis, as described in Carpentier (2017). Briefly, the sample (peptide-Mel or peptide alone) is loaded onto an acrylamide gel. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R-250 to quantify the free peptide in the gel. The binding of the peptide to melanin can be expressed as the following ratio: [amount of unbound peptide in the sample peptide-Mel / amount of peptide in the control sample containing only the peptide].

[0094] The immunostimulatory composition may further comprise another adjuvant as described above.In a preferred embodiment, the adjuvant is added to the resulting composition before administration, ie within one hour before administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 : Cross-reactivity of T cell responses obtained after immunization of transgenic SURE mice with SEQ 7. Mice were immunized with SEQ 7 as described in Table 1 and sacrificed on day 8. Splenocytes (5×10 5 cells / well) for 18 hours, and the number of IFNγ-SFCs (spot-forming cells) was measured after 18 hours of incubation. DETAILED DESCRIPTION

[0096] Example

[0097] To select an optimized TERT antigen that can be used in human patients, several peptides containing one of the known TERT immunotopes (SEQ ID NO: 1) were screened in a melanin-based vaccine as described in WO2021165306.

[0098] These peptides (SEQ ID NO: 4 to SEQ ID NO: 7; Table 2) all contain the UCP2 epitope (SEQ ID NO: 1) with an amino acid added to the NH2 terminus. As disclosed in WO2021165306, such modifications enhance the efficacy of melanin-based vaccines. The present application teaches that cysteine, acetylcysteine, methionine, proline, hydroxyproline, histidine, and lysine are preferred amino acids that can enhance peptide binding, with cysteine ​​being the preferred amino acid for optimal binding.

[0099] Table 2. List of sequences used and corresponding T cell responses obtained after subcutaneous immunization of transgenic Surel1 mice.

[0100]

[0101] *As described in Godet et al., Clin Can Res 2012 & Dosset et al., Clin Can Res 2012.

[0102] Example 1. Binding of peptides to melanin

[0103] L-DOPA (0.8 mg / ml) alone was polymerized at pH 8.5, 60°C, and stirred for 2 hours to form melanin. The reaction mixture was then filtered through a 10 kDa filter, and the retentate containing the synthesized melanin was resuspended in phosphate buffer, pH 7.5. Peptide was then added (melanin:peptide weight ratio: 2), and the mixture was incubated at room temperature for 18 hours.

[0104] Tricine-SDS-PAGE analysis was performed according to the method described in Carpentier 2017 (supra). Briefly, the sample (peptide-Mel or peptide alone) was loaded onto an acrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and imaged with ChemiDoc XRS+system (Bio-Rad Laboratory) to quantify the free peptide in the gel. The binding of the peptide to melanin can be expressed as the following ratio: [the amount of unbound peptide in the sample peptide-Mel / the amount of peptide in the control sample containing only the peptide]. As shown in Table 3, SEQ ID NO: 7 was observed to have binding ability, and unexpectedly, it was much higher than the binding observed for the unmodified epitope peptide SEQ ID NO: 1, and also higher than the binding observed for the modified peptides (e.g., SEQ ID NO: 5 and SEQ ID NO: 6) according to the preferred embodiment of WO2021165306.

[0105] Table 3: Binding of peptides to synthetic melanin.

[0106] 18 hours at 20℃ 3 weeks at 20℃ 1 hour at 60°C SEQ ID NO: 1 41％ 64％ 55％ SEQ ID NO: 4 59％ / / SEQ ID NO: 5 50％ / 55％ SEQ ID NO: 6 43％ / 54％ SEQ ID NO: 7 60％ 90％ 65％

[0107] L-DOPA (0.8 mg / ml) was oxidatively polymerized at 60°C for 2 hours under aerobic conditions at pH 8.5. The reaction mixture was then filtered through a 10 kDa filter, and the retentate containing the synthesized melanin was resuspended in pH 7.5. Peptide was then added (melanin:peptide weight ratio: 2), and the mixture was incubated at room temperature or 60°C for various times. Finally, the percentage of peptide bound to melanin was quantified by SPS-PAGE analysis.

[0108] Example 2. Immunogenicity of different peptides

[0109] The immunogenicity of these different peptides was screened by vaccination in HLA-A2 / DR1 mice (also known as the Surel 1 model), which are transgenic mice that mimic the human immune system.

[0110] For immunization, L-DOPA (0.8 mg / ml) was oxidatively polymerized at 60°C under aerobic conditions at pH 8.5 for 2 hours. The reaction mixture was then filtered through a 10 kDa filter, and the retentate containing the synthesized melanin was resuspended in water. The peptide (10 μg / mouse) was then added (peptide / L-DOPA weight ratio = 1 / 4), and after an 18-hour incubation period, the mixture was used for subcutaneous immunization of mice. Prior to immunization, the phosphorothioate oligonucleotide CpG-28 (5'-TAAACGTTATAACGTTATGACGTCAT, SEQ ID NO: 3) was added to the vaccine formulation (10 μg / mouse). Mice were sacrificed on day 8, and T cell responses were performed (as described in Carpentier, 2017). Briefly, splenocytes were restimulated in vitro with the corresponding peptide (not conjugated to melanin), and the number of IFNγ-SFCs (spot-forming cells) was measured and expressed as the mean ± SEM.

[0111] Surprisingly, it was observed that the peptide SEQ ID NO: 7 was highly effective in triggering an immune response after immunization in mice (Table 2). That is, compared to the most preferred modifications recommended in WO2021165306 (such as SEQ ID NO: 5 and SEQ ID NO: 6 with methionine or lysine at the NH2 terminus), SEQ ID NO: 7 was more effective and almost as effective as SEQ ID NO: 4 with a cysteine ​​at the NH2 terminus.

[0112] Under other conditions, SEQ ID NO: 7 also exhibited immunogenicity. Following immunization with 10 μg of antigen / mouse combined with the phosphorothioate oligonucleotide CpG-28 (SEQ ID NO: 3) or montanide on days 1 and 14, specific immune responses were observed on day 28: the CpG-28 and montanide groups produced 22 ± 5 and 15 ± 5 IFNγ-SFCs, respectively, compared to only 2 ± 1 in the epitope-independent control group (mean ± SEM, n = 8 / group, data from two independent experiments).

[0113] These experimental results indicate that SEQ ID NO: 7 carries an epitope that can be recognized by the immune system and is therefore immunogenic.

[0114] Example 3. Peptide stability and filterability in solution

[0115] For vaccine development, it is necessary to ensure that the drug substance has good stability (>95% change over time), because even trace amounts of peptide degradation products may cause undesirable toxicity when administered to patients, compromising the purity required for drug development.

[0116] To assess stability, the peptide was diluted in water at 0.5 mg / mL, adjusted to pH 7.4, and stored frozen at -20°C. On the designated date, the peptide was analyzed by HPLC (Hypersil GOLD C8 15 cm x 4.6 mm x 5 μm (ThermoFisher)) using an elution buffer of 15% CH3CN and 0.1% TFA. Peak areas were compared with those of reference standards.

[0117] As shown in Table 4, SEQ ID NO: 5 and SEQ ID NO: 4 are highly unstable at physiological pH (7.4) after oxidation; SEQ ID NO: 6 exhibits unexpectedly significant degradation under non-refrigerated conditions; whereas SEQ ID NO: 7 is very stable. In fact, after 7 days at -20°C, the peak area of ​​SEQ ID NO: 4 decreases by 53%, while SEQ ID NO: 7 is stable for at least 3 months (>95%) at -20°C, pH 7.4, and for at least 7 days at room temperature.

[0118] Table 4: Stability of peptides after 7 days of incubation in pH 7.4 solution at different temperatures (peak area on the specified day / peak area of ​​the same peptide in fresh solution) (average data of 2 or 3 different experiments + / - SD).

[0119] -20℃ 4℃ Room temperature SEQ ID NO: 1 100％+ / -0％ 99％+ / -1％ 96％+ / -4％ SEQ ID NO: 4 47％ Not conducted Not conducted SEQ ID NO: 5 100％+ / -0％ 84％+ / -22％ 67％+ / -43％ SEQ ID NO: 6 100％+ / -0％ 92％+ / -2％ 76％+ / -1％ SEQ ID NO: 7 98％+ / -2％ 100％+ / -0％ 100+ / -0％

[0120] Filterability of pharmaceutical preparations through a 0.2μ filter is crucial for obtaining sterile solutions. Surprisingly, SEQ ID NO: 7 showed particular relevance for further pharmaceutical development, as described in Example 2, because the peptide + melanin mixture did not precipitate, could be filtered through a 0.2μ filter, and remained suspended in an isotonic solution for over 18 hours, whereas SEQ ID NO: 6 precipitated under these conditions and could not be filtered.

[0121] Under preferred conditions, the weight ratio of the melanin of SEQ ID NO: 7: peptide mixture is higher than 2.5:1, preferably 2.7:1, to obtain optimal filterability on a 0.2μ filter.

[0122] In fact, lower ratios may result in partial precipitation accompanied by a slight decrease in the absorbance of the supernatant (measured by optical density at 220 nm).

[0123] Example 4. Verification of cross-reactivity of selected peptides

[0124] Therefore, SEQ ID NO: 7 exhibits good binding, immunogenicity, stability and filterability.

[0125] Since SEQ ID NO: 7 is different from the native epitope SEQ ID NO: 1, it was anticipated that this modification might generate a novel epitope that did not cross-react with the native epitope. Therefore, mice were immunized with SEQ ID NO: 7 to examine whether the T cell response triggered by SEQ ID NO: 7 could similarly recognize the native epitope SEQ ID NO: 1.

[0126] This is indeed the case ( Figure 1 ), thus demonstrating the cross-reactivity of the immune responses at the two epitopes.

[0127] References

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Claims

1. A peptide comprising SEQ ID NO:

7.

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

7.

3. The peptide according to claim 1, comprising a maximum of 50 amino acids. The peptide according to claim 1 , 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 the peptide according to claim 1 or the nucleic acid molecule according to claim 5 .

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

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

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

10. Use of the peptide according to any one of claims 1 to 7, the nucleic acid according to claim 5, or the vaccine composition according to any one of claims 6 to 8 for treating cancer.

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 glioblastoma, glioma, melanoma, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and thyroid cancer.

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

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

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