Vaccine for receptor-type protein tyrosine phosphatase Z1 (PTPRZ1)-positive tumors

Abstract

The present invention relates to a novel epitope of PTPRZ1 that exhibits good immunogenicity and is 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] PTPRZ1 is a protein tyrosine phosphatase (PTP) receptor expressed in brain tissues, including the cerebral cortex, cerebellum, hippocampus, and caudate nucleus. PTPRZ1 is located in glial cells and neurons.

[0003] The structure of PTPRZ1 consists of an N-terminal carbonic anhydrase domain, a fibronectin type III domain, a chondroitin sulfate (CS)-binding domain, a transmembrane segment, two phosphatase domains, and a C-terminal PDZ-binding motif. The extracellular region of PTPRZ1 is responsible for its binding to various ligands (e.g., PTN, MK, IL-34, and basic fibroblast growth factor), extracellular matrix proteins (e.g., tenascin-C and -R), and cell adhesion molecules. The CS chain consists of repeating disaccharide units of GlcA and GalNAc with sulfate groups at various positions on the sugar chain. Electrostatic repulsion between the negatively charged glycosaminoglycan moieties keeps PTPRZ1 as a monomer. PTN and MK contain positively charged regions that reduce the electrostatic repulsion of the CS chain after binding, thereby inducing dimerization and inactivating phosphatase activity. PTPRZ1 also plays a role in differentiation, cell motility, and myelination through binding to its ligands, and may play a role in demyelinating processes such as multiple sclerosis. The interaction of PTPRZ1 with PTN also influences the tumor microenvironment, namely M2 tumor-associated macrophages.

[0004] In cancer, especially glioma, PTPRZ1 can be involved in gene fusions (translocations); for example, 25 PTPRZ1 gene fusions have been described in various cancers (Woo et al., 2020). Gene fusions (PTPRZ1-MET, PTPRZ1-ETV1…) have been frequently reported in glioma (Woo et al., 2020).

[0005] Apart from fusion genes in cancer, several splice variants of PTPRZ1 have been described: a 9.5 kb long transmembrane form; a 6.4 kb short transmembrane form (lacking the extracellular domain); and two soluble secreted forms, also known as phosphacans, one short (4 kb) and one long (8.4 kb). These transcripts are differentially expressed in different cell types (Cannol 1996).

[0006] PTPRZ1 expression is particularly high in gliomas, present in melanoma, lung, and neck cancers, and lower in other cancer types. Immunohistochemistry has shown that PTPRZ1 is widely expressed throughout glioma tissue at both nuclear and cytoplasmic levels. PTPRZ1 expression was reported in 16 of 24 glioblastomas (66%), with coexpression of long and short PTPRZ1 in nearly all positive glioma samples (14 / 16) (Lorente et al., 2005). In gliomas, PTPRZ1 is overexpressed in glioma stem cells (GSCs) compared to non-tumor cells (Shy et al., 2017). Antibodies targeting PTPRZ1 have shown remarkable antitumor properties in several glioma models (Foehr et al., 2006; Shy et al., 2017), and knockdown experiments further support a role for PTPRZ1 in glioma tumorigenesis (Fujikawa et al., 2017).

[0007] Therefore, PTPRZ1 is considered a reasonable antigenic target for cancer vaccines. Hilf et al. (2019) described the epitope PTP-013 (MIWEHNVEV, SEQ ID NO: 4) and the fact that this epitope is naturally presented by HLA-A2 in patients. In the APVAC clinical trial for glioblastoma, reactive CD8+ T cells against this peptide were observed in some patients after immunization. Thus, this PTPRZ1-derived peptide was demonstrated to be an epitope capable of generating an immune response. In another clinical trial (IMA950), patients demonstrated CD8 T cell responses against various tumor peptides, including those derived from PTPRZ1. However, the immune response was weak and did not translate into clinical benefit (Migliorini et al., 2019). Other PTPRZ1 human epitopes have been described in the literature (Dutoit 2012; Neidert 2018). WO2015 / 063302 also disclosed that PTP-013 has some immunogenicity.

[0008] An optimal PTPRZ1 epitope for immunizing patients against PTPRZ1-positive tumors preferably exhibits the following two properties: - the epitope must be strongly immunogenic in humans, i.e., capable of inducing a strong immune response after immunization; and - The epitope should be located in a region that is common to the long and short isoforms, otherwise the tumor may escape the immune response by turning off the expression of the long isoform.

[0009] 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 achieved 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 difficult challenge. Finally, vaccine technologies such as emulsions, liposomes, nanoparticles, fusion molecules, or DNA and RNA vaccines can be either unstable or difficult to synthesize, resulting in high production costs. Adjuvants are commonly used to increase the immunogenicity of an administered antigen.

[0010] WO2017089529 discloses that melanin can be used as an adjuvant to increase the immune response to an antigen bearing the epitope.

[0011] WO2021165306 discloses that the addition of an amino acid having a nucleophilic residue to a peptide is useful for improving the immunogenicity of the peptide when complexed with melanin. Preferred amino acids are cysteine, acetylcysteine, methionine, proline, hydroxyproline, histidine, and lysine, with cysteine ​​being of particular interest. Summary of the Invention

[0012] The present inventors have modified the epitope PTP-013 disclosed by Hilf by mutating one of its residues, and have shown that this mutation increases the immune response generated when administered in vivo, and that T lymphocytes from animals immunized with this modified epitope can be re-stimulated with an unmodified peptide, thereby exhibiting cross-reactivity.The present inventors have shown that when the modified epitope is complexed with melanin, it generates a strong immune response by itself, especially after being modified by adding two lysines to its N-terminus.Using melanin as an adjuvant, the present inventors have shown that when the epitope is modified (even when modified by adding cysteine, lysine, or histidine to its terminus according to WO2021165306), immunogenicity is improved compared to the unmodified epitope.Notably, the addition of two lysines to the terminus of the modified epitope resulted in a very strong immune response.

[0013] In a first aspect, the present invention therefore relates to a polypeptide or peptide bearing this specific epitope. The present invention relates to a polypeptide or peptide comprising SEQ ID NO: 15 or a peptide consisting of SEQ ID NO: 15.

[0014] In another embodiment, the present invention relates to a polypeptide or peptide comprising or consisting of SEQ ID NO:14.

[0015] Both SEQ ID NO: 15 and SEQ ID NO: 14, which includes SEQ ID NO: 15, can be used to generate an immune response against PTPRZ1 that is useful in the treatment of cancer.

[0016] As shown below, a peptide preferably contains at most 100 amino acids, more preferably at most 50. If the peptide is larger than SEQ ID NO: 14 (resp. SEQ ID NO: 15), SEQ ID NO: 14 (resp. SEQ ID NO: 15) may be located at the N-terminus of the peptide or elsewhere within the peptide. However, a peptide may contain more than 50 or 100 amino acids. It may also be a protein.

[0017] In some embodiments, the polypeptide or peptide comprising SEQ ID NO: 14 or SEQ ID NO: 15 further comprises a CD4 or CD8 epitope of an antigen, particularly a cancer antigen, notably a cancer targeted by the immunogenic compositions disclosed herein. Other epitopes can be linked to SEQ ID NO: 14 or SEQ ID NO: 15 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 constructions. When melanin is used as an adjuvant, it is preferred if SEQ ID NO: 14 or SEQ ID NO: 15 is at the N-terminus of the polypeptide or peptide.

[0018] The other epitope may be another epitope derived from PTPRZ1, or may be an epitope derived from a protein other than PTPRZ1, the expression of which is associated with the same cancer as PTPRZ1.

[0019] 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 PTPRZ1 through a response to SEQ ID NO: 15.

[0020] 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, the nucleic acid can be protected from degradation by methods known in the art, particularly liposome encapsulation. It can be highly advantageous to incorporate the nucleic acid encoding SEQ ID NO:15 into a larger DNA or RNA sequence to be used as a vaccine, and which may encode other epitopes or an entire protein. Such constructs are within the scope of the present invention.

[0021] The polypeptides or peptides disclosed herein can be used to treat or prevent cancers in which PTPRZ1 expression is high, particularly glioblastoma, glioma, melanoma, lung and head and neck cancer, cervical cancer, or testicular cancer.

[0022] 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).

[0023] 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 injected into a mammal, particularly a human. In particular, administration can be intramuscular, intravenous, subcutaneous, intraperitoneal, 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 a patch.

[0024] 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 precursors) is used. In particular, the melanin is soluble melanin. In this embodiment, the peptide is conjugated to the melanin. The vaccine composition can be obtained by oxidative polymerization of melanin precursors in the presence of the peptide, as disclosed in WO2017089529, or by adding the peptide to already synthesized melanin, as disclosed in WO2021165306.

[0025] The present invention also relates to a polypeptide, peptide, nucleic acid, or vaccine composition disclosed herein for use in the treatment of cancer.

[0026] 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.

[0027] Vaccines can be prophylactic (i.e., intended to protect the recipient from developing disease) or therapeutic (i.e., intended to help the recipient combat existing disease), with the disease being linked to a target antigen (PTPRZ1) that is expressed or presented by cells during the course of the disease.

[0028] 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 PTPRZ1, which then attacks and eliminates tumor cells, thereby providing a therapeutic effect.

[0029] 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 cancer-associated PTPRZ1, wherein the immune response has a protective (prophylactic) effect against the cancer.

[0030] In particular, the cancer is a low-grade or high-grade glial tumor.

[0031] In another embodiment, the cancer is melanoma.

[0032] In another embodiment, the cancer is head and neck cancer.

[0033] In another embodiment, the cancer is lung cancer.

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

[0035] In another embodiment, the cancer is testicular cancer. DETAILED DESCRIPTION OF THE INVENTION

[0036] 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 without administration of the composition. An effective amount is an amount that provides therapeutic improvement while minimizing side effects or adverse effects. An effective amount of antigen can be 10 μg to 5 mg, preferably 100 μg to 500 μg. When melanin is used in a vaccine formulation, the amount of melanin that can be used may be 50 μg to 10 mg, particularly 500 μg to 2 mg, or 200 μg to 1 mg.

[0037] The resulting vaccine formulation can be used to protect animals from diseases that are implicated in (i.e., involve and / or involve) cells that internally express, surface express, or secrete PTPRZ1.

[0038] Alternatively, the polypeptide, peptide, nucleic acid, or immunostimulatory composition 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 against the antigen PTPRZ1 in the recipient. 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.

[0039] Therefore, the present invention relates to an in vitro method for priming or stimulating CD8 lymphocytes against PTPRZ1, which comprises contacting and incubating the polypeptide, peptide, or nucleic acid molecule disclosed herein, or immunogenic composition, 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 cancers associated with PTPRZ1.

[0040] The present invention also relates to methods for detecting an immune response to PTPRZ1 (particularly to the epitope set forth in SEQ NO: 15) 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 PTPRZ1 antigen present in the immunostimulatory composition. Such a response can be detected by measuring molecules secreted by lymphocytes, such as cytokines, particularly gamma-interferon.

[0041] The present invention also relates to a method for detecting an immune response to PTPRZ1 (particularly to the epitope set forth in SEQ ID NO: 15) 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 the specific immune response of the patient's lymphocytes to the PTPRZ1 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 therefore present the epitome set forth in SEQ ID NO: 15 on their surface via MHC I molecules.

[0042] These methods are carried out in vitro.

[0043] melanin " melaninIt 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 with hydrogen peroxide and are thus commercially available, for example, from Sigma-Aldrich.

[0044] In the context of the present application, the preferred melanin is eumelanin.

[0045] Process for obtaining synthetic melanin Synthetic melanin is obtained in vitro after oxidative polymerization of melanin precursors.

[0046] 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.

[0047] The mixture is also exposed to an oxidizing agent (oxidizer or oxidizing agent) as disclosed below to promote polymerization and yield synthetic melanin.

[0048] In particular, the skilled artisan can optimize various parameters such as the ratio of melanin precursor mixtures when used, the type of oxidizing agent, the pH, the buffer, the length of incubation, or the temperature of the reaction.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In a preferred embodiment, the synthetic melanin is a soluble melanin, ie, in the form of particles less than 500 nm.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Other melanin precursors or derivatives thereof are described in the art, such as the products described in WO2017089529.

[0059] 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.

[0060] 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.

[0061] Therefore, it is preferred if the oxidizing agent is selected from the group consisting of oxygen, hydrogen peroxide, ammonium persulfate, and ferric ions.

[0062] Vaccines, immunogenic or immunostimulatory compositions " Vaccines, 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.

[0063] However, the immunogenic composition is preferably used in a mammal, which is preferably a human, but may also be other mammals if the composition is used in the veterinary field, particularly to induce immunity in livestock such as cattle (cows), sheep, goats or horses, but also in pets such as dogs or cats.

[0064] 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.

[0065] 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).

[0066] The immunogenic composition may allow for better presentation of antigens present therein through the MHC1 or MHC2 pathway.

[0067] The immunogenic composition may comprise a polypeptide or peptide comprising SEQ ID NO: 14 or SEQ ID NO: 15. It may also be a mixture of polypeptides or peptides.

[0068] In some embodiments, the immunogenic composition comprises a nucleic acid encoding a polypeptide or peptide comprising SEQ ID NO: 14 or SEQ ID NO: 15. 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: 14 or SEQ ID NO: 15, and the resulting cell composition can be used as the immunogenic composition.

[0069] 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.

[0070] 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 preferably describes a receptor agonist, i.e., a substance that binds to and activates a receptor, in particular the TLR3 and TLR9 receptors. The melanin described herein acts as an adjuvant, and it is also possible to use another adjuvant.

[0071] When melanin is used and another adjuvant is added, it is preferably selected from the group consisting of TLR3 agonists and TLR9 agonists, and particularly when this further adjuvant is selected from polyinosinic:polycytidylic acid (poly I:C) and CpG oligonucleotides.

[0072] 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.

[0073] 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.).

[0074] 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.

[0075] Thus, in the context of the present invention, biologically active peptides should comprise the epitope shown in SEQ ID NO: 15 or SEQ ID NO: 14. As indicated, these sequences may be comprised in larger proteins or longer peptides, which may be further modified with glycosylation or end-blocking.

[0076] 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.

[0077] 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.

[0078] In particular, the polypeptide or peptide bearing the modified epitope is combined with a melanin, particularly a synthetic melanin as described herein.

[0079] 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, preferably at room temperature, before use. The resulting solution can be washed and resuspended in water or any suitable buffer.

[0080] 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:

[0081] 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]

[0082] [Figure 1] Cross-reactivity of T cell responses obtained after immunization with SEQ 14 in transgenic SURE mice. Mice were immunized against SEQ 14 as described in Table 1 and sacrificed on day 8. Spleen cells (5.10^5 cells / well) were restimulated in vitro with either SEQ 14 or the native epitope SEQ 4 (neither conjugated to melanin) at various concentrations (5 μg / ml to 0.3 ng / ml) for 18 hours, and the number of IFNg-SFCs (spot-forming cells) was measured after 18 hours of culture (representative experiment from three experiments). [Example]

[0083] Example 1. Peptide screening To select suitable PTPRZ1 antigens for use in human patients, several peptides containing various PTPRZ1 immune epitopes were screened. The immunogenicity of these various peptides was screened by vaccination in HLA-A2 / DR1 mice (also known as the Surel1 model). HLA-A2 / DR1 mice are transgenic mice that mimic the human immune system. As a vaccine procedure for screening, a melanin-based vaccine was used, as described in WO2021165306.

[0084] As previously described (Dutoit, 2012; Hilf, 2018; Neidert, 2018), a small number of potential CD8 epitopes were identified within the human PTPRZ1 sequence (SEQ ID NO: 1–SEQ ID NO: 4, Table 1). Three of these four epitopes were located in regions common to the long and short PTPRZ1 isoforms.

[0085] These epitopes were then screened using a melanin-based vaccine in HLA-A2 / DR1 mice using peptides (SEQ ID NO: 5 to SEQ ID NO: 8) containing the epitopes and an additional cysteine ​​at the NH2-terminus (as this modification was disclosed in WO2021165306 to enhance the efficacy of melanin-based vaccines).

[0086] Although SEQ ID NO: 5 induced a strong immune response, this epitope is not present in the shorter PTPRZ1 isoform.

[0087] SEQ ID NO: 8 was common to both the long and short isoforms and was able to elicit an immune response (Table 1).

[0088] Because the immune response obtained after immunization with SEQ 8 was weak, a few amino acids were modified within its NH2-terminal sequence.

[0089] The immunogenicity of the modified peptides (SEQ ID NO: 9 to SEQ ID NO: 14) was screened after immunization in mice.

[0090] Surprisingly and unexpectedly, it was observed that only peptide SEQ ID NO: 14 elicited a strong CD8 immune response after immunization in mice, despite the fact that the modifications involved part of the epitope sequence itself (SEQ ID NO: 4) (Table 1).

[0091] Table 1. List of sequences used and the corresponding T cell responses obtained after subcutaneous immunization of transgenic Surel1 mice. TIFF2026502065000001.tif1311381. Described in Dutoit 2012 and Hilf 2018. Not included in short isoforms. 2. Described in Neidert 2018. Located in the intracellular portion of PTPRZ1. 3. As described in Dutoit 2012 and Hilf 2018. 4. Described in Hilf 2018. Located in the intracellular portion of PTPRZ1.

[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 phosphate buffer. A peptide (10 μg / mouse) containing the epitope (underlined) was then added (peptide / L-dopa = 1 / 4 by weight), and the mixture was used for subcutaneous immunization in mice. The phosphorothioate oligonucleotide CpG-28 TIFF2026502065000002.tif4128 was added to the vaccine formulation (10 μg / mouse) immediately prior to immunization. Mice were sacrificed on day 8, and CD8 T cell responses were performed as described by Carpentier et al. (2017). Briefly, splenocytes were restimulated in vitro with the corresponding MHC class I epitope (not conjugated to melanin), and the number of IFNg-SFCs (spot-forming cells) was determined and expressed as mean + / - SEM.

[0093] Example 2. Verification of cross-reactivity of novel epitopes Because SEQ ID NO:14 has been modified from the natural epitope SEQ ID NO:4, it can be expected that this modification may result in an entirely new epitope that has no cross-reactivity with the native epitope.

[0094] Therefore, mice were immunized with SEQ ID NO: 14 to determine whether the T cell response elicited by SEQ ID NO: 14 could similarly recognize the mutant epitope SEQ ID NO: 15 and the native epitope SEQ ID NO: 4.

[0095] This was shown to be the case (Figure 1), thus demonstrating the cross-reactivity of the immune response to both epitopes.

[0096] Surprisingly, the reactivity of lymphocytes primed with the modified epitope (SEQ ID NO: 14) to the native epitope (SEQ ID NO: 4) was even higher than the reactivity to the modified epitope (SEQ ID NO: 14 or SEQ ID NO: 15) used to immunize the animals.

[0097] Modifying amino acids within epitopes to make them more immunogenic, particularly by changing amino acids located in the "anchor" region (which binds the epitope to MHC class I, usually the second and ninth positions of a 9-mer epitope), has been of interest for many years (Hebeisen M et al. 2013), but no clear rules for optimizing a given epitope have been defined.

[0098] However, in the present application, the reactivity of primed lymphocytes to the native epitope (SEQ ID NO: 4) was higher than that to the mutant epitope (SEQ ID NO: 14), suggesting a lower affinity for the latter (Figure 1).

[0099] These results indicate that the modifications described in SEQ ID NO:14 or SEQ ID NO:15 deviate from the classical anchor region and are not expected to increase epitope affinity when tested with binding prediction software, such as those disclosed in the IEDB (Immune Epitope Database and Analysis resource), an NIAID-funded resource that catalogs experimental data on antibody and T cell epitopes and hosts tools to aid in epitope prediction and analysis. Epitope generation is a collaborative service between CBS, ISIM, and LIAI, available at http: / / tools.immuneepitope.org / mhci / , which allows the generation of peptides of any length. services.healthtech.dtu.dk / service.php?NetMHC-4.0

[0100] This modification to SEQ ID NO:4 thus had entirely unexpected results.

[0101] Example 3. Immunogenicity of SEQ ID NO: 14 SEQ ID NO: 4 and SEQ ID NO: 14 were tested in HLA-A2 / DR1 mice in various adjuvant formulations.

[0102] The peptide (10 μg / mouse) was mixed with the TLR9 agonist CpG-28 (SEQ ID NO: 16) (10 μg / mouse), and the mixture was used for subcutaneous immunization in mice. Mice were sacrificed on day 8, and CD8 T cell responses were performed as described in Carpentier et al. (2017). Briefly, splenocytes were restimulated in vitro with the corresponding MHC class I epitope (not conjugated with melanin), and the number of IFNg-SFCs (spot-forming cells) was measured and expressed as mean + / - SEM.

[0103] It was observed that even in the absence of melanin, peptide SEQ ID NO: 14 elicited a stronger CD8 immune response after immunization in mice than (SEQ ID NO: 4).

[0104] Table 2. T cell responses obtained after subcutaneous immunization in mice. TIFF2026502065000003.tif35138

[0105] This data demonstrates that SEQ ID NO:14 is immunogenic and that such immunogenicity is not limited to use within melanin formulations.

[0106] References TIFF2026502065000004.tif184144

Claims

1. A peptide comprising SEQ ID NO:

15.

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

15.

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

14.

4. 4. The peptide of claim 3, consisting of SEQ ID NO:

14.

5. 4. The peptide of claim 1 or 3, comprising at most 50 amino acids.

6. 10. The peptide of claim 1, 3 or 5, further comprising an epitope of a cancer antigen.

7. A nucleic acid molecule encoding the peptide according to any one of claims 1 to 6.

8. A vaccine composition comprising a peptide according to any one of claims 1 to 6 or a nucleic acid molecule according to claim 7.

9. 9. The vaccine composition according to claim 8, comprising a peptide according to any one of claims 1 to 6 and an adjuvant.

10. 10. The vaccine composition of claim 9, wherein the adjuvant is melanin.

11. A peptide according to any one of claims 1 to 6, a nucleic acid according to claim 7 or a vaccine composition according to claim 9 or 10 for use as a medicament or as a vaccine.

12. A peptide according to any one of claims 1 to 6, a nucleic acid according to claim 7, or a vaccine composition according to claim 9 or 10, for use in the treatment of cancer.

13. 13. The peptide of any one of claims 1 to 6, the nucleic acid of claim 7, or the vaccine composition of claim 9 or 10, for use according to claim 12, wherein the cancer is selected from the group consisting of glioblastoma, glioma, melanoma, lung cancer, head and neck cancer, cervical cancer, and testicular cancer.

14. 13. The peptide of any one of claims 1 to 6, the nucleic acid of claim 7, or the vaccine composition of claim 9 or 10, for use according to claim 12, wherein the cancer is a low-grade or high-grade glial tumor.

15. An in vitro method for detecting, priming, or stimulating CD8 lymphocytes directed against PTPRZ1, comprising the step of contacting antigen-presenting cells and T lymphocytes with a peptide described in any one of claims 1 to 6 or a nucleic acid molecule described in claim 7, optionally together with an adjuvant.

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